阅读说明：本技术 人源抗人tigit抗体及其应用 (Human anti-human TIGIT antibody and application thereof ) 是由 杜鹏 杨志新 韩冬 徐银凤 岳俊杰 余云舟 陆建昇 于 2020-12-07 设计创作，主要内容包括：本发明公开了人源抗人TIGIT抗体及其应用。本发明公开的抗人TIGIT抗体,含有重链可变区V-H和轻链可变区V-L,V-H和V-L均由决定簇互补区和框架区组成；V-H和V-L的决定簇互补区均由CDR1、CDR2和CDR3组成；V-H的CDR1、CDR2和CDR3的氨基酸序列如SEQ ID No.10的第30-35位、第50-66位和第99-107位氨基酸所示；V-L的CDR1、CDR2和CDR3的氨基酸序列如SEQ ID No.9的第23-33位、第49-55位和第88-96位氨基酸所示。本发明的抗体能够高亲和力结合TIGIT,能够有效阻断CD155与TIGIT的结合,并具有抗肿瘤活性。(The invention discloses a human anti-human TIGIT antibody and application thereof. The invention discloses an anti-human TIGIT antibody, which contains a heavy chain variable region V H And light chain variable region V L ，V H And V L Both consist of a determinant complementary region and a framework region; v H And V L Each of the determinant complementary regions of (a) consists of a CDR1, a CDR2 and a CDR 3; v H The amino acid sequences of the CDR1, CDR2 and CDR3 of (A) are shown as amino acids at positions 30-35, 50-66 and 99-107 of SEQ ID No. 10; v L The amino acid sequences of the CDR1, CDR2 and CDR3 are shown in the 23 rd to 33 th, 49 th to 55 th and 88 th to 96 th positions of SEQ ID No.9Amino acid at position (v). The antibody disclosed by the invention can be combined with TIGIT (tungsten inert gas) with high affinity, can effectively block the combination of CD155 and TIGIT, and has antitumor activity.)

1. anti-TIGIT antibody comprising heavy metalVariable region V_HAnd light chain variable region V_LSaid V is_HAnd V_LBoth consist of a determinant complementary region and a framework region;

the V is_HAnd said V_LEach of the determinant complementary regions of (a) consists of a CDR1, a CDR2 and a CDR 3;

the V is_HThe amino acid sequence of CDR1 of (1) is shown as amino acids 30-35 of SEQ ID No. 10;

the V is_HThe amino acid sequence of CDR2 of (1) is shown as amino acids 50-66 of SEQ ID No. 10;

the V is_HThe amino acid sequence of CDR3 of (1) is shown as amino acids 99-107 of SEQ ID No. 10;

the V is_LThe amino acid sequence of CDR1 of (1) is shown as amino acids 23-33 of SEQ ID No. 9;

the V is_LThe amino acid sequence of CDR2 of (1) is shown as amino acids 49-55 of SEQ ID No. 9;

the V is_LThe amino acid sequence of CDR3 of (1) is shown as amino acids 88-96 of SEQ ID No. 9.

2. The antibody of claim 1, wherein: the V is_HThe amino acid sequence of (A) is shown as 1-118 of SEQ ID No.10 in the sequence table; the V is_LThe amino acid sequence of (A) is shown as 1-108 of SEQ ID No.9 in the sequence table.

3. The antibody of claim 1 or 2, characterized in that: the anti-TIGIT antibody is a) or b) or c) or d) as follows:

a) from V as described in claim 1 or 2_HAnd said V_LLinking the obtained single-chain antibody;

b) a fusion antibody comprising a) said single chain antibody;

c) comprising V as defined in claim 1 or 2_HAnd said V_LThe Fab of (1);

d) comprising V as defined in claim 1 or 2_HAnd said V_LThe whole antibody of (1).

4. The antibody of claim 3, wherein: a) the amino acid sequence of the single-chain antibody is shown as amino acids 1-247 of SEQ ID No. 6;

c) the Fab consists of a Fab heavy chain and a light chain, wherein the amino acid sequence of the Fab heavy chain is shown as 1 st to 221 th positions of SEQ ID No.10, and the amino acid sequence of the light chain is shown as SEQ ID No. 9;

d) the complete antibody consists of a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID No.10, and the amino acid sequence of the light chain is shown as SEQ ID No. 9.

5. The biomaterial related to anti-TIGIT antibody of any one of claims 1 to 4, being any one of B1) to B12):

B1) a nucleic acid molecule encoding the anti-TIGIT antibody of any one of claims 1-4;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic animal cell line comprising the nucleic acid molecule of B1);

B10) a transgenic animal cell line comprising the expression cassette of B2);

B11) a transgenic animal cell line containing the recombinant vector of B3);

B12) a transgenic animal cell line comprising the recombinant vector of B4).

6. The biomaterial of claim 5, wherein:

the V in the anti-TIGIT antibody_HThe coding sequence of CDR1 is shown in the sequence tableShown at positions 88-105 of SEQ ID No. 8;

the V in the anti-TIGIT antibody_HThe coding sequence of CDR2 is shown as position 148-198 of SEQ ID No.8 in the sequence table;

the V in the anti-TIGIT antibody_HThe coding sequence of CDR3 is shown as position 295-321 of SEQ ID No.8 in the sequence table;

the V in the anti-TIGIT antibody_LThe coding sequence of CDR1 is shown in 67-99 of SEQ ID No.7 of the sequence table;

the V in the anti-TIGIT antibody_LThe coding sequence of CDR2 is shown as position 145-165 of SEQ ID No.7 in the sequence table;

the V in the anti-TIGIT antibody_LThe coding sequence of CDR3 is shown as position 262-288 of SEQ ID No.7 in the sequence table;

further, in the present invention,

the V in the anti-TIGIT antibody_HThe coding sequence of (A) is shown as 1-354 of SEQ ID No.8 in the sequence table; the V in the anti-TIGIT antibody_LThe coding sequence of (A) is shown in 1-324 of SEQ ID No.7 in the sequence table.

7. The biomaterial according to claim 5 or 6, characterized in that:

the anti-TIGIT antibody is a single-chain antibody, and the coding sequence of the single-chain antibody is shown as 1 st-741 th bit of SEQ ID No.5 in a sequence table;

the anti-TIGIT antibody is Fab, the Fab consists of a Fab heavy chain and a light chain, the coding sequence of the Fab heavy chain is shown as the 1 st-663 th position of SEQ ID No.8, and the coding sequence of the light chain is shown as SEQ ID No. 7;

the anti-TIGIT antibody is a complete antibody, the complete antibody consists of a heavy chain and a light chain, the coding sequence of the heavy chain is shown as SEQ ID No.8, and the coding sequence of the light chain is shown as SEQ ID No. 7.

8. A composition, being any one of P1-P6:

p1, a composition consisting of the anti-TIGIT antibody of any one of claims 1-4 and an anti-PD-1 antibody;

p2, a composition consisting of a biomaterial according to any one of claims 5 to 7 and an anti-PD-1 antibody.

P3, a composition consisting of the anti-TIGIT antibody of any one of claims 1 to 4 and an immune cell;

p4, a composition consisting of the biomaterial of any one of claims 5-7 and an immune cell;

p5, a composition consisting of the anti-TIGIT antibody of any one of claims 1-4, an immune cell, and an anti-PD-1 antibody;

p6, a composition consisting of the biological material of any one of claims 5 to 7, an immune cell and an anti-PD-1 antibody.

9. A product for treating and/or preventing tumors, which comprises any one of M1-M8 as an active ingredient:

m1, the anti-TIGIT antibody of any one of claims 1 to 4;

m2, the biomaterial of any one of claims 5-7;

m3, a composition consisting of the anti-TIGIT antibody of any one of claims 1-4 and an anti-PD-1 antibody;

m4, a composition consisting of the biomaterial of any one of claims 5-7 and an anti-PD-1 antibody;

m5, a composition consisting of the anti-TIGIT antibody of any one of claims 1 to 4 and an immune cell;

m6, a composition consisting of the biomaterial of any one of claims 5-7 and an immune cell;

m7, a composition consisting of the anti-TIGIT antibody of any one of claims 1-4, an immune cell, and an anti-PD-1 antibody;

m8, a composition consisting of the biomaterial of any one of claims 5 to 7, an immune cell and an anti-PD-1 antibody.

10. Any of the following applications:

use of N1 or the anti-TIGIT antibody of any one of claims 1-4 in the preparation of a tumor suppressor or a tumor cell suppressor;

use of N2, the biomaterial of any one of claims 5 to 7 in the preparation of a tumor suppressor or a tumor cell suppressor;

the use of N3 or the composition of claim 8 for the preparation of a tumor suppressor or a tumor cell suppressor;

n4, the use of the product of claim 9 in the preparation of a tumor suppressor or a tumor cell suppressor.

Technical Field

The invention relates to a human anti-human TIGIT antibody and application thereof in the field of biomedicine.

Background

Tumor Immunotherapy (Cancer Immunotherapy) is a leading hot area of fundamental and clinical research in tumor therapy in recent years. In 2011, the CTLA-4 inhibitor Ipilimumab (Ipilimumab, also known as "Y drug") was approved for marketing, which exerts an anti-tumor effect by activating a T cell-mediated immune response; thus, a new era of tumor immunotherapy is opened. PD-1/PD-L1 antibodies such as Nivolumab (also called "O drug") and palboclizumab (also called "K drug") were developed in succession since 2014, and have achieved remarkable therapeutic effects, profoundly changing the paradigm of cancer therapy. CTLA-4 drug development facilitators James Allison, american scientists, and molecular discoverer PD-1/PD-L1, Tasuku Honjo, japan scientist, also have acquired a nobel physiology or medical prize in 2018 due to their contribution to "treating cancer by inhibiting negative immune regulation". The success of the first generation Immune Checkpoint Inhibitor (ICB) represented by CTLA-4 and PD-1/PD-L1 monoclonal antibodies not only drives the rapid increase of drug research and development investment, but also promotes the deep research of tumor Immune mechanism and immunotherapy strategy.

At the same time, the intensive search of clinical practice has also gradually revealed the outstanding problems faced by the first generation of immune checkpoint antibodies: beneficial patients haveLimited, poor effect of most tumors, drug tolerance, etc. In the case of PD-1/PD-L1 monoclonal antibody, for most tumor types, only less than 20% of patients actually benefit from treatment; at the same time, a significant fraction of patients with effective early treatment experience tumor recurrence during or after treatment. According to the differences of immunophenotype of Tumor Microenvironment (TME), the current mainstream theory divides tumors into four types of ' hot ' Tumor (inflammatory Tumor), ' cold ' Tumor (immune ' desert), ' immunosuppressive Tumor and ' immune-blocking Tumor ((TM))Galon and Daniela Brunii, nat. Rev. drug Discov.2019,18: 197-218.). Clinical research results show that the response rate of the 'hot' tumor with a large amount of immune cell infiltration in the tumor parenchyma to ICB antibody single drug treatment is high, and the congenital drug resistance probability is low. In addition, the selective pressure developed by immunotherapy remodels the tumor microenvironment, and then evolves a new immunosuppressive mechanism, causing a shift in tumor immunophenotype (immune-altered), which may be one of the important mechanisms for tumor immune escape and drug resistance. In this dilemma, the research focus of academia and industry has focused on three main directions: (1) discovery of a new mechanism and development of a new target; (2) the research of a combined medication mechanism and the innovative design of a clinical scheme; (3) development of new biomarkers; and the like.

T cell immunoglobulins and ITIM domain proteins (TIGIT, also known as WUCAM, Vstm3 or VSIG9) are checkpoint molecules that are expressed predominantly on the surface of immune cells such as NK cells, T cells, Treg cells, etc. Human TIGIT consists of an extracellular IgV-like domain, a type I transmembrane domain, and intracellular Immunoreceptor Tyrosine Inhibitory Motif (ITIM) and immunoreceptor tyrosine tail motif (ITT), which are typical inhibitory receptor proteins. TIGIT competes with the co-inhibitory receptor CD96, the co-stimulatory receptor CD226 (also known as DNAM-1) for binding to ligands CD155 (also known as PVR or necl-5) and CD112 (also known as nectin-2). CD155 is a high affinity core ligand for TIGIT, which interacts with TIGIT much higher than other partner molecules in the pathway. Structural biology studies have shown that molecular pairs such as CD155/TIGIT, CD155/CD226, CD155/CD96 interact through extracellular IgV-like domains in a conserved pattern called "Lock-Key" (Lock and Key); the interacting epitopes cover the entire spatial area between the "lock-key".

TIGIT is a novel immune checkpoint molecule, the regulatory mechanism of which is similar to PD-1/PD-L1. Under normal physiological conditions, negative immune regulation of CD155/TIGIT can be considered as another complement to MHC class I mechanisms to protect normal cells of epithelial origin (broadly expressing CD155) and the like from NK cells. However, this mechanism is also "stolen" by tumor cells to avoid the killing of the immune system and achieve immune escape. Early studies revealed high expression and tumor infiltration of TIGIT CD8⁺Correlation of T cell depletion and observed that TIGIT blocker reversed CD8 in a mouse tumor-bearing model⁺Good effects on T cell depletion and enhancement of antitumor activity; the TIGIT antibody and the PD-1/PD-L1 antibody are combined to show remarkable antitumor activity, and the synergy between the CD155/TIGIT and the PD-1/PD-L1 pathway is suggested. Meanwhile, in a large number of studies aiming at clinical patients, significant improvement of TIGIT expression level on the surfaces of T cells infiltrated by tumors and NK cells is also observed; the expression of CD155 in tumor tissues is also remarkably increased, and is closely related to the stage of TNM, the overall survival rate, the recurrence rate and the like.

With the progress of research, the action mechanism of TIGIT in the regulation of tumor immune negativity is gradually clarified, and can be summarized into three aspects: (1) TIGIT is highly expressed on the surfaces of NK cells and effector T cells related to tumor infiltration or migration, CD155 is highly expressed on the surface of the tumor cells, and the tumor cells directly act on the NK cells and the effector T cells through the combination of CD155/TIGIT to inhibit the activity of the NK cells and the effector T cells, so that the function of the NK cells and the effector T cells is exhausted; (2) treg cells in a tumor microenvironment inhibit inflammatory reaction by secreting anti-inflammatory factors such as IL-10 and the like; the tumor cells are combined with TIGIT with high expression on the surface of the Treg through CD155, the function of the Treg is promoted, and the inflammatory reaction of a tumor microenvironment is further inhibited; (3) the NK cell and the T effector cell surface TIGIT form a competitive competition for the combination of a co-stimulation receptor CD226(DNAM-1) and CD155 on the one hand, and inhibit the expression of the CD226 on the other hand, so that the function of the CD226 is lost, and the activity of immune effector cells is further inhibited. The reason why TIGIT has a clearly opposite regulatory function (suppression and promotion) on immune effector cells and Treg cells is not clear in the aforementioned (1) (2).

In addition, researches in a mouse model also show that the expression of TIGIT is closely related to NK cells, the T cell exhaustion is closely related to the NK cell exhaustion, and the therapeutic effects of the PD-1/PD-L1 antibody and the TIGIT antibody depend on the existence of the NK cells; the key role of NK cells in multiple immunosuppression and the great potential of TIGIT as an immunotherapy target are deeply revealed, and an important theoretical basis is provided for clinical combined use of immune checkpoint antibodies.

