阅读说明：本技术 一种vegf-crm197重组融合蛋白疫苗及其制备方法和应用 (VEGF-CRM197 recombinant fusion protein vaccine and preparation method and application thereof ) 是由 张文耀 陈国友 于 2021-12-13 设计创作，主要内容包括：本发明提供了一种VEGF-CRM197重组融合蛋白疫苗及其制备方法和应用。具体地,本发明提供了一种丧失VEGF生物学活力但保留免疫原性的VEGF截短体VEGF1-107抗原片段,并融合白喉毒素突变体CRM197重组表达,形成VEGF重组融合蛋白。所述VEGF重组融合蛋白与液体佐剂联合使用后可以诱导小鼠和恒河猴体内产生高滴度高抗体,阻滞VEGF-A与其受体结合,从而抑制VEGF-A对血管内皮细胞增殖促进作用。(The invention provides a VEGF-CRM197 recombinant fusion protein vaccine, a preparation method and application thereof. Specifically, the invention provides a VEGF truncation VEGF1-107 antigen fragment which loses VEGF biological activity but retains immunogenicity, and fusion diphtheria toxin mutant CRM197 is subjected to recombinant expression to form a VEGF recombinant fusion protein. After the VEGF recombinant fusion protein is used together with a liquid adjuvant, high-titer high antibodies can be induced to be generated in mice and rhesus monkeys, and VEGF-A is blocked from being combined with a receptor thereof, so that the effect of promoting the proliferation of vascular endothelial cells by the VEGF-A is inhibited.)

1. A recombinant fusion protein, wherein the structure of the fusion protein is represented by formula I:

Z1-Z2-Z3 （I）

wherein Z1 is a VEGF antigen fragment element;

z2 is a linker peptide element or nothing; and

z3 is a diphtheria toxin mutant CRM197 protein element;

"-" denotes a peptide bond or a peptide linker connecting the above elements;

the amino acid sequence of the VEGF antigen fragment is shown as SEQ ID NO. 5 or SEQ ID NO. 6, and the VEGF antigen fragment loses VEGF biological activity but retains immunogenicity; and the amino acid sequence of the diphtheria toxin mutant CRM197 protein is shown in SEQ ID NO 7.

2. The fusion protein of claim 1, wherein the amino acid sequence of the fusion protein is set forth in SEQ ID NO 1 or SEQ ID NO 2.

3. The fusion protein of claim 1, wherein the amino acid sequence of the fusion protein is set forth in SEQ ID NO 1.

4. A polynucleotide encoding the recombinant fusion protein of claim 1.

5. An expression vector comprising the polynucleotide of claim 4.

6. A host cell comprising the expression vector of claim 5 or having the polynucleotide of claim 4 integrated into its genome.

7. A method of producing a recombinant fusion protein according to claim 1, comprising the steps of:

(i) culturing the host cell of claim 6;

(ii) inducing the host cell to express the recombinant fusion protein using an inducing agent, thereby obtaining an expressed recombinant fusion protein;

(iii) isolating the expressed recombinant fusion protein, thereby obtaining an isolated recombinant fusion protein.

8. A pharmaceutical composition comprising the recombinant fusion protein of claim 1 and a pharmaceutically acceptable carrier.

9. Use of a recombinant fusion protein according to any one of claims 1-3, a polynucleotide according to claim 4, an expression vector according to claim 5, a host cell according to claim 6 and/or a pharmaceutical composition according to claim 8 for the preparation of a medicament for the treatment and/or prevention of a disease.

10. The use according to claim 9, wherein the disease is selected from the group consisting of: tumors or cancers, macular edema secondary to retinal vein occlusion, wet age-related macular degeneration, and diabetic macular edema, or combinations thereof.

Technical Field

The invention belongs to the field of biotechnology and medicine, and particularly relates to preparation and application of a Vascular Endothelial Growth Factor (VEGF) vaccine.

Background

The VEGF/VEGFR signaling pathway is a critical limiting step in tumor tissue angiogenesis and is also a key factor in promoting tumor metastasis. Vascular endothelial growth factor a (VEGF-a), the cytokine most closely involved in the execution of tumor angiogenesis and lymphangiogenesis, activates the VEGF/VEGFR signaling pathway, resulting in epithelial cell survival, mitosis, metastasis and differentiation, vascular permeability. VEGF-mediated vascular hyperpermeability has been shown to be closely associated with metastasis of malignant tumors. Research shows that the tumor tissue leads to the expression level of VEGF-A gene through tissue hypoxia, and the expression level of VEGF-A is remarkably up-regulated in a plurality of tumor tissues, such as non-small cell lung cancer, colorectal cancer, breast cancer and the like. Therefore, inhibition of angiogenesis by blocking the VEGF/VEGFR signaling pathway is a very promising therapeutic approach for cancer. The VEGF humanized monoclonal antibody has been widely applied to the treatment of metastatic colorectal cancer and lung cancer, and the clinical application proves that the VEGF monoclonal antibody can remarkably prolong the survival time of patients with metastatic colorectal cancer and lung cancer. Although tumor immunotherapy has made a major breakthrough in the fields of immunosuppressants and CAR-T, both immunosuppressant mabs and CAR-T therapy are very expensive. The monoclonal antibody has the factors of large drug dosage, high production cost and the like, and can easily cause severe anaphylactic reaction after long-term use.