According to the gradually clear action mechanism of TIGIT, through an immunotherapy antibody targeting TIGIT, the immunosuppression signal mediated by a CD155/TIGIT channel is blocked, and NK cells and CD8 capable of reversing tumor infiltration⁺The exhaustion of T cells releases the tumor killing activity of immune effector cells, and good anti-tumor curative effect is expected to be obtained.

Disclosure of Invention

The invention aims to provide an anti-TIGIT antibody.

The anti-TIGIT antibody provided by the invention contains a heavy chain variable region V_HAnd light chain variable region V_LSaid V is_HAnd V_LBoth consist of a determinant complementary region and a framework region;

the V is_HAnd said V_LEach of the determinant complementary regions of (a) consists of a CDR1, a CDR2 and a CDR 3;

the V is_HThe amino acid sequence of CDR1 of (1) is shown as amino acids 30-35 of SEQ ID No. 10;

the V is_HThe amino acid sequence of CDR2 of (1) is shown as amino acids 50-66 of SEQ ID No. 10;

the V is_HThe amino acid sequence of CDR3 of (1) is shown as amino acids 99-107 of SEQ ID No. 10;

the V is_LThe amino acid sequence of CDR1 of (1) is shown as amino acids 23-33 of SEQ ID No. 9;

the V is_LThe amino acid sequence of CDR2 of (1) is shown as amino acids 49-55 of SEQ ID No. 9;

the V is_LThe amino acid sequence of CDR3 of (1) is shown as amino acids 88-96 of SEQ ID No. 9.

In the above antibody, the V_HThe amino acid sequence of (A) can be shown as 1-118 of SEQ ID No.10 in the sequence table; the V is_LThe amino acid sequence of (A) can be shown as 1-108 of SEQ ID No.9 in the sequence listing.

The anti-TIGIT antibody may be a) or b) or c) or d) as follows:

a) from V as described in claim 1 or 2_HAnd said V_LLinking the obtained single-chain antibody;

b) a fusion antibody comprising a) said single chain antibody;

c) comprising V as defined in claim 1 or 2_HAnd said V_LThe Fab of (1);

d) comprising V as defined in claim 1 or 2_HAnd said V_LThe whole antibody of (1).

In the antibody, the amino acid sequence of the single-chain antibody of a) can be shown as amino acids 1-247 of SEQ ID No. 6;

c) the Fab consists of a Fab heavy chain and a light chain, wherein the amino acid sequence of the Fab heavy chain can be shown as the 1 st to 221 th positions of SEQ ID No.10, and the amino acid sequence of the light chain can be shown as the SEQ ID No. 9;

d) the complete antibody consists of a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain can be shown as SEQ ID No.10, and the amino acid sequence of the light chain can be shown as SEQ ID No. 9.

The invention also provides a biomaterial related to the anti-TIGIT antibody, wherein the biomaterial is any one of B1) to B12):

B1) a nucleic acid molecule encoding the anti-TIGIT antibody;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic animal cell line comprising the nucleic acid molecule of B1);

B10) a transgenic animal cell line comprising the expression cassette of B2);

B11) a transgenic animal cell line containing the recombinant vector of B3);

B12) a transgenic animal cell line comprising the recombinant vector of B4).

In the above biomaterial, the V in the anti-TIGIT antibody_HThe coding sequence of CDR1 of (A) can be represented by positions 88-105 of SEQ ID No.8 of the sequence Listing;

the V in the anti-TIGIT antibody_HThe coding sequence of CDR2 of (1) can be shown as position 148-198 of SEQ ID No.8 in the sequence Listing;

the V in the anti-TIGIT antibody_HThe coding sequence of CDR3 of (1) can be shown as position 295-321 of SEQ ID No.8 in the sequence Listing;

the V in the anti-TIGIT antibody_LThe coding sequence of CDR1 of (A) can be represented by positions 67-99 of SEQ ID No.7 of the sequence Listing;

the V in the anti-TIGIT antibody_LThe coding sequence of CDR2 of (1) can be shown as position 145-165 of SEQ ID No.7 of the sequence Listing;

the V in the anti-TIGIT antibody_LThe coding sequence of CDR3 of (1) can be represented by position 262-288 of SEQ ID No.7 of the sequence Listing.

Further, in the present invention,

the V in the anti-TIGIT antibody_HThe coding sequence of (A) can be shown as 1-354 in SEQ ID No.8 in the sequence table; the V in the anti-TIGIT antibody_LThe coding sequence of (A) can be shown as 1-324 of SEQ ID No.7 in the sequence listing.

Further, in the case of a liquid crystal display,

the anti-TIGIT antibody is a single-chain antibody, and a coding sequence of the single-chain antibody can be shown as 1 st to 741 th sites of SEQ ID No.5 in a sequence table;

the anti-TIGIT antibody is Fab, the Fab consists of a Fab heavy chain and a light chain, the coding sequence of the Fab heavy chain can be shown as the 1 st to 663 th position of SEQ ID No.8, and the coding sequence of the light chain can be shown as SEQ ID No. 7;

the anti-TIGIT antibody is a complete antibody, the complete antibody consists of a heavy chain and a light chain, the coding sequence of the heavy chain can be shown as SEQ ID No.8, and the coding sequence of the light chain can be shown as SEQ ID No. 7.

In the above biological material, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above-mentioned biomaterial, the expression cassette containing a nucleic acid molecule encoding the anti-TIGIT antibody (anti-TIGIT antibody gene expression cassette) according to B2) is DNA capable of expressing the anti-TIGIT antibody in the host cell, and the DNA may include not only a promoter that promotes transcription of the anti-TIGIT antibody gene but also a terminator that terminates transcription of the anti-TIGIT antibody gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the anti-TIGIT antibody gene expression cassette can be constructed by using the existing expression vector.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid can be pABL, pABG-Fab or pABK

In one embodiment of the invention, the anti-TIGIT antibody is Fab, and the Fab heavy chain expression vector of the Fab is obtained by replacing a DNA fragment between Afl II and Nhe I recognition sequences of an antibody Fab heavy chain expression vector pABG-Fab with the V shown in the 1 st to 354 th positions of SEQ ID No.8 in a sequence table_HThe Fab heavy chain expression vector contains the Fab heavy chain coding gene shown in the 1 st to 663 th position of SEQ ID No.8 and can express the Fab heavy chain;

the Fab light chain expression vector of the Fab is obtained by replacing a DNA fragment between BsrG I and Hind III recognition sequences of a vector pABL with the V shown in the 1 st to 324 th positions of SEQ ID No.7 in a sequence table_LThe recombinant vector obtained from the coding gene of (1)The Fab light chain expression vector contains a light chain coding gene shown in SEQ ID No.7 and can express the Fab light chain.

In another embodiment of the invention, the anti-TIGIT antibody is an intact antibody, the light chain expression vector of the intact antibody is the Fab light chain expression vector;

the heavy chain expression vector of the complete antibody is obtained by replacing a DNA fragment between Afl II and Nhe I recognition sequences of a vector pABG with the V shown in the 1-354 th site of SEQ ID No.8 in a sequence table_HThe heavy chain expression vector contains a heavy chain coding gene shown in SEQ ID No.8 and can express the heavy chain.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi.

In the above biological material, the transgenic animal cell line is a non-propagating material; the transgenic animal cell line can be obtained by introducing the nucleic acid molecule B1) or the recombinant vector B2) into animal cells (such as FreeStyle)^TM293-F cells or CHO cells).

The nucleotide sequence of the anti-TIGIT antibody of the invention may be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides that are artificially modified to have 75% or more identity to the nucleotide sequence of the anti-TIGIT antibody of the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the anti-TIGIT antibody and have anti-TIGIT antibody activity.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence that is 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of the protein consisting of the amino acid sequence indicated by the anti-TIGIT antibody of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 75%, 80%, 85%, 90% or 95% or more.

The invention also provides a composition, which is any one of P1-P6:

p1, a composition consisting of the anti-TIGIT antibody and an anti-PD-1 antibody;

p2, a composition consisting of said biomaterial and an anti-PD-1 antibody.

P3, a composition consisting of the anti-TIGIT antibody and an immune cell;

p4, a composition consisting of the biological material and immune cells;

p5, a composition consisting of the anti-TIGIT antibody, an immune cell and an anti-PD-1 antibody;

p6, a composition consisting of said biological material, immune cells and anti-PD-1 antibodies.

In one embodiment of the invention, the anti-PD-1 antibody is Nivolumab.

The composition can be used for treating and/or preventing tumors, or preparing tumor inhibitors or tumor cell inhibitors.

The invention also provides a product for treating and/or preventing tumors, wherein the active ingredient of the product is any one of M1-M8:

m1, the anti-TIGIT antibody;

m2, the biomaterial;

m3, a composition consisting of the anti-TIGIT antibody and an anti-PD-1 antibody;

m4, a composition consisting of the biomaterial and an anti-PD-1 antibody;

m5, a composition consisting of the anti-TIGIT antibody and an immune cell;

m6, a composition consisting of the biomaterial and immune cells;

m7, a composition consisting of the anti-TIGIT antibody, an immune cell and an anti-PD-1 antibody;

m8, a composition consisting of the biological material, immune cells and anti-PD-1 antibodies.

The product may be a medicament or a vaccine.

The invention also provides any of the following applications:

n1 and the application of the anti-TIGIT antibody in preparing a tumor inhibitor or a tumor cell inhibitor;

n2, the application of the biological material in preparing tumor inhibitors or tumor cell inhibitors;

n3, the application of the composition in preparing tumor inhibitors or tumor cell inhibitors;

n4, and application of the product in preparing tumor inhibitors or tumor cell inhibitors.

In the present invention, the immune cell may be a tumor immune cell. The tumor immune cells can be NK cells or effector T cells. In one embodiment of the invention, the tumor immune cells are NK-92MI cells.

The tumor may be a tumor with high expression of CD 155. The tumor can be lung cancer (such as non-small cell lung cancer), epidermal cancer, glioma, breast cancer, gastric cancer, intestinal cancer, renal cancer or melanoma.

The tumor cell can be a lung adenocarcinoma cell, an epidermal carcinoma cell, or a glioma cell. In one embodiment of the invention, the lung adenocarcinoma cell is a549, the epidermal carcinoma cell is a431, and the glioma cell is U251 MG.

The invention constructs a CHO cell strain with stable and high expression TIGIT, screens cells of a large-capacity fully-synthesized human single-chain antibody phage display library, and screens through multiple rounds of biological panning to obtain a single-chain antibody (scFv) of the specific human anti-human TIGIT. Further, a light and heavy chain full antibody expression vector is constructed by a molecular cloning technology, and eukaryotic expression and affinity purification are carried out. The obtained full antibody AET2010 has high expression level, good specificity, high purity, stable structure and good pharmacokinetic characteristics; can combine TIGIT of recombinant expression and TIGIT of cell surface natural state with high affinity; can effectively block the combination of CD155 and TIGIT; can promote and enhance the killing activity of NK-92MI on tumor cells cultured in vitro, and shows more excellent killing activity when being combined with the PD-1 monoclonal antibody; significantly enhanced antitumor activity was shown in CDX mouse models over-infused with NK-92MI cells. The molecular characteristics and the pharmacodynamic characteristics of the AET2010 are fully verified at the early development level, and good developability is expected. The features and advantages of the present invention are apparent from and are in particular described in the following detailed description and examples, which are not to be construed as limiting the invention. The contents of the references cited in the present invention and the disclosures of the patents and the like are all supplementary to the contents of the present invention.

Drawings

FIG. 1 shows the structural map of eukaryotic expression vectors pABG (A), pABL (B), pABK (C), pABG-Fab (D) and pABG4 (E). Wherein, pABG-Fab and pABG4 are obtained by modification on the basis of pABG.

FIG. 2 shows the flow assay results of TIGIT stable high expression cell line CHO-TIGIT.

FIG. 3 shows SDS-PAGE of hTIGIT-His, HSA-hTIGIT-His, hCD155-Fc and hCD155-His recombinant proteins under reducing conditions; m: protein molecular weight standard, 1: hTIGIT-His, 2: hCD155-Fc, 3: HSA-hTIGIT-His.

FIG. 4 shows an SDS-PAGE electrophoresis of recombinant antibody AET 2010; 1: reduction, 2: and (4) non-reduction.

Fig. 5 shows the HPLC purity identification results of the recombinant antibody AET 2010.

FIG. 6 shows the results of ELISA assay of the binding activity of AET2010 to recombinant HSA-hTIGIT-His.

FIG. 7 shows the results of cell ELISA detection of AET2010 binding activity to TIGIT in the natural state on the surface of CHO-TIGIT cells.

FIG. 8 shows the results of flow cytometry to detect the natural TIGIT binding activity of AET201 to CHO-TIGIT cell surface.

FIG. 9 shows a BIAcore^TM3000 representative sensorgrams of the affinity results for AET2010(A), MK7684(B) and antigen were determined.

FIG. 10 shows the results of the detection of the specificity of AET2010 by ELISA.

FIG. 11 shows an SDS-PAGE electrophoresis of AET2010-Fab and MK 7684-Fab; 1: AET2010-Fab, 2: MK 7684-Fab.

FIG. 12 shows the results of flow cytometry to detect the blocking of hCD155-Fc (rhCD155-Fc) by AET2010-Fab in binding to CHO-TIGIT cell surface native TIGIT.

FIG. 13 shows the results of ELISA detection of AET2010-Fab blocking of hCD155-Fc (rhCD155-Fc) binding to recombinant HSA-hTIGIT-His.

FIG. 14 shows the AET2010/TIGIT composite structural model (A) and the interaction interface (B). The key area of the lock-key structure is the key area of the complementary combination of TIGIT and CD 155.

FIG. 15 shows the results of ELISA assays for AET2010 accelerated placement stability in PBS (A) and FBS (B).

FIG. 16 shows the results of the high throughput protein stability analysis system Uncle detecting the particle size of AET2010 at 25 ℃.

FIG. 17 shows the results of the detection of pharmacokinetic profiles of AET2010 in nude mice; wherein A is the case of coating HSA-hTIGIT-His, and B is the case of coating donkey anti-human IgG/Fc antibody.

FIG. 18 shows the results of flow cytometry for the detection of CD155(A) and PD-L1(B) expression on the surface of tumor cells and TIGIT (C) and PD-1(D) expression on the surface of NK-92MI cells.

FIG. 19 shows the results of ELISA quantitative determination of the level of killer cytokines released by NK-92MI cells in vitro killing experiments. Concentrations indicated in the figure are antibody-corresponding concentrations; the results of the antibodies to be evaluated and isotype control antibodies at the same concentrations were analyzed for differences using the t-test, indicating p <0.05, p <0.01, p <0.001, and p < 0.0001.

FIG. 20 shows the results of ELISA quantitative determination of the level of killer cytokines released from NK-92MI cells in vitro killing experiments. Concentrations indicated in the figure are antibody-corresponding concentrations; the results of the antibodies to be evaluated and isotype control antibodies at the same concentrations were analyzed for differences using the t-test, indicating p <0.05, p <0.01, p <0.001, and p < 0.0001.

FIG. 21 shows the results of ELISA quantitative determination of the level of killer cytokines released from NK-92MI cells in vitro killing experiments. Concentrations indicated in the figure are antibody-corresponding concentrations; the results of the antibodies to be evaluated and isotype control antibodies at the same concentrations were analyzed for differences using the t-test, indicating p <0.05, p <0.01, p <0.001, and p < 0.0001. And (3) carrying out difference analysis on the combination condition of the antibody to be detected and Nivolumab and the result of Nivolumab used alone.

FIG. 22 shows the results of the detection of the killing activity of NK-92MI cells against tumor cells enhanced by antibodies. The tumor cells are A549(A), A431(B) and U251MG (C), respectively; the results of the antibodies to be evaluated and isotype control antibodies at the same concentrations were analyzed for differences using the t-test, indicating p <0.05, p <0.01, p <0.001, and p < 0.0001.

FIG. 23 shows the results of the evaluation of the pharmacological effects of the antibody in a549 tumor-bearing mouse model in which NK-92MI cells were adoptively infused; panel A is the tumor growth curve and panel B is the tumor weight at the end of the experiment. The results of the antibodies to be evaluated and isotype control antibodies at the end of the experiment were analyzed for differences using the t-test, indicating p <0.05, p <0.01, p <0.001, ns indicating no significant difference.

FIG. 24 shows the tumor morphology at the experimental end point, which is the result of the evaluation of the pharmacological effects of the antibody in the A549 tumor-bearing mouse model after adoptive infusion of NK-92MI cells.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

pABL and pABG in the following examples are described in "Du et al, A full human monoclonal antibodies with novel binding epitopes and excellent neutral activity to multiple human IFN-a subtypes A cardiac therapeutics for system viral antigens, mAbs 7:5,969-980; September/October 2015;2015 Taylor and Francis Group, LLC ", publicly available from the Applicant, is only used for repeating the experiments related to the invention and is not used for other purposes.

pABK in the following examples, the sequence of which is SEQ ID NO.21 of the sequence Listing.

Example 1 production of human anti-human TIGIT antibody

Materials (I) and (II)

1. Materials: the high-capacity fully-synthetic phage single-chain antibody library is constructed by bioengineering research institute (ZL200910091261.8) of original military medical academy of China (now military medical research institute of military scientific academy), and has a library capacity of 1.35 × 10¹⁰. The host strain for phage infection was XL1-Blue (Stratagene, USA), the plasmid amplification strain was Top10 (Beijing Quanjin Biotechnology Co., Ltd.), the helper phage was M13KO7 (Invitrogen, USA), and the horseradish catalase (HRP) -labeled anti-M13 KO7 antibody (11973-MM05T-H) was a product of Beijing Yi Qiao Hibiscus Biotechnology Co., Ltd. CHO cell, Transfection Reagent TransIntro EL Transfection Reagent (L20313) is Beijing Quanyu gold Biotechnology Ltd. Opti-MEM serum-free medium for transfection, 1640 medium for cell culture, 0.25% pancreatin and Fetal Bovine Serum (FBS) were all products of Gibco, and hygromycin B for screening high-expression cell lines was a product of WWR Life Science. APC-labeled mouse anti-human TIGIT antibody (372705) was produced by Biolegend, USA. Human TIGIT (HG10917-UT) and human serum albumin (HSA, HG10968-UT) cDNA cloning vectors are products of Beijing Yi Qiao Shenzhou biotechnology, Inc. The human TIGIT-His recombinant protein (10917-H08H) is a product of Beijing Yi Qian Shenzhou biotechnology limited. FreeStyle^TM293-F mammalian cell expression System (including the cell FreeStyle)^TM293-F, serum-free medium and transfection reagent) were all Invitrogene products. The structural maps of eukaryotic expression vectors pABL and pABG are shown in FIG. 1, and the protein purification systems AKTA Purifier 100 and HisTrap^TMFF pillar, HiTrap^TMMabselect SuRe column, HiTrap^TMThe desaling Columns are all products of GE company.

2. The method and the result are as follows:

2.1 construction of CHO-TIGIT Stable high expression cell line

0.25% pancreatin was used to digest and collect CHO cells in good culture state, and the cell density was adjusted to 1X 10⁵Perml, 2ml of the solution was inoculated into a 6-well plate at 37 ℃ with 5% CO₂Incubate overnight. Diluting 2 μ g of TIGIT expression plasmid (HG10917-UT) and 10 μ l of Transfection Reagent (TransIntro EL Transfection Reagent) with 100 μ l of Opti-MEM serum-free medium respectively, mixing well, standing for 15min, adding into cells, standing for 48h, discarding the cell culture medium, digesting with pancreatin, counting, and performing 1: 4, passage to a 48-well plate, adding 1640 culture medium (containing 10% FBS) containing 200 mu g/ml hygromycin B when the cells grow to full bottom area of 50-70%, and changing the culture solution every 3 d; observing and marking the monoclonal cell mass under a microscope, scraping the marked cell mass in an ultra-clean bench, adding the marked cell mass into a 96-well plate, adding 1640 culture medium containing 200 mu g/ml of hygromycin into each well, continuously culturing, and changing the culture solution every 3 days; after the cells in the hole are full, digesting and collecting the cells, and adjusting the density to 3 multiplied by 10⁵And/test, combining the cells with an APC-labeled mouse anti-human TIGIT antibody for 1 hour on ice, setting a control group without adding a flow antibody, setting 3 parallel controls for each group, detecting by a flow cytometer (BD C6 plus) after 2 times of complete culture medium washing by 1640, judging the TIGIT expression condition on the surface of the CHO cell according to the average fluorescence intensity (MFI) of the APC, and naming the obtained human TIGIT stable high-expression cell strain as CHO-TIGIT.

Through transfection, screening and identification, a CHO cell strain (CHO-TIGIT) which stably and highly expresses human TIGIT is successfully constructed. The positive rate of TIGIT on the cell surface is over 99 percent (figure 2) detected by flow cytometry, and the screening requirement is met. 1 strain (CHO-TIGIT 2-4) was selected for screening of phage antibody library.

2.2 construction, expression and purification of hTIGIT-His and HSA-TIGIT-His fusion protein expression vector

The amino acid sequence of the recombinant protein (hTIGIT-His) fused with 8 histidine labels in the human TIGIT ectodomain to be constructed is shown in SEQ ID NO. 1. And replacing the TIGIT signal peptide with pABG vector signal peptide (antibody heavy chain secretion signal peptide) by using a TIGIT cDNA cloning vector as a template. 3 upstream primers are designed, each primer has a sequence mutually overlapped with the previous primer, and TIGIT ectodomain genes are amplified by an overlapped PCR method, wherein relevant primers are listed in a table 1. The amino acid sequence of a recombinant protein (HSA-hTIGIT-His) formed by sequentially connecting a human serum albumin, a connecting peptide (4GS), a human TIGIT ectodomain and 8 histidine tags in series is shown as SEQ ID NO.3, a cDNA cloning vector of HSA and a cDNA cloning vector of TIGIT are respectively taken as templates, primers are respectively designed on the upstream and the downstream to amplify an HSA gene and a TIGIT ectodomain gene, a nucleotide sequence corresponding to the connecting peptide is introduced into a connecting part, then the HSA gene and the TIGIT ectodomain gene are subjected to fusion connection by adopting an overlapping PCR method, and related primers are listed in Table 1. And determining and recovering a target band by agarose gel electrophoresis, and cloning a target gene into a eukaryotic expression vector pABG through two enzyme cleavage sites of EcoR I and BamH I. Marking a recombinant vector containing a correct hTIGIT-His encoding gene (the sequence of which is shown in SEQ ID NO. 2) as pABG-hTIGIT-His, wherein the pABG-hTIGIT-His can express a recombinant protein hTIGIT-His shown in SEQ ID NO. 1; the recombinant vector containing the correct HSA-hTIGIT-His coding gene (the sequence of which is shown in SEQ ID NO. 4) is marked as pABG-HSA-hTIGIT-His, and the pABG-HSA-hTIGIT-His can express the recombinant protein HSA-hTIGIT-His shown in SEQ ID number 3. pABG-hTIGIT-His and pABG-HSA-hTIGIT-His can also be obtained by artificially synthesizing an hTIGIT-His coding gene containing EcoR I and BamH I recognition sequences and protective bases thereof, and replacing a small segment between the EcoR I and BamH I recognition sequences of a eukaryotic expression vector pABG by enzyme digestion after synthesizing an HSA-hTIGIT-His coding gene containing EcoR I and BamH I recognition sequences and protective bases thereof.

pABG-hTIGIT-His and pABG-HSA-hTIGIT-His are transfected into FreeStyle with good state respectively^TM293-F cells at 37 ℃ and 120rpm with 5% CO₂Shaking the flask by a horizontal shaking table for 3-4 days, and centrifuging at a high speed to collect an expression supernatant. Based on AKTA system, HisTrap is adopted^TMFF affinity purification, collection of sample eluted with 500mM imidazole, HiTrap^TMThe Desalting Columns replaced the purified proteins into PBS buffer pH7.4 to obtain fusion proteins hTIGIT-His and HSA-hTIGIT-His, respectively. Protein purity was identified by SDS-PAGE and protein concentration was quantified spectrophotometrically.

TABLE 1 primer List for TIGIT expression vector construction

The molecular cloning constructs the eukaryotic expression vectors of hTIGIT-His and HSA-hTIGIT-His, and the recombinant protein is obtained by expression and purification. The SDS-PAGE identification result is shown in figure 3, under the reducing condition, the size of hTIGIT-His is about 18KD, the size of HSA-hTIGIT-His is about 70KD, the molecular weight is in line with the expectation, and the protein purity is higher.

2.3 CHO-TIGIT cell screening and monoclonal identification of phage antibody library

The phage antibody library (sub library) is diluted into 1ml PBS milk (PBS containing 2% skimmed milk powder), and is blocked at 4 ℃ for 2h to obtain the blocked phage antibody library. CHO and CHO-TIGIT cells (i.e., CHO-TIGIT 2-4, supra) were collected by sequential digestion (2X 10)⁷) Respectively re-suspending in 5ml of precooled PBS milk, sealing for 1h at 4 ℃ (rotating at uniform speed to prevent cell aggregation, the same below), centrifuging at 4 ℃ and 1000rpm multiplied by 5min to collect cells, and respectively obtaining sealed CHO cells and sealed CHO-TIGIT cells; resuspending the blocked CHO cells by using a blocked phage antibody library, combining the cells at 4 ℃ for 4 hours, and centrifuging to obtain a supernatant; resuspending the sealed CHO-TIGIT cells by using the obtained supernatant, combining the cells at 4 ℃ for 4h, centrifuging the cells at 4 ℃ and 1000rpm multiplied by 5min, removing the supernatant, and collecting the cells; resuspend the fine using 1ml of precooled PBSAfter the cells are treated, centrifuging at 4 ℃ and 1000rpm multiplied by 5min, removing supernatant, collecting the cells, and repeatedly washing the cells for 10 times; resuspending the cell pellet with 500. mu.l PBS containing pancreatin at 10. mu.g/ml, allowing to act at room temperature for 30min, adding 500. mu.l PBS containing serum to a total volume of 1ml, and terminating the reaction; then adding 9ml of escherichia coli XL1-Blue in logarithmic phase, standing and infecting for 30min at room temperature, and culturing for 1h at 37 ℃ and 150rpm to obtain bacterial liquid; the bacterial suspension was applied to 2 XYT-CTG plates (containing 50. mu.g/ml chloramphenicol [ phagemid resistance ]) in a total volume of 1% and 0.1% respectively]10. mu.g/ml tetracycline [ E.coli resistance ]]And 2 XYT medium of 0.5% glucose) for calculation of yield, and the remaining bacterial liquid was applied to a 2 XYT-CTG plate after centrifugal concentration and cultured overnight at 37 ℃; the bacterial colony is collected by a scraper, the phage antibody is presented, the PEG8000 precipitate is used for recovering the phage antibody, and a certain amount of the phage antibody is taken to be put into the next round of screening. Repeating the steps, finishing 2-3 rounds of screening, and performing monoclonal presentation and identification according to the output condition; wherein, the cell input amount is reduced by turns, and the washing times are increased by turns.

Selecting the monoclonal colony produced by screening, and culturing the colony in a 96-well plate containing a 2 XYT-CTG culture medium at 37 ℃ and 220rpm overnight until the colony is saturated; transferring the saturated bacteria solution to a new 96-well plate (2 XYT-CT culture medium) according to a ratio of 1:10, culturing at 37 ℃ and 220rpm for 1.5h until OD600 is about 0.5, adding a helper phage M13KO7 according to the infection complex MOI of 50, standing at room temperature for 30min, culturing at 37 ℃ and 150rpm for 1h, adding kanamycin to a final concentration of 20 mu g/ml and IPTG to a final concentration of 0.15mM, and inducing at 30 ℃ and 200rpm for 12 h; after the supernatant was collected by centrifugation, the binding activity of the phage antibody was detected by ELISA using the target antigen (TIGIT-His) and irrelevant control antigen (IFN-. alpha. -His fusion protein prepared and stored in this chamber and CHO cell disruption supernatant (i.e., supernatant collected by centrifugation after disruption of CHO cells)), and monoclonal colonies whose identification result was positive in specificity were sequenced to obtain variable region genes of the candidate humanized antibody.

According to the method 2.3, the phage single-chain antibody library after CHO cell subtraction is combined with CHO-TIGIT, and is subjected to three rounds of progressively enhanced screening, the phage antibodies produced by screening are subjected to monoclonal identification, and the specific phage single-chain antibody 2011-4-D5 is obtained by enrichment, wherein the nucleotide and amino acid sequences of the single-chain antibody are respectively shown in SEQ ID NO.5 and SEQ ID number 6.

In SEQ ID NO.6, the positions 1-108 are the light chain variable region, the positions 23-33, 49-55 and 88-96 are the CDR1, CDR2 and CDR3 of the light chain variable region, the positions 130 and 247 are the heavy chain variable regions, and the positions 159 and 164, 179 and 195 and 228 and 236 are the CDR1, CDR2 and CDR3 of the heavy chain variable region.

In SEQ ID NO.5, the light chain variable region gene is located at positions 1-324, and the heavy chain variable region gene is located at position 388-741.

2.4 phage Single chain antibody conversion to Total antibody

The vectors pABG and pABL are used to clone antibody heavy chain and light chain variable region genes and artificially synthesize V_H5V was amplified from the variable region gene (i.e., the heavy chain variable region gene of scFv 2011-4-D5) using primers H5F and HR (Table 2), respectively_H5A variable region gene; artificially synthesized V_λ3V amplification of variable region gene (i.e. light chain variable region gene of single-chain antibody 2011-4-D5, adjusted by partial sequence), L3F and LR_λ3A variable region gene. V_λ3The variable region gene is cloned into the vector pABL, V by using the restriction sites BsrG I and Hind III_H5The variable region gene is cloned into a vector pABG by utilizing enzyme cutting sites Afl II and Nhe I. And respectively transforming the obtained recombinant vectors into escherichia coli TOP10, and sequencing and identifying the recombinant vectors to obtain the correct constructed light and heavy chain expression vectors of the full antibody. After extracting plasmid, mixing light and heavy chain expression vectors according to the molar ratio of 2:1, and co-transfecting FreeStyle with good state^TM293-F cells at 37 ℃ and 120rpm with 5% CO₂Shaking the flask by a horizontal shaking table for 3-4 days, and centrifuging at a high speed to collect an expression supernatant. Based on AKTA system, HiTrap is adopted^TMAffinity purification is carried out on MabSelect Sure pre-packed column, a sample is eluted and collected by 0.1M sodium citrate buffer solution (pH3.0), and HiTrap is used^TMThe desaling Columns replace the antibody with PBS buffer solution to obtain the whole antibody, which is named as AET 2010. Antibody concentration was quantified spectrophotometrically, and antibody purity was identified by SDS-PAGE and HPLC.