In recent years, with the rapid development and cross-penetration of related subjects such as oncology, immunology, molecular biology and the like, the treatment of cancer with tumor vaccines that activate body-specific anti-tumor immunity has become a research hotspot in the field of malignant tumor treatment. The tumor vaccine activates the specific immune reaction of the tumor through the tumor-associated antigen, achieves the aims of killing and eliminating tumor cells, and is a therapeutic active immunotherapy method. The cancer vaccine strategies mainly comprise polypeptide vaccines, DNA vaccines, antigen-shock dendritic cells and the like. Until now, a few anti-angiogenesis vaccines targeting VEGF or VEGFR have been studied in early stage, and a good growth inhibition effect is obtained in preclinical studies. However, the conventional polypeptide vaccine lacks enough immunogenicity and cannot induce the in vivo production of neutralizing antibodies, and the VEGF vaccine containing the space epitope is generally considered to introduce a VEGF active component during the vaccine injection process due to the biological activity of the VEGF. Cancer vaccines that target inhibition of angiogenesis that have been reported to date have taken two main strategies. One VEGF vaccine is VEGF121 containing three mutations of VEGFR2 binding sites R82E, K82E and H82E, and the biological activity of the mutant VEGF is greatly weakened. However, the results of clinical trials show that the vaccine induces weak neutralizing antibodies, and cannot effectively block the binding of VEGF165 to the receptor thereof.

Another reported VEGF vaccine is hVEGF26-104 synthetic polypeptide vaccine, which contains mutations at C51A-C60A to ensure the absence of VEGF biological activity. Recent reports show that although a certain anti-VEGF antibody titer can be observed in cynomolgus monkeys immunized by the hVEGF26-104 synthetic polypeptide vaccine, no significant anti-human VEGF165 antibody is induced by the polypeptide vaccine in phase I clinic, no reduction in VEGF concentration level is caused, and no clinical benefit is observed. Therefore, the design and preparation of VEGF vaccines are faced with the important problem of breaking immune tolerance, stimulating homologous proteins to generate immune response, and generating neutralizing antibodies against VEGF. On the one hand, polypeptide vaccines have the defect of low immunogenicity, and on the other hand, whether VEGF polypeptide epitopes can stimulate enough anti-VEGF neutralizing antibodies has not been clinically confirmed.

Therefore, the development of a vaccine which has no biological activity of VEGF, high immunogenicity and better effect of the induced anti-VEGF antibody is urgently needed in the field.

Disclosure of Invention

The invention aims to provide a VEGF fusion protein vaccine without VEGF biological activity and with high immunogenicity, which comprises recombinant fusion protein fused by VEGF antigen fragments (1-107) and a diphtheria toxin mutant CRM197, has no VEGF biological activity but strong immunogenicity, can break immune tolerance of an immune organism after being combined with a liquid adjuvant, and induces the organism to continuously generate anti-VEGF neutralizing antibodies.

In a first aspect of the present invention, there is provided a recombinant fusion protein having a structure represented by formula I:

Z1-Z2-Z3 （I）

wherein Z1 is a VEGF antigen fragment element;

z2 is a linker peptide element or nothing; and

z3 is a diphtheria toxin mutant CRM197 protein element;

"-" denotes a peptide bond or a peptide linker connecting the above elements;

the VEGF antigen fragment has an amino acid sequence shown as SEQ ID NO. 5 or SEQ ID NO. 6, loses VEGF biological activity and retains immunogenicity; and the diphtheria toxin mutant CRM197 protein has the amino acid sequence shown in SEQ ID No. 7.

In another preferred embodiment, the VEGF antigen fragment has the amino acid sequence shown in SEQ ID NO. 5.

In another preferred embodiment, the VEGF antigen fragment has an amino acid sequence as shown in SEQ ID NO 6.

In another preferred embodiment, the peptide linker is 0-10 amino acids, preferably 0-5 amino acids in length.

In another preferred embodiment, the amino acid sequence of the fusion protein has at least 90% sequence identity with the amino acid sequence as set forth in SEQ ID NO. 1.

In another preferred embodiment, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1.

In another preferred embodiment, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 2.

In another preferred embodiment, the amino acids of the fusion protein have at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 2.

In another preferred embodiment, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 9.

In another preferred embodiment, the fusion protein has the following characteristics:

(1) the VEGF antigen fragment is obtained by cutting 14 amino acid residues at the C tail end on the basis of the amino acid sequence of VEGF121 shown as SEQ ID NO. 8;

(2) the VEGF antigen fragment does not have VEGF biological activity;

(3) the VEGF antigen fragment can induce the production of anti-VEGF 165 antibodies and neutralizing antibodies in an organism.

In a second aspect of the invention, there is provided a polynucleotide encoding a recombinant fusion protein according to the first aspect of the invention.

In another preferred embodiment, the polynucleotide is selected from the group consisting of: a DNA sequence, an RNA sequence, or a combination thereof.

In another preferred embodiment, said polynucleotide additionally comprises an auxiliary element selected from the group consisting of: a signal peptide, a secretory peptide, a tag sequence (e.g., 6His), or a combination thereof.

In another preferred embodiment, the polynucleotide encodes a fusion protein having the structure of formula I:

Z1-Z2-Z3 （I）

wherein, Z1 is a VEGF antigen fragment;

z2 is a linker peptide or nothing; and

z3 is diphtheria toxin mutant CRM197 protein;

"-" denotes a peptide bond or a peptide linker connecting the above elements.