The method comprises the steps of respectively amplifying 2011-4-D5 light and heavy chain genes by utilizing PCR, carrying out enzyme digestion, connection and cloning to pABL and pABG expression vectors, and constructing a full antibody (the full antibody is marked as AET2010) expression vector in an IgG1 form, namely a light and heavy chain expression vector, wherein the light and heavy chain expression vector respectively contains light and heavy chain genes shown in SEQ ID NO.7 and SEQ ID NO.8 and can respectively express the light and heavy chains of the AET2010, and the amino acid sequences of the light and heavy chains of the AET2010 are respectively shown in SEQ ID NO.9 and SEQ ID NO. 10.

In SEQ ID NO.9, positions 1-108 are the light chain variable region, positions 23-33, 49-55 and 88-96 are the CDR1, CDR2 and CDR3 of the light chain variable region, respectively, and positions 109-213 are the constant region CL.

In SEQ ID NO.10, positions 1-118 are the heavy chain variable region, positions 30-35, 50-66 and 99-107 are the CDRs 1, 2 and CDR3 of the heavy chain variable region, positions 119 and 216 are the CH1, positions 217 and 231 are the hinge region, positions 232 and 341 are the CH2, and positions 342 and 448 are the CH 3.

The AET2010 holoantibody sample is obtained by eukaryotic expression and purification by the method. AET2010 expression levels were approximately 30 mg/L; SDS-PAGE electrophoresis results show that the band is correct and clear, and the purity is higher (FIG. 4); further HPLC analysis showed the AET2010 sample to be unimodal, free of peaks and 100% pure (fig. 5).

TABLE 2 complete antibody light and heavy chain expression vector construction related primer list

Example 2 binding Activity of AET2010

Materials and methods

1. Materials: anti-TIGIT monoclonal antibody MK7684 is obtained from America Moshadong, or synthesized light and heavy chain genes thereof and cloned into pABK (FIG. 1) and pABG vectors prepared according to the method for preparing AET2010 of example 1 (patent No.: US20160355589A1), the light chain and heavy chain amino acid sequences of MK7684 are shown as SEQ ID NO.11 and SEQ ID NO.12, respectively, the light chain and heavy chain gene sequences of MK7684 are shown as SEQ ID NO.13 and SEQ ID NO.14, respectively, and MK7684 is used as a control antibody.

Wherein, the light chain expression vector of MK7684 is a recombinant vector obtained by replacing the recognition sequence between Xba I and Nar I of the pABK expression vector with the light chain variable region gene shown in 1-327 th position of SEQ ID NO.13, the recombinant vector contains the light chain gene shown in SEQ ID NO.13, and can express the light chain of MK7684 shown in SEQ ID NO. 11.

The heavy chain expression vector of MK7684 is a recombinant vector obtained by replacing the recognition sequence between Afl II and Nhe I of pABG expression vector with the heavy chain variable region gene shown in 1-357 of SEQ ID No.14, contains the heavy chain gene shown in SEQ ID No.14, and can express the heavy chain of MK7684 shown in SEQ ID No. 12.

The goat anti-human IgG antibody marked by the HRP is a product of Beijing Zhonghua Jinqiao biotechnology limited. The CM5 chip is a product of GE company, anti-human immunoglobulin Fc capture sensor (AHC Biosensor) and streptavidin sensor (SA Biosensor) are all products of Eliza biotechnology (Shanghai) Limited, mTIGIT protein (i.e., mouse TIGIT protein) is a product of Acro company, hTIGIT-HIS (human TIGIT fusion HIS tag), mTIGIT-HIS (mouse TIGIT fusion HIS tag), hCSF1R-HIS (human colony stimulating factor 1 receptor fusion HIS tag), hFRC (human transferrin receptor fusion HIS tag), hLAG3 (human lymphocyte activating gene 3), hCD73 (human CD73), hCD38 (human CD38), and hNectin (human Nectin) proteins are prepared according to conventional methods. Other sources of material were the same as in example 1.

2. The method and the result are as follows:

2.1 ELISA detection of AET2010 binding Activity

Gradient dilution of PBST milk (PBS containing 2.5% skim milk powder and 1% tween 20) AET 2010: starting concentration 2.5. mu.g/ml, 3-fold gradient dilution 11 gradients were serially performed to obtain antibody dilutions of different concentrations.

The HSA-hTIGIT-His recombinant protein of example 1 was diluted with PBS to 1. mu.g/ml, added to a 96-well plate at 100. mu.l/well, and coated overnight at 4 ℃. The coating solution was discarded and the cells were blocked with PBS milk (PBS containing 2.5% skim milk powder) at 37 ℃ for 1 h. Disposable sealing liquidAdding antibody diluent into each well at a concentration of 100 μ l/well, and combining at 37 deg.C for 1 hr; PBST was washed 5 times, and 100. mu.l/well of a diluted HRP-labeled goat anti-human IgG antibody in PBST milk (PBST containing 2.5% skim milk powder) was added and bound at 37 ℃ for 45 min. PBST is washed for 5 times, TMB color developing solution is added, after standing at room temperature and developing for 10min, 2M H is added according to 100 mul/hole₂SO₄Terminating; the microplate reader detects the light absorption value at 450nm (595nm is used as a control wavelength), the Graphd Prism Software 5.0 plots the antibody concentration-light absorption value (OD) curve, and the half maximal effective concentration (EC50) is calculated.

AET2010 was replaced with anti-TIGIT monoclonal antibody MK7684 as a control according to the method described above.

The ELISA results show (FIG. 6) that AET2010 is in a dose-dependent relationship with the binding of HSA-hTIGIT-His fusion protein in a certain concentration range, the EC50 value is 3ng/ml, the EC50 value of the control antibody MK7684 is 2.2ng/ml, and the binding activity of the AET2010 and the control antibody is equivalent to that of the HSA-hTIGIT-His recombinant protein.

2.2 cell ELISA for detection of AET2010 binding Activity

1640 medium (10% FBS) diluted AET 2010: the initial concentration was 2.5. mu.g/ml, and 11 concentrations were serially diluted in a 3-fold gradient to obtain antibody dilutions of different concentrations.

CHO-TIGIT cells (i.e., CHO-TIGIT 2-4 of example 1) or CHO cells in the logarithmic growth phase were collected by digestion and cell density was adjusted to 1X 10 with growth medium⁵Cells/ml, 100. mu.l/well into 96-well cell culture plates, 37 ℃ with 5% CO₂Incubate until the cells are spread over the well plate. Add 1640 medium (containing 10% FBS) at 120. mu.l/well and block for 1h on ice; adding the antibody diluent after being sealed into the waste liquid of the 96-pore plate, and combining for 2 hours on ice; discarding the solution, washing 3 times with ice-precooled 1640 medium (containing 10% FBS), adding HRP-labeled goat anti-human IgG antibody diluted by the 1640 medium (containing 10% FBS) into 100 μ l/hole, and combining for 1h on ice; discarding liquid, washing with precooled 1640 culture medium (containing 10% FBS) for 3 times, washing with PBS for 2 times, adding TMB color developing solution at 100 μ l/hole, standing at room temperature for 10-20min, and adding 2M H at 100 μ l/hole₂SO₄Terminating; the microplate reader detects the light absorption value (595nm is used as the reference wavelength) with the wavelength of 450nm, Graphd Prism Software 5.0 plots antibody concentration-absorbance (OD) curves and calculates the half maximal effective concentration (EC 50).

AET2010 was replaced with anti-TIGIT monoclonal antibody MK7684 as a control according to the method described above.

2.3 flow cytometry detection of AET2010 binding Activity

The flow cytometer was BD C6 plus. Digesting and collecting CHO-TIGIT or CHO cells in logarithmic growth phase, and adjusting cell density to 5 × 10 with growth medium⁶Cells/ml, and dividing into several parts according to 100 mul/part; dilution of AET2010 and MK7684 in pre-cooled 1640 medium: the initial concentration is 1 mug/ml, 10 gradients are continuously diluted by 3 times of gradients, and the 10 gradients are mixed with a cell sample in equal volume and incubated for 2 hours at 4 ℃; adding 1ml of precooled 1640 medium (containing 10% FBS) into each sample, slightly reversing the mixture up and down for 4-6 times, centrifuging the mixture at the temperature of 4 ℃ and the speed of 1000rpm for 5min, discarding the supernatant, and repeatedly washing the supernatant for three times; FITC-labeled goat anti-human IgG antibody was diluted in pre-cooled 1640 medium (containing 10% FBS), and the cells were resuspended in 200. mu.l/sample solution and incubated at 4 ℃ for 1 h; after 3 washes, 100 μ l of pre-cooled 1640 medium (containing 10% FBS) was resuspended in cells, flow cytometric analysis, graph Prism Software 5.0 plotted antibody concentration-Mean Fluorescence Intensity (MFI) and half maximal effective concentration (EC50) was calculated.

AET2010 was replaced with anti-TIGIT monoclonal antibody MK7684 as a control according to the method described above.

The detection results of cell ELISA (figure 7) and flow cytometry (figure 8) show that AET2010 can be efficiently combined with TIGIT on the surface of CHO-TIGIT cells, and the AET2010 shows good dose dependence, and EC50 of the AET is equivalent to a control antibody MK 7684; wherein the EC50 of AET2010 and MK7684 cell ELISA is 3.9ng/ml and 8.8ng/ml respectively, and the EC50 of flow cytometry detection is 3.9ng/ml and 8.8ng/ml respectively. Both of them had binding activity equivalent to TIGIT in a natural state on the cell surface.

2.4 SPR determination of AET2010 affinity

Kinetic parameters of Antibody-antigen interactions were determined by Multi-cycle kinetics (Multi-cycle kinetics) method using a Human Antibody Capture Kit (GE Healthcare, BR-1008-39, USA) to couple Capture antibodies to the surface of a CM5 chip based on the BIAcore 3000 system. Purification ofThe latter AET2010 or MK7684 was diluted to 1. mu.g/mL with HBS-EP buffer (GE Healthcare, USA), and 5. mu.l/min. times.1 min was captured and immobilized on the chip surface. Recombinant protein TIGIT-His (Beijing Yiqian Shenzhou biotechnology, Inc., 10917-H08H) antigen is used as a mobile phase, and the concentration range is 120 nM-1.875 nM; HBS-EP + buffer serially diluted the antibody to be tested in 2-fold gradient for at least 5 dilutions. The test conditions were 25 ℃ and 30. mu.l/min; binding for 3min, and dissociation for 6 min. One cycle of repeated measurements was set for quality control and two cycles of blank controls were set to correct for systematic variation. Regeneration conditions were 3M MgCl₂30. mu.l/min. times.30 s. Analysis of results was performed using BIAevaluation Software, selecting 1: 1Langmuir binding pattern the data were fitted and kinetic constants for antigen-antibody binding were calculated; key data were measured in triplicate independently and the results are expressed as "mean ± standard deviation".

Representative kinetic sensorgrams obtained from the assays are shown in fig. 9, with the average of three independent replicates being listed in table 3. Affinity constant K of AET2010_D3.74. + -. 0.12nM, MK7684 and affinity constant K_DIt was 0.11. + -. 0.02 nM. MK7684 has a much higher affinity for recombinant TIGIT-His than AET2010, about 34 fold.

Specifically, the SPR experiment uses a mode of fixing an antibody, and the measured result is the one-arm binding ability, i.e., affinity (affinity), of the antibody. In the case of the ELISA, the cell ELISA and the flow assay, a mode of immobilizing an antigen (or a cell) is used, and the assay result represents the double-arm binding ability, i.e., avidity (avidity), of an antibody. The results in both forms show that the immobilized antibody AET2010, at much lower affinity than MK7684, has comparable avidity to MK7684 for immobilized antigen; it can be speculated that AET2010 will have a better functional activity if the epitope is appropriate.

TABLE 3 BIAcoreTM 3000 determination of affinity of the antibody for TIGIT interaction

2.5 ELISA detection of the binding specificity of AET2010

Diluting antigens hTIGIT-HIS, mTIGIT-HIS, hCSF1R-HIS, hFRC, hLAG3, hCD73, hCD38 and hNectin protein to 1 mu g/ml by PBS, adding 96-well enzyme linked plates according to 100 mu l/well, setting 3 multiple wells for each antigen, and coating at 4 ℃ overnight by using PBS, 2% BSA and 0.5CASIN as controls; discarding the coating solution, and sealing the 96-well plate with PBS milk; PBST milk dilution blocking AET2010 to 10 μ g/ml, adding 96-well enzyme linked plates according to 100 μ l/well, and combining for 1h at 37 ℃; PBST is washed for 5 times, goat anti-human IgG antibody diluent marked by HRP is added according to 100 mul/hole, and the mixture is combined for 45min at 37 ℃; PBST was washed 5 times, TMB color developing solution was added at 100. mu.l/well, and after standing at room temperature for 10min, 2M H was added at 100. mu.l/well₂SO₄Terminating; the absorbance at 450nm was measured by a microplate reader (595nm was used as a control wavelength), and the average absorbance was calculated and the data is expressed as "mean. + -. standard deviation".

The results are shown in fig. 10, AET2010 specifically binds human TIGIT with high affinity, and there is no cross-reaction with murine TIGIT or other detection antigens.

Example 3 blocking Activity of AET2010

Materials and methods

1. Materials: the cDNA cloning vector of human CD155(HG29682-UT) is a product of Beijing Yiwangshan Biotechnology Limited. HiTrap for protein purification^TMThe Q-Sepharose FF anion adsorption column is a product of GE company, the FITC marked mouse anti-human IgG/Fc flow type antibody is a product of Biolegend company, and the HRP marked goat anti-human IgG/Fc antibody is a product of Beijing Baixinyi biological technology Limited company. The isotype control antibody (C103S) was produced by Beijing Yi-Qiao Shen Biotechnology, Inc. Other sources of materials are the same as in examples 1 and 2.

2. The method comprises the following steps:

2.1 preparation of AET2010-Fab, MK7684-Fab and CD155-Fc recombinant proteins

The heavy chain expression vector of the antibody Fab segment is a pABG vector without a CH2 and CH3 gene sequence, the name of the vector is pABG-Fab (figure 1, the sequence of the vector is SEQ ID NO.22 in a sequence table), and the heavy chain amino acid sequences of AET2010-Fab (namely, a Fab antibody of AET2010) and MK7684-Fab (namely, a Fab antibody of MK 7684) to be prepared are respectively 1-221 th position of SEQ ID NO.10 and 1-222 th position of SEQ ID NO. 12. Through PCR amplification and enzyme cutting of sites Afl II and Nhe I, the AET2010 and MK7684 variable region (VH) genes are cloned into an antibody Fab heavy chain expression vector pABG-Fab, the obtained recombinant vector for expressing the AET2010-Fab heavy chain amino acid is recorded as pABG-Fab-AET2010, and the obtained recombinant vector for expressing the MK7684-Fab heavy chain amino acid is recorded as pABG-Fab-MK 7684. pABG-Fab-AET2010 contains the gene coding for the AET2010-Fab heavy chain amino acid shown in positions 1-663 of SEQ ID NO.8, and pABG-Fab-MK7684 contains the gene coding for the MK7684-Fab heavy chain amino acid shown in positions 1-666 of SEQ ID NO. 14.

pABG-Fab-AET2010 is a recombinant vector obtained by replacing the DNA fragment between the Afl II and Nhe I recognition sequences of pABG-Fab with the DNA fragment shown in positions 1-354 of SEQ ID NO. 8. pABG-Fab-MK7684 is a recombinant vector obtained by replacing the DNA fragment between the Afl II and Nhe I recognition sequences of pABG-Fab with the DNA fragment shown in positions 1-666 of SEQ ID NO. 14.

pABG-Fab-AET2010 and AET2010 light chain expression vector of example 1 were mixed at a molar ratio of 1:1 to cotransfect a well-conditioned FreeStyle^TM293-F cells at 37 ℃ and 120rpm with 5% CO₂Shake-flask culture for 3-4 days in horizontal shaking bed, high-speed centrifuging to collect expression supernatant, ultrafiltering and concentrating to a certain volume, and HiTrap^TMThe Desalting Columns replaced the buffer system with PH5.8 PBS; by HiTrap^TMAnd purifying the purified solution by using a Q-Sepharose FF anion adsorption column, collecting a flow-through solution containing the Fab, and replacing the flow-through solution into a PBS buffer solution with the pH of 7.4 by using a desalting column to obtain the AET 2010-Fab. Protein purity was identified by SDS-PAGE and protein concentration was quantified spectrophotometrically.

MK7684-Fab was prepared according to the method described above using pABG-Fab-MK7684 and the light chain expression vector for MK7684 of example 2. Protein purity was identified by SDS-PAGE and protein concentration was quantified spectrophotometrically.

The amino acid sequence of the human CD155 ectodomain and human IgG1 Fc fragment fusion protein (hCD155-Fc) to be prepared is shown in SEQ ID NO. 15. Respectively taking a cDNA cloning vector of CD155 and pABG as templates, amplifying an ectodomain gene and an Fc segment gene of CD155 by using primers listed in Table 4 through PCR, splicing the PCR and fusing the CD155 and the Fc gene, identifying and recovering a correct target band through agarose gel electrophoresis, cloning the target gene into an expression vector pABG through two enzyme cleavage sites of EcoR I and BamH I, marking the obtained recombinant vector with a correct sequence as pABG-hCD155-Fc, wherein the pABG-hCD155-Fc contains an hCD155-Fc coding gene shown in SEQ ID NO.16, and can express the hCD155-Fc shown in SEQ ID NO. 15.

FreeStyle with good pABG-hCD155-Fc transfection status^TM293-F cells at 37 ℃ and 120rpm with 5% CO₂Shaking the flask by a horizontal shaking table for 3-4 days, and centrifuging at a high speed to collect an expression supernatant. Based on AKTA system, HiTrap is adopted^TMAffinity purification is carried out on MabSelect Sure pre-packed column, a sample is eluted and collected by 0.1M sodium citrate buffer solution (pH3.0), and HiTrap is used^TMThe Desalting Columns displaced the purified protein into PH7.4 PBS buffer to give hCD 155-Fc. Protein purity was identified by SDS-PAGE and protein concentration was quantified spectrophotometrically.

TABLE 4 primer List used for CD155-Fc expression vector construction

The SDS-PAGE identification result of the Fab antibody is shown in figure 11, under the reducing condition, the AET2010-Fab and the MK7684-Fab have the size of about 25KD, the molecular weight is in accordance with the expectation, and the bands are clear and the purity is high. The SDS-PAGE identification result of the hCD155-Fc is shown in figure 3, and the molecular weight is about 75KD under the reducing condition, which is in line with the expectation, and the band is clear and has higher purity.

2.2 flow cytometry detection of blocking Activity of antibodies against CD155 binding to TIGIT

CHO-TIGIT cells in the logarithmic growth phase (i.e., CHO-TIGIT 2-4 of example 1) were collected by digestion and growth medium adjusted to a cell density of 5X 10⁶Cells/ml, 100. mu.l/portion divided into several portions. 1640 medium (10% FBS) diluted AET 2010-Fab: get upThe initial concentration is 3.2 mu M, and 6 gradients are continuously diluted by 2 times of gradients to obtain an antibody diluent; an equal volume (50. mu.l each) of the antibody dilutions was added to an equal volume of hCD155-Fc to obtain a protein mixture, wherein the concentration of hCD155-Fc in the protein mixture was 400 nM. Mixing the protein mixed solution with a corresponding cell sample, and incubating for 2h at 4 ℃; adding 1ml of 1640 culture medium (containing 10% FBS) into each sample, slightly reversing the mixture from top to bottom for 4-6 times, centrifuging the mixture at 4 ℃ and 1000rpm for 5min, removing supernatant, and repeatedly washing the mixture for three times; 200 μ l of FITC-labeled goat anti-human IgG/Fc antibody solution diluted in 1640 medium (containing 10% FBS) was added to each sample, and the cells were resuspended at 4 ℃ for 1 h; wash 3 times, resuspend cells in 100 μ l 1640 medium (containing 10% FBS), and flow cytometer load detect MFI for each group.

hCD155-Fc was replaced with isotype control antibody as a negative control as described above. CD155-Fc without the addition of AET2010-Fab was set as a positive control.

Calculating the blocking rate (MFI)_{Experimental group}-MFI_{Negative control group})/(MFI_{Positive control group}-MFI_{Negative control group}) X 100%. And drawing an antibody Fab concentration-blocking rate curve, and determining the blocking activity of the antibody Fab on the TIGIT/CD155-Fc interaction.

The blocking activity of MK7684-Fab was tested by replacing the AET2010-Fab with MK7684-Fab as described above.

The result is shown in FIG. 12, AET2010-Fab and MK7684-Fab can effectively block the combination of hCD155-Fc and TIGIT in the natural state of the cell surface in a certain concentration range, and the blocking rate is in a dose-dependent relationship; and the blocking rate of AET2010-Fab is equivalent to that of MK 7684-Fab.

2.3 ELISA detection of blocking Activity of antibodies against CD155 binding to TIGIT

The HSA-hTIGIT-His recombinant protein was diluted to 1. mu.g/ml in PBS, and a 96-well plate was added at 100. mu.l/well and coated overnight at 4 ℃. Discard the coating solution, and seal with PBS milk at 37 ℃ for 1 h. PBST milk gradient dilution AET 2010-Fab: initial concentration 3.0 μ M, 2-fold gradient dilution 11 gradients in series; adding equal volume (50 mu l of each concentration) of recombinant hCD155-Fc to obtain a protein mixed solution, wherein the concentration of the hCD155-Fc in the obtained protein mixed solution is 400nM, adding the protein mixed solution to a 96-well plate after sealing, and binding for 1h at 37 ℃; PBST was washed 5 times and a dilution of PBST milk (PBST containing 2.5% skim milk powder) with HRP-labeled goat anti-human IgG antibody was added at 100. mu.l/well. The subsequent washing, color development and light absorption value detection steps are the same as 2.1 in example 2. Graph Prism Software 5.0 plots Fab protein concentration-light absorption (OD) curves and calculates the half maximal inhibitory concentration (IC 50).

The half maximal inhibitory concentration of MK7684-Fab was determined by replacing the AET2010-Fab with MK7684-Fab as described above.

The results are shown in FIG. 13, AET2010-Fab and MK7684-Fab can effectively block the combination of CD155-Fc and recombinant HSA-hTIGIT-His in a certain concentration range and are in a dose-dependent relationship; the IC50 values for the AET2010-Fab and MK7684-Fab were 26.22 and 48.18nM, respectively, which were comparable.

Example 4, epitope of AET2010

Materials and methods

1. Materials: molecular simulation software adoptionDiscover Studio 3.0 (DS 3.0 for short). forteBIO^TM Octet QK^eThe system is matched with Snesor which is a product of Beijing Topu biotechnology limited. The Dpn I enzyme is a product of NEB company; the rest of the materials are the same as the previous embodiment.

2. The method comprises the following steps:

2.1 prediction and analysis of AET2010 and TIGIT composite structural model

2.1.1 homologous modeling building AET2010 variable region structural model

Searching a homologous sequence of AET2010 in a PDB database Blast, selecting a sequence and a structure with high light-heavy chain Homology as a homologous template, performing homologous Modeling by adopting a Build Homology model function in a DS 3.0Homology Modeling module, and optimizing a light-heavy chain CDR3 of an antibody by adopting Loop reference (MODELER); the structural rationality of the optimization model was evaluated by empirical review, in combination with the "environmental match score for residues (Profile-3D)" and the "Rarmchandar Plot".

2.1.2 construction of antigen-antibody Complex Structure model by molecular docking

The AET2010 variable region structure is the homologous modeling result of 2.1.1, and the TIGIT structure is derived from PDB:3 UCR; limiting the range of the interaction epitope according to the blocking experiment result and the characteristics of the antibody; adopting Dock Proteins (ZDOCK) in a DS 3.0 Dock Proteins module to butt TIGIT and AET2010, constructing a compound structure model, and carrying out cluster analysis and ZDOCK and ZRank scoring on the result; preferred clustering was optimized using Refine Dock Proteins (RDOCKs) in DS 3.0 Dock Proteins module and the final AET2010/TIGIT complex structural model was determined based on ZDock Score and ZRank Score in combination with empirical review and determination of interacting interface residues based on analysis of the complex structural model (antibody variable region sequence site numbering using Kabat rules, see Kabat et al, J Immunol 1991,147: 1709-19).

2.2 HSA-hTIGIT-His and AET2010 alanine scanning and mutant expression vector construction, expression and purification

According to the prediction results, the interfacial residues directly involved in the AET2010/TIGIT interaction were mutated to alanine. Using the HSA-hTIGIT-His expression vector (pABG-HSA-hTIGIT-His) of example 1 and the heavy and light chain expression vectors of AET2010 as templates, site-directed mutagenesis primers were designed as listed in Table 5, and an expression vector of a mutant was constructed by a site-directed mutagenesis PCR method. Sequencing-correct mutant plasmid transfection FreeStyle^TM293-F cells were subjected to expression of mutant antibodies as well as mutant antigens. SDS-PAGE identifies the purity of the purified mutants and the mutant concentration is quantified spectrophotometrically.

TABLE 5 construction of primer List with HSA-hTIGIT-His and AET2010 alanine scanning mutants

Note: in "mutant name column" of Table 5, the number indicates the mutation position of the original protein, the letter before the number is the pre-mutation amino acid, and the letter after the number indicates the post-mutation amino acid.

2.3 forteBIO^TMSystematic detection of changes in mutant binding activity

First, the change in binding activity of the HSA-hTIGIT-His mutant to AET2010 was examined. And (3) diluting AET2010, the HSA-hTIGIT-His parent and the mutant thereof to 100nM by using HBS-EP + buffer solution, and adding the diluted parent and the mutant to corresponding positions of a 96-well plate for detection according to a program design, wherein each position is 200 mu L/well. Performing Baseline>Loading>Baseline>Asscociation>The Disassocination process adopts AHC (Anti-Human IgG Conjugated) Biosensor to capture and fix AET2010, and the Asscoocination time is set to be 3min and the Disassocination time is set to be 6 min. The result is processed and fitted by Data Analysis software to obtain the affinity constant K of AET2010 and HSA-hTIGIT-His mutant_DAnd compared to the parent HSA-hTIGIT-His.

The AET2010 mutant was then tested for changes in binding activity to HSA-hTIGIT-His. HSA-hTIGIT-His, AET2010 parent and mutant are respectively diluted to 100nM by HBS-EP + buffer solution, and are added to the corresponding position of a 96-well plate for detection according to the program design, and 200 mu L/well. Baseline was performed using an AHC (Anti-Human IgG Conjugated) Biosensor>Loading>Baseline program, capture the antibodies to be tested one by one to the target response value (RU). Performing Baseline>Asscociation>The Disassocition process is used for detecting the dynamic characteristics of the interaction between the antibody to be detected and HSA-hTIGIT-His in real time, wherein the Association time is 3min, and the Disassocition time is 6 min. The result is processed and fitted by Data Analysis software to obtain the affinity constant K of AET2010 and mutant thereof and HSA-hTIGIT-His_DAnd compared.

The AET2010 mutant is a mutant antibody obtained by combining three mutants of the AET2010 light chain in table 5 with the AET2010 heavy chain or combining the AET2010 light chain with two mutants of the AET2010 heavy chain.

Second, result in

Prediction results of AET2010/TIGIT composite structure model and interaction

Blast search determined the homologous template for the AET2010 variable region to be PDB:6A3W (resolution of) The sequence consistency of the A chain and the B chain with AET2010 is 83.6%, the sequence similarity is 89.8%, 5 AET2010 variable region structure models are preliminarily constructed through homologous modeling, CDR regions of the sequencing optimal model are optimized to obtain 5 optimized models, and the AET2010 variable region structure models with reasonable structures and reliable quality are obtained through empirical inspection and combination of Profile-3D scores and Rarmchandar Plot (Rarmchandar Plot) conditions.

AET2010 variable region structural model and TIGIT (from PDB:3UCR, resolution of) The molecular docking of (c) produced 2000 complex conformations (Pose) and were subjected to cluster analysis and ZDock, ZRank scoring. Empirical examination of the Center conformation (Cluster Center) interaction characteristics of the ten clusters with the largest capacity (Cluster Size) revealed that the conformation of the two docking molecules in the Cluster with the largest capacity (i.e., Cluster 1) was also more rational. Further, the complete conformation of Cluster1 was structurally optimized using Refine Dock Proteins (RDOCK). Three-dimensional mapping (3D Point Plot) was performed based on ZDock Score and ZRank Score values for each conformation; empirically inspecting the structure of the conformation with the ZDock Score being more than or equal to 42 and the ZRank Score being less than or equal to-50, and finally determining the optimized Pose152 as an AET2010/TIGIT compound docking model by referring to the E _ RDOCK value, wherein the result is shown as A in FIG. 14; the distribution of amino acid residues directly involved in the interaction is shown in FIG. 14B and Table 6.

TABLE 6 AET2010/TIGIT Complex Structure model interaction interface residues with Hydrogen bonds

Note: the criterion for the interatomic distance of hydrogen bond interactions in the analysis is

2. Alanine scanning verification compound structure model

And (3) a mutant obtained by performing alanine substitution for the aforementioned predicted interacting interface residue. forteBIO^TMThe statistical results of the systematic determination of the affinity changes are shown in table 7, and the substitution of alanine for the Y32, E50 and K91 of AET2010 light chain and the Q56 site of TIGIT has a large influence on the binding activity, suggesting that these sites play an important role in the interaction, and also verifying the reliability of the model to a certain extent.

Table 7, results of evaluation of affinity change of TIGIT and AET2010 mutants

Note: "-" indicates a 2-10 fold decrease in affinity, "-" indicates a 10-100 fold decrease in affinity, "- -" indicates a complete loss of affinity, and "+/-" indicates a change in affinity within a factor of 2.

From the complex structure model (fig. 14), it can be seen that the binding epitope of AET2010 to TIGIT covers the "Lock-Key" (Lock-Key) region of the TIGIT molecule surface, the complementary binding region of CD 155; explains the molecular mechanism of good blocking activity of AET 2010.

Example 5 stability and pharmacokinetic characteristics of AET2010

Materials and methods

1. Materials: BALB/c nude mice (male, 18-22d) are products of Wintonlihua, donkey anti-human IgG/Fc monoclonal antibody is a product of Sigma, and fetal bovine serum is a product of Gibco. Other sources of material are as described in the previous embodiments.