In another preferred embodiment, the VEGF antigen fragment has the amino acid sequence shown in SEQ ID NO. 5.

In another preferred embodiment, the VEGF antigen fragment has an amino acid sequence as shown in SEQ ID NO 6.

In another preferred embodiment, the diphtheria toxin mutant CRM197 protein has the amino acid sequence shown in SEQ ID NO. 7.

In another preferred embodiment, the polynucleotide encodes a recombinant fusion protein as shown in SEQ ID NO. 1 having a nucleotide sequence as shown in SEQ ID NO. 3.

In another preferred embodiment, the polynucleotide encodes a recombinant fusion protein as shown in SEQ ID NO. 2 having a nucleotide sequence as shown in SEQ ID NO. 4.

In another preferred example, the polynucleotide encodes a recombinant fusion protein shown as SEQ ID NO. 9, and the N-terminal of the fusion protein contains a sumo tag which has a nucleotide sequence shown as SEQ ID NO. 10.

In a third aspect of the invention, there is provided an expression vector comprising a polynucleotide sequence according to the second aspect of the invention.

In another preferred embodiment, the expression vector is used for expressing the recombinant fusion protein.

In another preferred embodiment, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

In another preferred embodiment, the expression vector is a prokaryotic expression vector.

In another preferred example, the expression vector is a eukaryotic expression vector, and can be applied to the construction of a eukaryotic cell line expressing the VEGF fusion protein vaccine.

In another preferred embodiment, the vector comprises two open reading frames, one of which comprises the polynucleotide sequence of the second aspect of the invention and a nucleotide sequence encoding a sumo tag 5' of the polynucleotide sequence, the other of which comprises a nucleotide sequence encoding an escherichia coli disulphide isomerase DsbC.

In another preferred embodiment, the expression vector is an expression vector comprising two open reading frames, wherein one open reading frame comprises a nucleotide sequence shown as SEQ ID NO. 10, and the other open reading frame comprises a nucleotide sequence coding for E.coli disulfide isomerase DsbC, shown as SEQ ID NO. 11.

In a fourth aspect of the present invention, there is provided a host cell comprising an expression vector according to the third aspect of the present invention or a polynucleotide according to the second aspect of the present invention integrated into its genome.

In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

In another preferred embodiment, the host cell is a prokaryotic cell.

In another preferred embodiment, the host cell is E.coli.

In another preferred embodiment, the host cell is selected from the group consisting of: coli, insect cells, SF9, Hela, HEK293, CHO, yeast cells, or combinations thereof.

In another preferred embodiment, the host cell is selected from the group consisting of: BL21 (DE 3), Rosetta (DE 3), Origami B (DE 3).

In another preferred embodiment, the host cell is a chinese hamster ovary Cell (CHO).

In a fifth aspect of the present invention, there is provided a method for preparing a recombinant fusion protein according to the first aspect of the present invention, comprising the steps of:

(i) culturing the host cell of the fourth aspect of the invention;

(ii) inducing the host cell to express the recombinant fusion protein using an inducing agent, thereby obtaining an expressed recombinant fusion protein;

(iii) isolating the expressed recombinant fusion protein, thereby obtaining an isolated recombinant fusion protein.

In another preferred embodiment, in the host cell, the expressed recombinant fusion protein is expressed as an inclusion body of the recombinant fusion protein.

In another preferred embodiment, the isolation of step (iii) comprises inclusion body denaturation and renaturation of the recombinant fusion protein.

In another preferred embodiment, the method for inclusion body denaturation and renaturation of the recombinant fusion protein comprises the following steps:

(i) washing the recombinant fusion protein inclusion bodies with a washing buffer;

(ii) uniformly mixing the recombinant fusion protein inclusion body with process water with the mass volume not more than that of the recombinant fusion protein inclusion body, and dissolving the recombinant fusion protein inclusion body by using a denaturing solution to obtain a dissolved recombinant fusion protein;

(iii) renaturing the dissolved renaturation solution for the recombinant fusion protein at the renaturation temperature of 16-25 ℃ to obtain the renaturated recombinant fusion protein.

In another preferred embodiment, the method for preparing the recombinant fusion protein comprises the following steps:

(1) culturing a BL21 (DE 3) strain host cell according to the fourth aspect of the invention to an OD600 value of 10-20, and adding an inducer, thereby obtaining a BL21 (DE 3) strain containing recombinant VEGF fusion protein inclusion bodies;

(2) treating the BL21 (DE 3) strain containing the recombinant VEGF fusion protein inclusion body with a bacterium breaking buffer solution, collecting the recombinant VEGF fusion protein inclusion body, washing the inclusion body with the bacterium breaking buffer solution, and separating the recombinant VEGF fusion protein through denaturation, renaturation and purification.

In another preferred example, the medium in step (1) is a total synthetic medium;

the total synthetic culture medium contains 1-4g/L KH₂PO₄，1-5g/L K₂HPO₄•3H₂O，2-10g/L (NH₄)₂SO₄，0.1-2g/L MgSO₄•7H₂O, 10-50wt% glucose, and is supplemented with 10-5-wt% glycerol and 10-200g/L ammonium sulfate during the culture process.

In another preferred embodiment, the lysis buffer comprises 0.4-2mol/L urea, 0.1-1.0% Triton X-100, and 0.1-1.0% Triton X-114.

In another preferred example, the number of washing in step (2) is 2 to 5.