2. The method comprises the following steps:

2.1 standing accelerated test evaluation of in vitro stability of AET2010

Filtering through a 0.22-micron filter membrane, namely AET2010 for sterilization under the aseptic condition, and diluting AET2010 to 15 mu g/ml through aseptic PBS or Fetal Bovine Serum (FBS); sealing, placing in 37 deg.C incubator, taking out samples at 0 days, 1 day, 3 days, 7 days, 10 days and 14 days, and freezing at-20 deg.C for detection. The binding activity of the antibody was measured at each time point according to the ELISA method of the previous examples: coating HSA-hTIGIT-His, and using 0.05 mu g/ml as an initial concentration and 2-fold gradient to serially dilute 8 gradients of AET2010 samples to be tested at each time point.

2.2 AET2010 structural stability evaluation

The thermostability of the antibodies was determined using the Uncle high throughput protein stability assay System from Uncariained Labs. 1.85mg/ml of the purified AET2010 sample is detected on a machine: a Tm & Tagg with optional DLS program was used, the temperature interval was set at 25-95 ℃ and the sample was set with a duplicate well. Protein particle size, particle size distribution and thermal stability were analyzed from 9 parameters of analytical results Tm (protein melting temperature), Tagg266 (protein aggregation temperature), Tagg473, Z-ave.dia (protein mean diameter), PDI (particle size dispersion coefficient), Pk1.mode Dia (hydration kinetic diameter of major protein component in solution), Pk1 Mass% (Pk1 ratio), Pk2.mode Dia (hydration kinetic diameter of other protein component in solution), Pk2 Mass% (Pk2 ratio).

2.3 preliminary evaluation of AET2010 pharmacokinetic features

BALB/c nude mice (male, 18-22d), single tail vein injection of AET2010, 150 μ g/mouse, 3 mice per group. Tail veins were bled and sera collected at 1, 3, 6, 12, 24, 48, 72, 96, 144, 192 post injection and stored at-20 ℃ for testing. Antibody content in serum was quantified by ELISA at each time point: HSA-hTIGIT-His and donkey anti-human IgG/Fc monoclonal antibody are respectively coated, serum samples are serially diluted for 6 gradients according to 2-fold gradient at each time point (the initial dilution of samples for 1, 3, 6 and 12 hours is 1:10000, the initial dilution of samples for 24, 48, 72 and 96 hours is 1:5000, and the initial dilution of samples for 144 and 192 hours is 1:2000), and a standard curve is drawn by taking the reserved sample with the concentration of 500 mug/ml as a standard product. And detecting and calculating the AET2010 blood concentration at each time point, drawing a blood concentration-time curve, and preliminarily evaluating the pharmacokinetic characteristics of the AET 2010.

Second, result in

Stability of AET2010

Results of the accelerated standing experiment as shown in fig. 15, AET2010 placed at 37 ℃ in PBS (a in fig. 15) and FBS (B in fig. 15) still had little decreased binding activity by day 14, showing good standing stability.

The result of the Uncle particle size detection at 25 ℃ is shown in FIG. 16, the average particle size of the AET2010 sample is 79.12nm, and the particle size dispersion coefficient PDI is between 0.1 and 0.2, which indicates that the sample is slightly aggregated; the main protein component has a hydration kinetic diameter (Pk1 model Dia.) of 10.41nm, and belongs to the conventional particle size range of monoclonal antibodies; the Mass distribution was a single peak, and the Mass fraction ratio of the major particle diameter component (Pk1 Mass (%)) was 100%, indicating that the Mass fraction ratio of the aggregation peak was negligible.

The results of the Uncle stability test are listed in Table 8, and AET2010 has higher dissolution temperature and thermal aggregation temperature, which indicates that AET has good structural stability.

TABLE 8 stability results of AET2010 testing by Uncle

Preliminary pharmacokinetic characterization of AET2010

The results of the detection of the pharmacokinetic characteristics of AET2010 in nude mice are shown in fig. 17, no obvious abnormal characteristics are seen, and the stability of AET2010 is further confirmed.

Example 6 in vitro functional evaluation of AET2010

Materials (I) and (II)

The NK-92MI cell is a product of Wuhan Punuoist Life technologies, human lung adenocarcinoma cell A549, human epidermal carcinoma cell A431 and human glioma cell U251 MG. The alpha-MEM culture medium and the fetal bovine serum are products of Gibco company, the RPMI-1640 culture medium and the DMEM high-sugar culture medium, and the horse serum is a product of Zhongsheng Oxbang company. ELISA detection kits for perforin, interferon gamma (IFN-gamma) and tumor necrosis factor (TNF-alpha) are all products of Elapscience company, CCK8 reagent is a product of Nippon Hojindo chemical company, PE-labeled mouse anti-human CD155(337609), APC-labeled mouse anti-human PD-1(329907) and PD-L1(329707) flow-type antibodies are all products of Biolegend company, and isotype-independent control antibodies (anti-IFN-alpha human IgG1 antibodies) are prepared and stored for the applicant, and the preparation method is described in patent ZL 201510685200.X for the GGE mutant. Other sources of material are contemplated as in the previous embodiments.

2. The method comprises the following steps:

2.1 preparation of anti-PD-1 antibody Nivolumab

The Nivolumab (patent No. WO2006121168A1) antibody was promised by Wang Shuangbo, and consists of a light chain and a heavy chain. The construction method of the light and heavy chain vector is the same as that of MK7684 light and heavy chain vector in example 2, the light chain vector is pABK, and the heavy chain vector is pABG4 (FIG. 1, the sequence of which is SEQ ID NO.23 in the sequence table). Expression purification methods are also consistent with AET2010 or MK 7684. The light chain amino acid sequence is shown as SEQ ID NO.17 (the 1 st amino acid is a part of the carrier secretion signal peptide, the obtained antibody product does not contain the amino acid), and the gene coding sequence is shown as SEQ ID NO. 18; the heavy chain amino acid sequence is shown as SEQ ID NO.19 (the 1 st to 4 th amino acids are a part of the carrier secretion signal peptide, the obtained antibody product does not contain the 4 amino acids), and the gene coding sequence is shown as SEQ ID NO. 20.

Wherein the light chain expression vector of the Nivolumab is a recombinant vector obtained by replacing a recognition sequence between Xba I and Nar I of the pABK expression vector with a light chain variable region gene shown in 1-332 th position of SEQ ID NO.18, the recombinant vector contains the light chain gene shown in SEQ ID NO.18, and can express the light chain of the Nivolumab shown in 2-215 th position of SEQ ID NO. 17.

The Nivolumab heavy chain expression vector is a recombinant vector obtained by replacing a recognition sequence between Afl II and Nhe I of a pABG4 expression vector with a heavy chain variable region gene shown in the 1 st to 351 th sites of SEQ ID NO.20, contains the heavy chain gene shown in the SEQ ID NO.20, and can express the Nivolumab heavy chain shown in the 5 th to 444 th sites of SEQ ID NO. 19.

2.2 detection of expression of CD155 and PD-L1 on the surface of tumor cells

Digesting and collecting A549, A431 and U251MG cells in logarithmic growth phase, and adjusting cell density to 3 × 10⁵cells/test, coupled with PE-labeled mouse anti-human CD155 (5. mu.l/test) or APC-labeled mouse anti-human PD-L1 (5. mu.l/test) on ice for 1hEach group was set with 3 duplicate wells and no antibody as a blank control, washed 2 times with DMEM medium (containing 10% FBS) after binding was completed, detected by flow cytometry, and the cell surface CD155 or PD-L1 expression level was determined from the mean fluorescence intensity.

2.3 detection of NK-92MI cell surface TIGIT and PD-1 expression conditions

Collecting NK-92MI cells in logarithmic growth phase and adjusting cell density to 3 × 10⁵And/test, combining the cells with an APC-labeled mouse anti-human TIGIT antibody (5 mu l/test) or a PD-1 antibody (5 mu l/test) for 1h on ice, setting 3 multiple wells in each group, setting no antibody as a blank control group, washing the cells for 2 times by using a DMEM medium (containing 10% FBS), detecting the cells by using a flow cytometer, and determining the TIGIT and PD-1 expression conditions on the surfaces of the NK-92MI cells according to the average fluorescence intensity.

2.4 cell killing experiments and evaluation of antibody function

Three tumor cells, namely A549, A431 and U251MG, are used as target cells, NK-92MI is used as effector cells, an in vitro killing experiment is carried out, and the functional activity of the antibody is evaluated according to the experiment.

The target cells in good state are 5X 10³Cells/ml × 100 μ l/well are inoculated in 96-well plates and cultured for 24-48 h until they are full. AET2010 was diluted with DMEM medium (containing 10% fetal bovine serum): the initial concentration is 200 mug/ml, 6 gradients are serially diluted by 2 times of gradients, and the diluted solution is added into the corresponding position of a 96-well plate according to 50 mug/hole; the growth medium adjusts the effector cells with good state to a specific density, and the effector cells are added into the corresponding position of a 96-well plate according to 50 mul/well; the effective target (E: T) ratios used were 1:1(NK-92 MI: A549), 2: 1(NK-92 MI: A431) and 0.25:1(NK-92 MI: U251 MG); 3 duplicate wells were set for each experimental condition, and separate target cell control wells and effector cell control wells (also subjected to subsequent supernatant aspiration and wash steps, equivalent to a blank medium control) were set; an irrelevant human IgG1 antibody was used as an isotype control (negative), and MK7684 was used as a positive control, and diluted under the same conditions. Standing the cell culture box for 5-12h (A549 and A431 are 12h, and U251MG is 5h), sucking the supernatant, transferring the supernatant to a 96-hole deep-hole plate, centrifuging at 1500rpm for 15min, and taking the supernatant for quantitative detection of the levels of the killer cytokines such as perforin, TNF-alpha, IFN-gamma and the like. Detection methodThe corresponding ELISA detection kit was used.

The 96-well cell culture plate after the supernatant was aspirated was washed twice with PBS, the CCK8 reagent was diluted to a final concentration of 10% with DMEM medium (containing 10% fetal bovine serum) and added at 100 μ l/well, the cell culture chamber was left standing at 37 ℃ for 2.5 hours, absorbance at a wavelength of 450nm (595nm is a reference wavelength) was measured with a microplate reader, and the cell killing rate was calculated: the cell killing rate is [1- (absorbance of experiment group-absorbance of effector cell control group)/(absorbance of target cell control group-absorbance of blank medium) ] × 100%; concentration-kill curves were plotted and EC50 values were calculated.

For TIGIT and PD-1 double-positive target cells U251MG, a single anti-PD-1 antibody Nivolumab group and an equal ratio combined group respectively with AET2010 and MK7684 are further arranged on the basis of the above, the concentration of AET2010 is the same as that of AET2010, and the concentration of MK7684 is the same as that of AET 2010.

Second, result in

1. Flow assay of cell surface molecules

As shown in a in fig. 18, the cell surface CD155 positivity of target cells a549, a431 and U251MG was 99% or more; as shown in B in FIG. 18, the positive rate of PD-L1 on the cell surface of U251MG is more than 90%, while the positive rates of PD-L1 on the cell surfaces of A549 and A431 are lower. As shown in C and D in FIG. 18, the positive rates of two inhibitory receptors, TIGIT and PD-1, on the surface of NK-92MI of effector cells were 21.1% and 19.7%, respectively; it is thus presumed that the target cells have a certain inhibitory effect on the killing of NK-92MI, and that the addition of antibodies is blocked to promote and enhance the killing activity of NK-92MI, thereby evaluating the functional activity of the antibodies.

Effect of AET2010 on NK-92MI cell in vitro killing tumor cell Activity

As shown in FIGS. 19-21, compared with an unrelated human IgG1 antibody control (isotype control antibody), AET2010 and MK7684 can increase the levels of IFN-gamma of NK-92MI cells releasing perforin and TNF-alpha within a certain concentration range, and the difference has statistical significance.

As shown in FIG. 22, compared with isotype-independent control antibody, AET2010 and MK7684 can increase the killing rate of NK-92MI cells on tumor cells within a certain concentration range, and show a dose-dependent relationship. For A549 cells (A), the EC50 values corresponding to AET2010 and MK7684 were 30.70 μ g/ml and 27.43 μ g/ml, respectively; for A431 cells (B), the EC50 values for AET2010 and MK7684 were 24.93 μ g/ml and 24.25 μ g/ml, respectively; for U251MG cells (C), the EC50 values corresponding to AET2010 and MK7684 were 23.70 μ g/ml and 29.94 μ g/ml, respectively, and the promotion effects of the two on the in vitro tumor cell killing activity of NK-92MI cells were equivalent.

Meanwhile, for U251MG cells with double positive CD155 and PD-L1, the promotion effect of the combination of AET2010, MK7684 and Nivolumab on the killing activity of NK-92MI is also obviously stronger than that of the single use (figure C); the release levels of the corresponding killer cytokines were also significantly elevated (fig. 21).

At an antibody concentration of 100. mu.g/ml, the killing rate of the combination of AET2010 and Nivolumab on U251MG is 72.52 +/-0.05%, and the killing rate of the combination of MK7684 and Nivolumab on U251MG is 70.12 +/-2.60%; at an antibody concentration of 200. mu.g/ml, the killing rate of the combination of AET2010 and Nivolumab on U251MG was 71.56 + -0.10%, and the killing rate of the combination of MK7684 and Nivolumab on U251MG was 70.02 + -0.58%.

Example 7 evaluation of in vivo drug efficacy of AET2010

Materials and methods

1. Materials: BALB/c nude mice (male, 4 weeks old) were a product of Witongli, Inc., and other experimental materials were the same as in the previous examples.

2. The method comprises the following steps: 4-week-old Balb/c nude mice (N ═ 20) were inoculated subcutaneously in the back with A549 tumor cells (5X 10)⁶cells/100 μ l/cell), tumor growth was observed periodically, and tumor volume was measured (V ═ long diameter × short diameter)²/2). When the tumor grows to 300 +/-100 mm³At this time, groups were randomized into four groups of 5, 3 experimental groups and 1 control group. The groups were dosed as follows: AET2010 panel was injected with NK-92MI cells (1X 10) via tail vein adoptively⁷cells/200. mu.l/mouse) and AET2010 (300. mu.g/mouse), MK7684 panels were injected with NK-92MI cells (1X 10 cells) via tail vein⁷cells/200. mu.l/mouse) and MK7684 (300. mu.g/mouse), independent antibody panel was injected by tail vein adoptively with NK-92MI cells (1)×10⁷cells/200. mu.l/mouse) and isotype-independent control antibody (human IgG1 antibody against IFN-. alpha.) (300. mu.g/mouse); the control group was injected with PBS buffer (200. mu.l/mouse) via tail vein. The frequency of treatment administration was once every 5 days. During the experiment, mice survival status was observed and recorded periodically weekly, tumor volumes were measured and recorded, and tumor growth (tumor volume-time) curves were plotted. Tumor volume in mice over 2000m on day 30 after initiation of treatment³Mice were euthanized, tumor masses were removed, weighed and photographed.

Second, result in

A CDX mouse model inoculated with A549 cells subcutaneously is taken as a research object, and the effect of the antibody on promoting the activity of NK-92MI in vivo and further killing tumors is evaluated by adoptively infusing NK-92MI cells and applying antibody drug therapy, so that the in vivo efficacy of the antibody is preliminarily determined by the model. In the experimental process, the tumor volume of the PBS control group of mice exceeds 2000mm on the 20 th day³Then the patient is sacrificed; mice were sacrificed at the experimental end-point (day 30 after treatment initiation) and tumors were weighed. The results are shown in fig. 23-24, where AET2010 and MK7684 had significant inhibition of tumor growth (P < 0.0001) compared to isotype control antibodies, and the antitumor effects of both were comparable.