In another preferred embodiment, the inclusion bodies are first mixed with process water not exceeding the equivalent mass volume of the inclusion bodies, and then dissolved in the denaturation solution as follows:

in another preferred embodiment, the denaturing solution comprises 6 mol/L guanidine hydrochloride and 1-50 mM DTT.

In another preferred example, the denaturant is diluted by 8 mol/L urea in a ratio of 1:4, and then the renaturation method is carried out by diluting in the renaturation solution.

In another preferred embodiment, the renaturation solution contains 0.1% -0.5% of PEG 4000.

In another preferred embodiment, the renaturation temperature is 16-25 ℃.

In another preferred embodiment, the purification is a purification using anion exchange chromatography, and the anion exchange medium used is Q sepharose.

In another preferred embodiment, the recombinant fusion protein is expressed in soluble form by addition of a tag or co-expression with a chaperone that aids in protein folding.

In another preferred embodiment, the method for preparing the recombinant fusion protein comprises the following steps:

(1) culturing the host cell according to the fourth aspect of the present invention, and inducing the host cell to express the recombinant fusion protein using an inducer, thereby obtaining an expressed recombinant fusion protein;

(2) crushing thallus, collecting expression supernatant, and purifying through affinity chromatography;

(3) cutting off the label by using specific protease, and removing the label and the added protease by using affinity chromatography;

(4) and purifying by anion exchange chromatography and/or molecular sieve chromatography to obtain the high-purity recombinant fusion protein.

In another preferred embodiment, the method for preparing the recombinant fusion protein comprises the following steps:

(1) constructing a Chinese Hamster Ovary (CHO) cell line for expressing the recombinant VEGF fusion protein by applying the eukaryotic expression vector provided by the third aspect of the invention;

(2) high-density fermentation culture of the CHO cells for 7-21 days at a fermentation temperature of 30-38 deg.C until the cell density is 10 × 10⁶-20×10⁶/ml；

(3) Collecting the fermentation supernatant obtained in the step (2), and completing purification by respectively using hydrophobic chromatography, anion exchange chromatography and cation exchange chromatography.

In a sixth aspect of the invention, there is provided a pharmaceutical composition comprising a recombinant fusion protein according to the first aspect of the invention and a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutically acceptable carrier comprises a liquid, preferably water, saline or a buffer.

In another preferred embodiment, the carrier further comprises auxiliary substances, preferably fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like.

In another preferred embodiment, the vector further comprises a cell transfection reagent.

In another preferred embodiment, the composition is a vaccine composition.

In another preferred embodiment, the vaccine composition comprises the recombinant fusion protein according to the first aspect of the present invention and a vaccinally acceptable carrier, preferably a pharmaceutically acceptable carrier.

In another preferred embodiment, the vaccine composition may be a bivalent vaccine or a multiple vaccine.

In another preferred embodiment, the vaccine composition further comprises an adjuvant.

In another preferred embodiment, the adjuvant comprises: particulate and non-particulate adjuvants.

In another preferred embodiment, the particulate adjuvant is selected from the group consisting of: an aluminum salt, a water-in-oil emulsion, an oil-in-water emulsion, a nanoparticle, a microparticle, a liposome, an immunostimulatory complex, or a combination thereof;

in another preferred embodiment, the non-particulate adjuvant is selected from the group consisting of: muramyl dipeptide and its derivatives, saponin, lipid A, cytokine, derivative polysaccharide, bacterial toxin, microorganism and its product such as mycobacteria (Mycobacterium tuberculosis, Bacillus Calmette-Guerin), Bacillus pumilus, Bordetella pertussis, propolis, or combinations thereof.

In another preferred embodiment, the adjuvant is selected from the group consisting of: montanide ISA 51 VG, aluminum phosphate adjuvant, MF59, AS04, or combinations thereof.

In another preferred embodiment, the amount of the VEGF recombinant fusion protein per dose of the vaccine composition is 0.1-5 mg.

In another preferred embodiment, the vaccine composition is in an injectable dosage form.

In a seventh aspect of the present invention, there is provided a recombinant fusion protein according to the first aspect of the present invention, a polynucleotide according to the second aspect of the present invention, an expression vector according to the third aspect of the present invention, a host cell according to the fourth aspect of the present invention, and/or a pharmaceutical composition according to the sixth aspect of the present invention for use in the preparation of a medicament for the treatment and/or prevention of a disease.

In another preferred embodiment, the disease is selected from the group consisting of: tumor (or cancer), macular edema secondary to retinal vein occlusion, wet age-related macular degeneration, and diabetic macular edema, or a combination thereof.

In another preferred embodiment, the tumor (or cancer) comprises a solid tumor.

In another preferred embodiment, the solid tumor is selected from the group consisting of: lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, liver cancer, stomach cancer, esophageal cancer, pancreatic cancer, melanoma, kidney cancer, prostate cancer, cervical cancer, ovarian cancer, nasopharyngeal cancer, oral cancer, osteosarcoma, brain glioma, bladder cancer, or a combination thereof.

In an eighth aspect of the present invention, there is provided a method for treating and/or preventing a disease, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to the sixth aspect of the present invention.

In another preferred embodiment, the disease is selected from the group consisting of: tumor (or cancer), macular edema secondary to retinal vein occlusion, wet age-related macular degeneration, and diabetic macular edema, or a combination thereof.

In another preferred embodiment, the tumor (or cancer) comprises a solid tumor.