AET2010 showed enhanced antitumor activity in a CDX mouse model with adoptive infusion of NK-92MI cells, whose potential pharmacodynamic characteristics were expected.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> military medical research institute of military science institute of people's liberation force of China

<120> human anti-human TIGIT antibody and application thereof

<160> 23

<170> PatentIn version 3.5

<210> 1

<211> 142

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 1

Met Asp Phe Gly Leu Ser Leu Val Phe Leu Val Leu Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser

20 25 30

Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr

35 40 45

Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu

50 55 60

Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys

65 70 75 80

Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu

85 90 95

Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro

100 105 110

Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser

115 120 125

Val Ala Glu His Gly Ala Arg Phe His His His His His His

130 135 140

<210> 2

<211> 426

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

atggatttcg gcctgagcct ggtgttcctg gtgctgatcc ttaagggcgt gcagtgcatg 60

atgacaggca caatagaaac aacggggaac atttctgcag agaaaggtgg ctctatcatc 120

ttacaatgtc acctctcctc caccacggca caagtgaccc aggtcaactg ggagcagcag 180

gaccagcttc tggccatttg taatgctgac ttggggtggc acatctcccc atccttcaag 240

gatcgagtgg ccccaggtcc cggcctgggc ctcaccctcc agtcgctgac cgtgaacgat 300

acaggggagt acttctgcat ctatcacacc taccctgatg ggacgtacac tgggagaatc 360

ttcctggagg tcctagaaag ctcagtggct gagcacggtg ccaggttcca ccatcaccac 420

catcat 426

<210> 3

<211> 739

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 3

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala

20 25 30

His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu

35 40 45

Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val

50 55 60

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp

65 70 75 80

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp

85 90 95

Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala

100 105 110

Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln

115 120 125

His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val

130 135 140

Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys

145 150 155 160

Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro

165 170 175

Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys

180 185 190

Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu

195 200 205

Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys

210 215 220

Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val

225 230 235 240

Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser

245 250 255

Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly

260 265 270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile

275 280 285

Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu

290 295 300

Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp

305 310 315 320

Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser

325 330 335

Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly

340 345 350

Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val

355 360 365

Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys

370 375 380

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu

385 390 395 400

Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys

405 410 415

Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu

420 425 430

Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val

435 440 445

Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His

450 455 460

Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val

465 470 475 480

Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg

485 490 495

Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe

500 505 510

Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala

515 520 525

Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu

530 535 540

Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys

545 550 555 560

Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala

565 570 575

Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe

580 585 590

Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly

595 600 605

Leu Gly Gly Gly Gly Ser Met Met Thr Gly Thr Ile Glu Thr Thr Gly

610 615 620

Asn Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu

625 630 635 640

Ser Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp

645 650 655

Gln Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro

660 665 670

Ser Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu

675 680 685

Gln Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His

690 695 700

Thr Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu

705 710 715 720

Glu Ser Ser Val Ala Glu His Gly Ala Arg Phe His His His His His

725 730 735

His His His

<210> 4

<211> 2217

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60

gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa 120

gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca gtgtccattt 180

gaagatcatg taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat 240

gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa attatgcaca 300

gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360

gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg 420

agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac atttttgaaa 480

aaatacttat atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc 540

tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600

tgcctgttgc caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 660

agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc atgggcagta 720

gctcgcctga gccagagatt tcccaaagct gagtttgcag aagtttccaa gttagtgaca 780

gatcttacca aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac 840

agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag taaactgaag 900

gaatgctgtg aaaaacctct gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat 960

gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaaagtaa ggatgtttgc 1020

aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga 1080

aggcatcctg attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact 1140

ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt gttcgatgaa 1200

tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga gctttttgag 1260

cagcttggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa gaaagtaccc 1320

caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 1380

tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag aagactatct atccgtggtc 1440

ctgaaccagt tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt caccaaatgc 1500

tgcacagaat ccttggtgaa caggcgacca tgcttttcag ctctggaagt cgatgaaaca 1560

tacgttccca aagagtttaa tgctgaaaca ttcaccttcc atgcagatat atgcacactt 1620

tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagctcgt gaaacacaag 1680

cccaaggcaa caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagag 1740

aagtgctgca aggctgacga taaggagacc tgctttgccg aggagggtaa aaaacttgtt 1800

gctgcaagtc aagctgcctt aggcttaggt ggaggcggta gcatgatgac aggcacaata 1860

gaaacaacgg ggaacatttc tgcagagaaa ggtggctcta tcatcttaca atgtcacctc 1920

tcctccacca cggcacaagt gacccaggtc aactgggagc agcaggacca gcttctggcc 1980

atttgtaatg ctgacttggg gtggcacatc tccccatcct tcaaggatcg agtggcccca 2040

ggtcccggcc tgggcctcac cctccagtcg ctgaccgtga acgatacagg ggagtacttc 2100

tgcatctatc acacctaccc tgatgggacg tacactggga gaatcttcct ggaggtccta 2160

gaaagctcag tggctgagca cggtgccagg ttccaccatc accaccatca tcaccat 2217

<210> 5

<211> 741

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

agctacgaac tgacccagcc gccgagcgtg tcggtggcgc cgggtcagac cgcgcgtatc 60

acctgctcgg gcgatgcgct gggcgataaa tacgcgagct ggtatcagca gaaaccgggt 120

caggcaccgg tgctggtgat ttacgaagat tctaaacgcc cgtctggcat cccggaacgc 180

tttagcggct cgaattcggg caacaccgcg accctgacca ttagcggcac ccaggcggag 240

gatgaggcgg actattactg ctcggcgaag gatgccagct ttaattctgt gtttggcggt 300

ggcaccaaac tgaccgtgct gggcagcggc ggctcgacca taacttcgta taatgtatac 360

tatacgaagt tatcgagctc gggcagcgaa gttcaactgg ttcaaagtgg tgcggaagtg 420

aagaaaccgg gcgaaagtct gaaaattagt tgcaaaggct ctggttattc ttttacgtct 480

tattggatcg gctgggttcg tcagatgccg ggtaaaggtc tggaatggat gggtattatt 540

tatccgggtg atagtgatac gcgttattct ccgagttttc agggtcaggt tactattagt 600

gcagataaaa gcatcagcac cgcgtatctg cagtggagtt ctctgaaagc gagtgatacc 660

gcgatgtatt attgcgcacg tgctgcgtgg tggcaggggt ttgatcactg gggtcagggc 720

actctggtga ccgtgtcgag c 741

<210> 6

<211> 247

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 6

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Gly Asp Lys Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Glu Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Lys Asp Ala Ser Phe Asn Ser

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Gly Gly Ser

100 105 110

Thr Ile Thr Ser Tyr Asn Val Tyr Tyr Thr Lys Leu Ser Ser Ser Gly

115 120 125

Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

130 135 140

Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser

145 150 155 160

Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp

165 170 175

Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser

180 185 190

Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala

195 200 205

Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr

210 215 220

Cys Ala Arg Ala Ala Trp Trp Gln Gly Phe Asp His Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 7

<211> 639

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

agctacgaac tgacccagcc gccgagcgtg tcggtggcgc cgggtcagac cgcgcgtatc 60

acctgctcgg gcgatgcgct gggcgataaa tacgcgagct ggtatcagca gaaaccgggt 120

caggcaccgg tgctggtgat ttacgaagat tctaaacgcc cgtctggcat cccggaacgc 180

tttagcggct cgaattcggg caacaccgcg accctgacca ttagcggcac ccaggcggag 240

gatgaggcgg actattactg ctcggcgaag gatgccagct ttaattctgt gtttggcggt 300

ggcaccaagc ttaccgtcct aggtcagccc aaggctgccc cctcggtcac tctgttcccg 360

ccctcctctg aggagcttca agccaacaag gccacactgg tgtgtctcat aagtgacttc 420

tacccgggag ccgtgacagt ggcctggaag gcagatagca gccccgtcaa ggcgggagtg 480

gagaccacca caccctccaa acaaagcaac aacaagtacg cggccagcag ctatctgagc 540

ctgacgcctg agcagtggaa gtcccacaga agctacagct gccaggtcac gcatgaaggg 600

agcaccgtgg agaagacagt ggcccctaca gaatgttca 639

<210> 8

<211> 1344

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

gaagttcaac tggttcaaag tggtgcggaa gtgaagaaac cgggcgaaag tctgaaaatt 60

agttgcaaag gctctggtta ttcttttacg tcttattgga tcggctgggt tcgtcagatg 120

ccgggtaaag gtctggaatg gatgggtatt atttatccgg gtgatagtga tacgcgttat 180

tctccgagtt ttcagggtca ggttactatt agtgcagata aaagcatcag caccgcgtat 240

ctgcagtgga gttctctgaa agcgagtgat accgcgatgt attattgcgc acgtgctgcg 300

tggtggcagg ggtttgatca ctggggtcag ggcactctgg tgaccgtgtc gagcgctagc 360

accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420

gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480

tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540

tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600

tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960

aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 1080

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200

tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 1260

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320

agcctctccc tgtccccggg taaa 1344

<210> 9

<211> 213

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 9

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Gly Asp Lys Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Glu Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Lys Asp Ala Ser Phe Asn Ser

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala

100 105 110

Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala

115 120 125

Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala

130 135 140

Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val

145 150 155 160

Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser

165 170 175

Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr

180 185 190

Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala

195 200 205

Pro Thr Glu Cys Ser

210

<210> 10

<211> 448

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 10

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Ala Ala Trp Trp Gln Gly Phe Asp His Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 11

<211> 214

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr Ser Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 12

<211> 449

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 12

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 13

<211> 642

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

gacattcaga tgacccagag ccccagcagc ctgagcgcca gcgtgggaga cagagtgacc 60

atcacctgca gagccagcga gcacatctac tcctacctgt cctggtacca gcagaaaccc 120

ggcaaagccc caaaactgct gatctacaac gccaagaccc tggccgaagg cgtgcccagc 180

agattctcag gcagcggctc cggcaccgac ttcaccctga ctatcagcag cctgcagccc 240

gaagatttcg ccacctacta ctgccagcac cacttcggca gccccctgac cttcggacag 300

ggcaccagac tggagatcaa gagaaccgtg gcggcgccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggtaccgct agcgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

<210> 14

<211> 1347

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

gaggtgcagc tggtgcagag cggagccgag gtgaagaagc caggagccag cgtgaaggtg 60

agctgtaagg ctagtggata cacattcagc agctacgtga tgcactgggt gagacaggcc 120

cctggacagg gactggagtg gattggatat atcgacccct acaatgacgg cgccaagtac 180

gcccagaaat tccagggcag agtgaccctg accagcgaca agagcaccag caccgtgtat 240

atggagctga gcagcctgag aagcgaggac accgccgtgt actactgcgc cagaggagga 300

ccctacggct ggtatttcga tgtgtggggc cagggaacca ccgtgacagt gagtagcgct 360

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcctct ccctgtcccc gggtaaa 1347