In another preferred embodiment, the solid tumor is selected from the group consisting of: lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, liver cancer, stomach cancer, esophageal cancer, pancreatic cancer, melanoma, kidney cancer, prostate cancer, cervical cancer, ovarian cancer, nasopharyngeal cancer, oral cancer, osteosarcoma, brain glioma, bladder cancer, or a combination thereof.

In a ninth aspect of the present invention, there is provided a method for vaccination with a VEGF recombinant fusion protein vaccine, comprising the steps of:

(i) mixing the VEGF recombinant fusion protein of the first aspect of the invention with an adjuvant and then emulsifying to obtain an emulsified VEGF recombinant fusion protein vaccine;

(ii) and (3) inoculating the emulsified VEGF recombinant fusion protein vaccine to a subject to be inoculated.

In another preferred embodiment, the adjuvant is a liquid adjuvant.

In another preferred example, the liquid adjuvant is Montanide ISA 51 VG;

in another preferred embodiment, the "emulsification after mixing of the VEGF recombinant fusion protein and the adjuvant" is specifically that the VEGF recombinant fusion protein and Montanide ISA 51 VG are mixed by connecting two injectors through a joint according to the volume ratio of 1 (0.5-2), and the mixture is pushed back and forth at a slow speed for 10-30 times and then pushed back and forth at a fast speed for 30-60 times;

in another preferred example, the inoculation mode is as follows: the immunization is carried out 1 time per week and 4 times in total according to the dosage of 0.05-2 mg/kg.

In another preferred example, the liquid adjuvant is an aluminum phosphate adjuvant, and the recombinant VEGF fusion protein and the aluminum phosphate adjuvant are mixed and emulsified according to the volume ratio of 1 (0.2-5).

In another preferred example, the liquid adjuvant is MF59, and the recombinant VEGF fusion protein and the MF59 adjuvant are mixed and emulsified according to the volume ratio of 1 (0.2-5).

In another preferred embodiment, the liquid adjuvant is AS04, and the recombinant VEGF fusion protein and the AS04 adjuvant are mixed and emulsified according to the volume ratio of 1 (0.2-5).

In another preferred example, the subject to be vaccinated is a human or non-human mammal.

In another preferred embodiment, the non-human mammal is selected from the group consisting of: mouse, rat, rabbit, rhesus monkey.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the results of VEGF biological activity assays for VEGF fragments of varying lengths.

FIG. 2 shows the results of immunogenicity testing of VEGF fragments of different lengths.

FIG. 3 shows a recombinant expression whole bacterial protein map of the recombinant fusion protein vaccine.

FIG. 4 shows a SDS-PAGE purity scheme of the recombinant fusion protein vaccine after preparation.

FIG. 5 shows a schematic representation of the RP-HPLC purity of the recombinant fusion protein vaccine after preparation.

FIG. 6 shows the result of the measurement of the serum antibody titer of the mice immunized with the recombinant fusion protein vaccine

FIG. 7 shows the result of detecting neutralizing antibodies in the serum of mice immunized with the recombinant fusion protein vaccine.

FIG. 8 shows the results of the serum inhibition assay of VEGF-stimulated vascular endothelial cell proliferation in mice immunized with the recombinant fusion protein vaccine.

FIG. 9 shows the result of detecting the serum antibody titer of the mice immunized with the recombinant fusion protein vaccine (SEQ ID NO: 2).

FIG. 10 shows the sequence alignment of the recombinant fusion protein vaccine sequences SEQ ID NO 1 and SEQ ID NO 2.

FIG. 11 shows the results of antibody titer detection of rhesus monkey immunized by the recombinant fusion protein vaccine.

FIG. 12 shows the detection results of the neutralizing antibody of the rhesus monkey antibody immunized by the recombinant fusion protein vaccine.

FIG. 13 shows the result of the antibody significantly inhibiting tumor growth after immunization with the recombinant fusion protein vaccine, in which C021 is the recombinant fusion protein vaccine (comprising the sequence shown in SEQ ID NO: 1).

FIG. 14 shows that the antibody significantly extends the survival of tumor-bearing mice after immunization with the recombinant fusion protein vaccine.

FIG. 15 shows the comparison of antibody titer detection after VEGF121-CRM197 and VEGF107-CRM197 immunized mice.

Detailed Description

The present inventors have conducted extensive and intensive studies and, for the first time, have unexpectedly found that VEGF synthetic polypeptides cannot stimulate the production of high antibody titers in animals, while the truncated VEGF1-107 fragment of VEGF121, which loses VEGF biological activity but retains immunogenicity, can stimulate the production of high titer antibodies in animals. The recombinant fusion protein prepared by fusing the VEGF antigen fragment (1-107) and the diphtheria toxin mutant CRM197 has strong immunogenicity, and can break immune tolerance of immune organisms after being combined with a liquid adjuvant and induce the organisms to continuously generate anti-VEGF neutralizing antibodies. Experiments prove that compared with VEGF synthetic polypeptide, the VEGF recombinant fusion protein has stronger affinity with a receptor protein kinase domain receptor, and the titer of antibodies generated in an animal body after the animal is immunized is higher. On the basis of this, the present invention has been completed.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the terms "fragments of VEGF 1-107", "fragments of VEGF antigen (1-107)" and "VEGF 107" are used interchangeably.