<210> 15

<211> 557

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 15

Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln

1 5 10 15

Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln

20 25 30

Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg

35 40 45

His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro

50 55 60

Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly

65 70 75 80

Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu

85 90 95

Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser

100 105 110

Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr

115 120 125

Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala

130 135 140

Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His

145 150 155 160

Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu

165 170 175

Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser

180 185 190

Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe

195 200 205

Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro

210 215 220

Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn

225 230 235 240

Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly

245 250 255

Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala

260 265 270

Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr

275 280 285

Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu

290 295 300

Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Met

305 310 315 320

Ser Arg Asn Ala Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

325 330 335

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

340 345 350

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

355 360 365

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

370 375 380

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

385 390 395 400

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

405 410 415

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

420 425 430

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

435 440 445

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

450 455 460

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

465 470 475 480

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

485 490 495

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

500 505 510

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

515 520 525

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

530 535 540

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

545 550 555

<210> 16

<211> 1671

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

tggccacccc caggaaccgg ggacgtcgtc gtgcaggcgc ccacccaggt gcccggcttc 60

ttgggcgact ccgtgacgct gccctgctac ctacaggtgc ccaacatgga ggtgacgcat 120

gtgtcacagc tgacttgggc gcggcatggt gaatctggca gcatggccgt cttccaccaa 180

acgcagggcc ccagctattc ggagtccaaa cggctggagt tcgtggcagc cagactgggc 240

gcggagctgc ggaatgcctc gctgaggatg ttcgggttgc gcgtagagga tgaaggcaac 300

tacacctgcc tgttcgtcac gttcccgcag ggcagcagga gcgtggatat ctggctccga 360

gtgcttgcca agccccagaa cacagctgag gttcagaagg tccagctcac tggagagcca 420

gtgcccatgg cccgctgcgt ctccacaggg ggtcgcccgc cagcccaaat cacctggcac 480

tcagacctgg gcgggatgcc caatacgagc caggtgccag ggttcctgtc tggcacagtc 540

actgtcacca gcctctggat attggtgccc tcaagccagg tggacggcaa gaatgtgacc 600

tgcaaggtgg agcacgagag ctttgagaag cctcagctgc tgactgtgaa cctcaccgtg 660

tactaccccc cagaggtatc catctctggc tatgataaca actggtacct tggccagaat 720

gaggccaccc tgacctgcga tgctcgcagc aacccagagc ccacaggcta taattggagc 780

acgaccatgg gtcccctgcc accctttgct gtggcccagg gcgcccagct cctgatccgt 840

cctgtggaca aaccaatcaa cacaacttta atctgcaacg tcaccaatgc cctaggagct 900

cgccaggcag aactgaccgt ccaggtcaaa gagggacctc ccagtgagca ctcaggcatg 960

tcccgtaacg ctagcgagcc caaatcttgt gacaaaactc acacatgccc accgtgccca 1020

gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 1080

ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 1140

cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 1200

ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 1260

caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 1320

cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 1380

ctgcccccat cccgggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 1440

ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1500

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta tagcaagctc 1560

accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1620

gctctgcaca accactacac gcagaagagc ctctccctgt ccccgggtaa a 1671

<210> 17

<211> 215

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 17

Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

1 5 10 15

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro

85 90 95

Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 18

<211> 444

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 18

Gly Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val

1 5 10 15

Gln Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr

20 25 30

Phe Ser Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly

35 40 45

Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr

50 55 60

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

65 70 75 80

Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 19

<211> 645

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

ggcgagatcg tgctgaccca gtctcctgcc acactgtccc tgtctcctgg agagagagcc 60

acactgtcct gcagagcctc tcagtccgtg tcctcctacc tggcctggta ccagcagaag 120

cctggacagg ctcctcggct gctgatctac gacgcctcca acagagccac aggcatccct 180

gctcggttct ctggctctgg ctctggcaca gacttcaccc tgaccatctc ctctctggag 240

cctgaggact tcgccgtgta ctactgccag cagtcctcca actggcctag gaccttcgga 300

cagggcacca aggtggagat caagcggacc gtggcggcgc catctgtctt catcttcccg 360

ccatctgatg agcagttgaa atctggtacc gctagcgttg tgtgcctgct gaataacttc 420

tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480

caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540

acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600

ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 645

<210> 20

<211> 1332

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

ggcgtgcagt gccaggtgca gctggtggag tctggaggtg gagtggtgca gcctggacgg 60

tctctgagac tggactgcaa ggcctctggc atcaccttct ccaactctgg catgcactgg 120

gtgagacagg ctcctggcaa gggactggag tgggtggctg tgatctggta cgacggctcc 180

aagcggtact acgccgactc cgtgaaggga cggttcacca tctctcggga caactccaag 240

aacaccctgt tcctgcagat gaactccctg agagccgagg acaccgctgt gtactactgc 300

gccaccaacg acgactactg gggacagggc acactggtga ccgtgtcctc cgctagcacc 360

aagggcccat ccgtcttccc cctggcgccc tgctccagga gcacctccga gagcacagcc 420

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacgaagac ctacacctgc 600

aacgtagatc acaagcccag caacaccaag gtggacaaga gagttgagtc caaatatggt 660

cccccatgcc caccatgccc agcacctgag ttcctggggg gaccatcagt cttcctgttc 720

cccccaaaac ccaaggacac tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 780

gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 840

gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 900

agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 960

tccaacaaag gcctcccgtc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 1020

cgagagccac aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtc 1080

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 1140

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1200

ttcttcctct acagcaggct aaccgtggac aagagcaggt ggcaggaggg gaatgtcttc 1260

tcatgctccg tgatgcatga ggctctgcac aaccactaca cacagaagag cctctccctg 1320

tctctgggta aa 1332

<210> 21

<211> 4735

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 60

cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 120

ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 180

tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 240

taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 300

caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 360

agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 420

gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 480

gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 540

ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 600

acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 660

tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 720

ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt 780

ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga 840

ccgagcgcag cgagtcagtg agcgaggaag cgtacattta tattggctca tgtccaatat 900

gcccgggatg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 960

tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1020

gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 1080

cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 1140

tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta 1200

aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt 1260

acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg 1320

ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 1380

ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc 1440

cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 1500

tagtgaaccg tcagatcctc actctcttcc gcatcgctgt ctgcgagggc cagctgttgg 1560

gctcgcggtt gaggacaaac tcttcgcggt ctttccagta ctcttggatc ggaaacccgt 1620

cggcctccga acggtactcc gccaccgagg gacctgagcg agtccgcatc gaccggatcg 1680

gaaaacctct cgagaaaggc gtctaaccag tcacagtcgc aaggtaggct gagcaccgtg 1740

gcgggcggca gcgggtggcg gtcggggttg tttctggcgg aggtgctgct gatgatgtaa 1800

ttaaagtagg cggtcttgag acggcggatg gtcgaggtga ggtgtggcag gcttgagatc 1860

cagctgttgg ggtgagtact ccctctcaaa agcgggcatt acttctgcgc taagattgtc 1920

agtttccaaa aacgaggagg atttgatatt cacctggccc gatctggcca tacacttgag 1980

tgacaatgac atccactttg cctttctctc cacaggtgtc cactcccagg tccaagttta 2040

aacggatctc tagcgaattc gccgccacca tggacttcca ggtgcagatc ttcagcttcc 2100

tgctgatgag cgccagcgtg atcatgtcta gaggccacgt ggcggcgcca tctgtcttca 2160

tcttcccgcc atctgatgag cagttgaaat ctggtaccgc tagcgttgtg tgcctgctga 2220

ataacttcta tcccagagag gccaaagtac agtggaaggt ggataacgcc ctccaatcgg 2280

gtaactccca ggagagtgtc acagagcagg acagcaagga cagcacctac agcctcagca 2340

gcaccctgac gctgagcaaa gcagactacg agaaacacaa agtctacgcc tgcgaagtca 2400

cccatcaggg cctgagctcg cccgtcacaa agagcttcaa caggggagag tgttagggat 2460

cccccgacct cgacctctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg 2520

aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt ggtcgagatc 2580

cccgggatct ctagctagag gatcgatccc cgccccggac gaactaaacc tgactacgac 2640

atctctgccc cttcttcgcg gggcagtgca tgtaatccct tcagttggtt ggtacaactt 2700

gccaactgaa ccctaaacgg gtagcatatg cttcccgggt agtagtatat actatccaga 2760

ctaaccctaa ttcaatagca tatgttaccc aacgggaagc atatgctatc gaattagggt 2820

tagtaaaagg gtcctaagga acagcgatgt aggtgggcgg gccaagatag gggcgcgatt 2880

gctgcgatct ggaggacaaa ttacacacac ttgcgcctga gcgccaagca cagggttgtt 2940

ggtcctcata ttcacgaggt cgctgagagc acggtgggct aatgttgcca tgggtagcat 3000

atactaccca aatatctgga tagcatatgc tatcctaatc tatatctggg tagcataggc 3060

tatcctaatc tatatctggg tagcatatgc tatcctaatc tatatctggg tagtatatgc 3120

tatcctaatt tatatctggg tagcataggc tatcctaatc tatatctggg tagcatatgc 3180

tatcctaatc tatatctggg tagtatatgc tatcctaatc tgtatccggg tagcatatgc 3240

tatcctaata gagattaggg tagtatatgc tatcctaatt tatatctggg tagcatatac 3300

tacccaaata tctggatagc atatgctatc ctaatctata tctgggtagc atatgctatc 3360

ctaatctata tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc 3420

ctaatctata tctgggtagt atatgctatc ctaatttata tctgggtagc ataggctatc 3480

ctaatctata tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc 3540

ctaatctgta tccgggtagc atatgctatc ctcatgcata agctgtcaaa catgagaatt 3600

aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 3660

ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 3720

tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 3780

atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 3840

attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 3900

gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 3960

agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 4020

aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt 4080

cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 4140

cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 4200

actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 4260

cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 4320

ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 4380

ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 4440

gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 4500

gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 4560

ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 4620

cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 4680

caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaa 4735

<210> 22

<211> 4708

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 60

cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 120

ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 180

tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 240

taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 300

caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 360

agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 420

gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 480

gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 540

ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 600

acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 660

tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 720

ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt 780

ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga 840

ccgagcgcag cgagtcagtg agcgaggaag cgtacattta tattggctca tgtccaatat 900

gcccgggatg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 960

tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1020

gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 1080

cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 1140

tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta 1200

aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt 1260

acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg 1320

ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 1380

ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc 1440

cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 1500

tagtgaaccg tcagatcctc actctcttcc gcatcgctgt ctgcgagggc cagctgttgg 1560

gctcgcggtt gaggacaaac tcttcgcggt ctttccagta ctcttggatc ggaaacccgt 1620

cggcctccga acggtactcc gccaccgagg gacctgagcg agtccgcatc gaccggatcg 1680

gaaaacctct cgagaaaggc gtctaaccag tcacagtcgc aaggtaggct gagcaccgtg 1740

gcgggcggca gcgggtggcg gtcggggttg tttctggcgg aggtgctgct gatgatgtaa 1800

ttaaagtagg cggtcttgag acggcggatg gtcgaggtga ggtgtggcag gcttgagatc 1860

cagctgttgg ggtgagtact ccctctcaaa agcgggcatt acttctgcgc taagattgtc 1920

agtttccaaa aacgaggagg atttgatatt cacctggccc gatctggcca tacacttgag 1980

tgacaatgac atccactttg cctttctctc cacaggtgtc cactcccagg tccaagttta 2040

aacggatctc tagcgaattc gccgccacca tggatttcgg cctgagcctg gtgttcctgg 2100

tgctgatcct taagggcgct agcaccaagg gcccatcggt cttccccctg gcaccctcct 2160

ccaagagcac ctctgggggc acagcggccc tgggctgcct ggtcaaggac tacttccccg 2220

aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac accttcccgg 2280

ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg ccctccagca 2340

gcttgggcac ccagacctac atctgcaacg tgaatcacaa gcccagcaac accaaggtgg 2400

acaagaaagt tgagcccaaa tcttgttgag gatcccccga cctcgacctc tggctaataa 2460

aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg 2520

acatatggga gggcaaatca tttggtcgag atccccggga tctctagcta gaggatcgat 2580

ccccgccccg gacgaactaa acctgactac gacatctctg ccccttcttc gcggggcagt 2640

gcatgtaatc ccttcagttg gttggtacaa cttgccaact gaaccctaaa cgggtagcat 2700

atgcttcccg ggtagtagta tatactatcc agactaaccc taattcaata gcatatgtta 2760

cccaacggga agcatatgct atcgaattag ggttagtaaa agggtcctaa ggaacagcga 2820

tgtaggtggg cgggccaaga taggggcgcg attgctgcga tctggaggac aaattacaca 2880

cacttgcgcc tgagcgccaa gcacagggtt gttggtcctc atattcacga ggtcgctgag 2940

agcacggtgg gctaatgttg ccatgggtag catatactac ccaaatatct ggatagcata 3000

tgctatccta atctatatct gggtagcata ggctatccta atctatatct gggtagcata 3060

tgctatccta atctatatct gggtagtata tgctatccta atttatatct gggtagcata 3120

ggctatccta atctatatct gggtagcata tgctatccta atctatatct gggtagtata 3180

tgctatccta atctgtatcc gggtagcata tgctatccta atagagatta gggtagtata 3240

tgctatccta atttatatct gggtagcata tactacccaa atatctggat agcatatgct 3300

atcctaatct atatctgggt agcatatgct atcctaatct atatctgggt agcataggct 3360

atcctaatct atatctgggt agcatatgct atcctaatct atatctgggt agtatatgct 3420

atcctaattt atatctgggt agcataggct atcctaatct atatctgggt agcatatgct 3480

atcctaatct atatctgggt agtatatgct atcctaatct gtatccgggt agcatatgct 3540

atcctcatgc ataagctgtc aaacatgaga attaattctt gaagacgaaa gggcctcgtg 3600

atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 3660

acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 3720

atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 3780

agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 3840

cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 3900

gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 3960

cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 4020

tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 4080

ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 4140

ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 4200

atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 4260

cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 4320

atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 4380

gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 4440

cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 4500

tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 4560

tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 4620

gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 4680

gatttaaaac ttcattttta atttaaaa 4708

<210> 23

<211> 5380

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 60

cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 120

ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 180

tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 240

taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 300

caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 360

agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 420

gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 480

gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 540

ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 600

acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 660

tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 720

ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt 780

ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga 840

ccgagcgcag cgagtcagtg agcgaggaag cgtacattta tattggctca tgtccaatat 900

gcccgggatg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 960

tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1020

gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 1080

cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 1140

tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta 1200

aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt 1260

acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg 1320

ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 1380

ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc 1440

cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 1500

tagtgaaccg tcagatcctc actctcttcc gcatcgctgt ctgcgagggc cagctgttgg 1560

gctcgcggtt gaggacaaac tcttcgcggt ctttccagta ctcttggatc ggaaacccgt 1620

cggcctccga acggtactcc gccaccgagg gacctgagcg agtccgcatc gaccggatcg 1680

gaaaacctct cgagaaaggc gtctaaccag tcacagtcgc aaggtaggct gagcaccgtg 1740

gcgggcggca gcgggtggcg gtcggggttg tttctggcgg aggtgctgct gatgatgtaa 1800

ttaaagtagg cggtcttgag acggcggatg gtcgaggtga ggtgtggcag gcttgagatc 1860

cagctgttgg ggtgagtact ccctctcaaa agcgggcatt acttctgcgc taagattgtc 1920

agtttccaaa aacgaggagg atttgatatt cacctggccc gatctggcca tacacttgag 1980

tgacaatgac atccactttg cctttctctc cacaggtgtc cactcccagg tccaagttta 2040

aacggatctc tagcgaattc gccgccacca tggatttcgg cctgagcctg gtgttcctgg 2100

tgctgatcct taagggcgct agcaccaagg gcccatccgt cttccccctg gcgccctgct 2160

ccaggagcac ctccgagagc acagccgccc tgggctgcct ggtcaaggac tacttccccg 2220

aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac accttcccgg 2280

ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg ccctccagca 2340

gcttgggcac gaagacctac acctgcaacg tagatcacaa gcccagcaac accaaggtgg 2400

acaagagagt tgagtccaaa tatggtcccc catgcccacc atgcccagca cctgagttcc 2460

tggggggacc atcagtcttc ctgttccccc caaaacccaa ggacactctc atgatctccc 2520

ggacccctga ggtcacgtgc gtggtggtgg acgtgagcca ggaagacccc gaggtccagt 2580

tcaactggta cgtggatggc gtggaggtgc ataatgccaa gacaaagccg cgggaggagc 2640

agttcaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag gactggctga 2700

acggcaagga gtacaagtgc aaggtctcca acaaaggcct cccgtcctcc atcgagaaaa 2760

ccatctccaa agccaaaggg cagccccgag agccacaggt gtacaccctg cccccatccc 2820

aggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc ttctacccca 2880

gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac aagaccacgc 2940

ctcccgtgct ggactccgac ggctccttct tcctctacag caggctaacc gtggacaaga 3000

gcaggtggca ggaggggaat gtcttctcat gctccgtgat gcatgaggct ctgcacaacc 3060

actacacaca gaagagcctc tccctgtctc tgggtaaatg aggatccccc gacctcgacc 3120

tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc 3180

tcactcggaa ggacatatgg gagggcaaat catttggtcg agatccccgg gatctctagc 3240

tagaggatcg atccccgccc cggacgaact aaacctgact acgacatctc tgccccttct 3300

tcgcggggca gtgcatgtaa tcccttcagt tggttggtac aacttgccaa ctgaacccta 3360

aacgggtagc atatgcttcc cgggtagtag tatatactat ccagactaac cctaattcaa 3420

tagcatatgt tacccaacgg gaagcatatg ctatcgaatt agggttagta aaagggtcct 3480

aaggaacagc gatgtaggtg ggcgggccaa gataggggcg cgattgctgc gatctggagg 3540

acaaattaca cacacttgcg cctgagcgcc aagcacaggg ttgttggtcc tcatattcac 3600

gaggtcgctg agagcacggt gggctaatgt tgccatgggt agcatatact acccaaatat 3660

ctggatagca tatgctatcc taatctatat ctgggtagca taggctatcc taatctatat 3720

ctgggtagca tatgctatcc taatctatat ctgggtagta tatgctatcc taatttatat 3780

ctgggtagca taggctatcc taatctatat ctgggtagca tatgctatcc taatctatat 3840

ctgggtagta tatgctatcc taatctgtat ccgggtagca tatgctatcc taatagagat 3900

tagggtagta tatgctatcc taatttatat ctgggtagca tatactaccc aaatatctgg 3960

atagcatatg ctatcctaat ctatatctgg gtagcatatg ctatcctaat ctatatctgg 4020

gtagcatagg ctatcctaat ctatatctgg gtagcatatg ctatcctaat ctatatctgg 4080

gtagtatatg ctatcctaat ttatatctgg gtagcatagg ctatcctaat ctatatctgg 4140

gtagcatatg ctatcctaat ctatatctgg gtagtatatg ctatcctaat ctgtatccgg 4200

gtagcatatg ctatcctcat gcataagctg tcaaacatga gaattaattc ttgaagacga 4260

aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag 4320

acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 4380

atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 4440

tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 4500

gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 4560

gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 4620

gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 4680

ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat 4740

tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 4800

acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 4860

cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 4920

catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 4980

cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa 5040

ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 5100

ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 5160

ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 5220

atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 5280

gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat 5340

atactttaga ttgatttaaa acttcatttt taatttaaaa 5380