The VEGF1-107 fragment is obtained by cutting 14 amino acid residues at the C terminal on the basis of the VEGF121 amino acid sequence shown in SEQ ID NO. 8, and reserving the 107 th amino acid from the N terminal to the C terminal of the VEGF antigen fragment. The obtained shorter VEGF1-107 fragment loses VEGF biological activity but retains immunogenicity, reduces safety risks caused by VEGF biological activity to the maximum extent, improves the immunopotency of the vaccine, and can reduce the generation of nonspecific antibodies to a certain extent.

VEGF121 is a secreted vascular endothelial growth factor, is a VEGF spliceosome with the smallest molecular weight naturally existing in a human body, and has the functions of stimulating VEGF receptors and inducing the growth of vascular endothelial cells through signal transduction.

In another preferred embodiment, the VEGF1-107 fragment of the invention has an amino acid sequence as shown in SEQ ID NO. 5.

In another preferred embodiment, the VEGF1-107 fragment of the invention further comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 5, for example, the VEGF1-107 fragment has the amino acid sequence shown in SEQ ID No. 6, which has three amino acid mutations compared to SEQ ID No. 5, specifically, Arg at position 82 is mutated to Glu, Lys at position 84 is mutated to Glu, and His at position 86 is mutated to Glu.

The VEGF1-107 fragment of the invention has the following amino acid sequence:

APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPK（SEQ ID NO:5）；

APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMEIEPEQGQHIGEMSFLQHNKCECRPK（SEQ ID NO:6）。

CRM197

as used herein, the term "CRM 197" refers to the diphtheria toxin mutant CRM197, specifically the mutation of glycine at position 52 of diphtheria toxin to glutamic acid, which has the amino acid sequence shown in SEQ ID NO. 7. The mutant toxin A fragment cannot be combined with elongation factor II in nucleus, so that the cytotoxic effect of the mutant toxin A fragment is lost, but the antigenicity and the immunogenicity of the mutant toxin A fragment are still basically consistent with those of natural diphtheria toxin.

Fusion proteins

As used herein, the terms "recombinant VEGF fusion protein", "VEGF recombinant fusion protein" and "VEGF fusion protein" and "recombinant fusion protein of the invention" are used interchangeably and refer to the recombinant fusion protein VEGF107-CRM197 made by fusing fragments (1-107) of the VEGF antigen (fragments VEGF 1-107) to the diphtheria toxin mutant CRM 197.

In another preferred embodiment, the fusion protein has a structure shown as Z1-Z2-Z3 (formula I),

wherein, Z1 is a VEGF antigen fragment element; z2 is a linker peptide element or nothing; and Z3 is a diphtheria toxin mutant CRM197 protein element; "-" denotes a peptide bond or a peptide linker connecting the above elements.

In another preferred embodiment, the coding sequence of the fusion protein is shown in SEQ ID NO. 1 or SEQ ID NO. 2.

As used herein, the term "fusion protein" also includes variants of the fusion protein (sequences shown as SEQ ID NO:1 or SEQ ID NO: 2) having the above-described activity. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 3 (usually 1 to 2, more preferably 1) amino acids, and addition or deletion of one or 15 (usually up to 3, preferably up to 2, more preferably up to 1) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).

The invention also includes active fragments, derivatives and analogs of the above fusion proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of a fusion protein of the invention. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which an antigenic peptide is fused to another compound (such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (a fusion protein in which a leader sequence, a secretory sequence or a tag sequence such as 6 × His is fused). Such fragments, derivatives and analogs are well within the purview of those skilled in the art in view of the teachings of the present invention.

A preferred class of reactive derivatives refers to those having at most 3, preferably at most 3, amino acid sequences compared to the amino acid sequence of formula I

2, more preferably at most 1 amino acid is replaced by a qualitatively similar or analogous amino acid to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

The invention also provides analogs of the fusion proteins of the invention. The analogs may differ from the polypeptide of any of SEQ ID No. 1-2 by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

Expression vectors and host cells

The invention also relates to vectors comprising polynucleotides encoding the fusion proteins of the invention, as well as genetically engineered host cells produced with the vectors of the invention or the coding sequences of the fusion proteins of the invention, and methods for producing the fusion proteins of the invention by recombinant techniques.

The polynucleotide sequences of the present invention may be used to express or produce recombinant fusion proteins by conventional recombinant DNA techniques. Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a fusion protein of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) separating and purifying protein from culture medium or cell.

In the present invention, the polynucleotide sequence encoding the fusion protein may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Any plasmid or vector may be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences encoding the fusion proteins of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters capable of controlling gene expression in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell (e.g., E.coli), or a lower eukaryotic cell, or a higher eukaryotic cell, such as a yeast cell or a mammalian cell (including human and non-human mammals). Representative examples are: escherichia coli, insect cells, SF9, Hela, HEK293, CHO, yeast cells, etc. In a preferred embodiment of the present invention, Escherichia coli (e.g., BL21 (DE 3), Rosetta (DE 3), JM109, etc.) is selected as the host cell. In another preferred embodiment of the invention, the CHO cell is selected as the host cell.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene. Examples include the SV40 enhancer at the late side of the replication origin at 100 to 270 bp, the polyoma enhancer at the late side of the replication origin, and adenovirus enhancers.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide or fusion protein encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Peptide linker

The invention provides a fusion protein, which optionally contains a peptide linker. The size and complexity of the peptide linker may affect the activity of the protein. In general, the peptide linker should be of sufficient length and flexibility to ensure that the two proteins being linked have sufficient degrees of freedom in space to function. Meanwhile, the influence of alpha helix or beta sheet formation in the peptide linker on the stability of the fusion protein is avoided.

In a preferred embodiment of the invention, the peptide linker is generally 0-10 amino acids, preferably 0-5 amino acids in length.

Pharmaceutical composition

The invention also provides a pharmaceutical composition. The pharmaceutical composition contains the fusion protein, and pharmaceutically acceptable carriers, diluents, stabilizers and/or thickeners, and can be prepared into medicament types such as freeze-dried powder, tablets, capsules, syrup, solution or suspension.

"pharmaceutically acceptable carrier or excipient (excipient)" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of being combined with the active ingredients of the present invention and with each other without significantly diminishing the efficacy of the active ingredient.

The composition may be a liquid or a solid, such as a powder, gel or paste. Preferably, the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.

Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The compositions may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intraperitoneal, intravenous, or topical administration. The pharmaceutical composition is for use (a) in the treatment or prevention of cancer or a tumour (especially a solid tumour); (b) used for treating or preventing macular edema, wet age-related macular degeneration, diabetic macular edema, and other diseases due to retinal vein occlusion; (c) inducing the production of neutralizing antibodies that block the binding of VEGF to the receptor.

The solid tumor is selected from the group consisting of: lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, liver cancer, stomach cancer, esophageal cancer, pancreatic cancer, melanoma, kidney cancer, prostate cancer, cervical cancer, ovarian cancer, nasopharyngeal cancer, oral cancer, osteosarcoma, brain glioma, bladder cancer, or a combination thereof.

Furthermore, the pharmaceutical composition may be administered to a subject in need thereof, alone or in combination with other pharmaceutical preparations, for the treatment or prevention of the disease.

Vaccine composition

The pharmaceutical composition provided by the invention is preferably a vaccine composition. The vaccine composition comprises the recombinant fusion protein of the first aspect of the invention and a vaccinally acceptable carrier, preferably a pharmaceutically acceptable carrier.

In another preferred embodiment, the vaccine composition further comprises an adjuvant, preferably a liquid adjuvant. After the vaccine composition of the invention is mixed and emulsified with a liquid adjuvant, the vaccine composition can be inoculated into a human or non-human mammal to induce the production of anti-VEGF neutralizing antibodies in vivo.

The invention also provides a VEGF recombinant fusion protein vaccine immunization method, which comprises the following steps:

(i) mixing and emulsifying the VEGF recombinant fusion protein and an adjuvant to obtain an emulsified VEGF recombinant fusion protein vaccine;

(ii) and (3) inoculating the emulsified VEGF recombinant fusion protein vaccine to a subject to be inoculated.

In another preferred embodiment, the adjuvant is selected from the group consisting of: montanide ISA 51 VG, aluminum phosphate adjuvant, MF59, AS04, or combinations thereof.

Compared with the prior art strategy, the invention mainly has the following advantages:

(1) the invention selects VEGF1-107 fragment which does not have VEGF biological activity but has strong immunogenicity as antigen, and the antigen is easier to induce and generate neutralizing antibody which blocks the combination of VEGF and receptor in vivo;

(2) the invention provides a recombinant VEGF fusion protein vaccine expressed by fusion of a human VEGF1-107 fragment and a diphtheria toxin mutant CRM197, wherein the CRM197 can obviously improve the immunogenicity of the vaccine antigen;

(3) the invention provides a plurality of preparation methods of the VEGF fusion protein vaccine;

(4) the immunogenicity of the vaccine antigen can be further improved by emulsifying the recombinant VEGF fusion protein vaccine and a liquid adjuvant.

The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Example 1: screening of VEGF antigen fragment with low VEGF biological activity and high immunogenicity

VEGF121 (1-121), VEGF107 (1-107) and VEGF82 (24-105) were prepared separately by E.coli expression systems and three VEGF fragments were detected using VEGF-responsive luciferase reporter cell lines.

And (3) paving the VEGF response cell strain in a 96-well plate, respectively adding three VEGF fragments after the cells are fixed, diluting the VEGF fragments from a 500ng/ml multiple ratio in a gradient manner, incubating and culturing for 24 hours, and adding a luciferase substrate to detect a luminescence value.

As shown in the detection results of fig. 1, VEGF121 at higher concentration can induce luciferase expression by binding VEGFR2 on the cell membrane of the response cell strain through signal pathway conduction, and the concentration and the fluorescence value are in an S-shaped curve; neither VEGF107 nor VEGF82 at the highest concentration of 500ng/ml induced luciferase expression, indicating that VEGF107 and VEGF82 have no VEGF biological activity.

Mice were immunized with VEGF121, VEGF107 and VEGF82, respectively, with complete freund's adjuvant 10 μ g each, 8 mice per group, 4 immunizations a week, and blood was collected 1 week after the second immunization and 1 week after the fourth immunization, respectively, and antibody titers were detected with VEGF 165.

As shown in FIG. 2, after 1 week of the secondary immunization, 100% of V107 groups were positive-transformed, 1 positive-transformed V121 and V82 were both positive-transformed, and none of V33 groups were positive-transformed. By 1 week after four immunizations, the antibody titers were highest in group V107, and none of the positive-transgenic mice in group V82. This result indicates that VEGF107 is most immunogenic.

VEGF107 (containing 1-107 amino acid sequences) is found to have no biological activity but strong immunogenicity through screening, and the VEGF fragment is very suitable for preparing VEGF vaccine.

Example 2: construction and expression of plasmid pCDFDuet-1- (sumoVEGF107-CRM197) -DsbC

The DNA coding sequence (shown as SEQ ID NO: 10) of sumoVEGF107-CRM197 is completely synthesized and constructed into the first open reading frame of pCDFDuet-1 expression plasmid by using gene synthesis means, wherein the amino acid sequence for coding sumo is positioned at the N end of VEGF107-CRM197 (SEQ ID NO: 1), and the amino acid sequence of sumoVEGF107-CRM197 is shown as SEQ ID NO: 9; on the basis of the previous construction, the DNA coding sequence of the E.coli disulfide isomerase DsbC (shown as SEQ ID NO: 11) is completely synthesized by gene synthesis means and constructed into the second open reading frame of the pCDFDuet-1 expression plasmid. The pCDFDuet-1- (sumoVEGF107-CRM197) -DsbC construction was completed upon completion of the identification.

2 mu L of the constructed formed pCDFDuet-1- (sumoVEGF107-CRM197) -DsbC plasmid is taken and added into BL21 (DE 3) expression engineering bacteria, the cells are incubated on ice for 30 minutes, heat shock is carried out at 42 ℃ for 90 seconds, 500 mu L of LB culture medium without antibiotics is added after incubation on ice for 5 minutes, shaking culture is carried out at 37 ℃ for 30 minutes, 50 mu L of the plasmid is taken and laid in LB solid culture medium containing 100 mu g/ml streptomycin, and culture is carried out overnight at 37 ℃.

Single clones on a plate after pCDFDuet-1- (sumoVEGF107-CRM197) -DsbC plasmid transformation are picked and placed into an LB liquid culture medium, the culture is carried out at 37 ℃, when OD600 of the culture medium is 10-20, glycerol and ammonium sulfate are slowly supplemented, IPTG with the final concentration of 1mM is added, expression thalli are collected after 8 hours of induction at 25 ℃, and the expression condition is identified by an SDS-PAGE method, wherein sumoVEGF107-CRM197 is expressed in a supernatant component as shown in figure 3.

Example 3: preparation of recombinant fusion protein VEGF107-CRM197

The expressed cells of example 2 were collected, disrupted, and the supernatant was immediately subjected to Ni affinity column chromatography under the conditions of 10% Buffer B (containing about 50mM imidazole) and 50% Buffer B (containing about 250mM imidazole). After the affinity chromatography elution product is subjected to enzyme digestion by sumo label specific protease Ulp1, Ni sepharose FF affinity chromatography is used again, the components of the flow-through liquid are collected, and the sumo label still with the His label, the target protein without the cut label and the label with high affinity of part of the target protein and the Ni column are removed.

Concentrating the digested and tag-free VEGF107-CRM197 flow-through component, continuing to perform fine purification by Sephacryl S200 molecular sieve chromatography, collecting target protein peaks, wherein the SDS-PAGE identification picture of the protein sample is shown in figure 4, the RP-HPLC analysis picture is shown in figure 5, and the analytical purities of the protein sample and the protein sample are both more than 95%. Thus obtaining the recombinant fusion protein vaccine VEGF107-CRM 197.

Example 4: the recombinant fusion protein VEGF107-CRM197 and liquid adjuvant Montanide ISA 51 VG are emulsified and immunized on mice

The recombinant fusion protein VEGF107-CRM197 prepared in example 3 was diluted to 0.2mg/ml, 0.5ml of the diluted fusion protein was taken out into a 2ml syringe, 0.5ml of the liquid adjuvant Montanide ISA 51 VG was taken out into another 2ml syringe, the two syringes were connected by a joint, and the emulsification was completed by pushing back and forth for 15 rounds at a slow speed and then 30 times at a fast speed as much as possible. After the emulsification was complete, all emulsions were pushed into one side syringe.

The mice were divided into test and control groups of 8 mice each, weighing more than 18 g. The test group mice were injected subcutaneously with the emulsified emulsion in an amount of 100 μ L/mouse, and the control group was injected subcutaneously with VEGF antigen, immunized 1 time per week, and blood was collected 1 week after the 4 th immunization to determine the anti-VEGF 165 antibody titer and the anti-VEGF 165 neutralizing antibody.

4.1 detection of anti-VEGF antibody titer in mouse serum after immunization with recombinant fusion protein vaccine (containing the sequence shown in SEQ ID NO: 1):

(1) coating: taking ELISA to detect 96-well plate, and adding Na to VEGF165 protein₂CO₃-NaHCO₃And diluting the coating buffer solution with the pH of 9.6 to 0.5 mug/ml, respectively taking 100 mug L of diluted VEGF165 protein, adding the diluted VEGF165 protein into each hole of a 96-well plate, and incubating overnight at the temperature of 2-8 ℃.

(2) And (3) sealing: the next day, discarding the coating solution, adding 150 μ L PBS blocking solution containing 0.05% (v/v) Tween-20 and 1% BSA into each well, and incubating and blocking for 1 hour at 37 ℃;

(3) washing: after the blocking solution is discarded, repeatedly washing each well for 3 times by using 200 microliter of PBS containing 0.05% (v/v) Tween-20, and sucking the liquid in the well;

(4) diluting the collected mouse serum, adding 100 muL diluted serum into the leftmost side hole of a 96-well plate, diluting each mouse by a multiple ratio from left to right in a way of 1 row, diluting by 12 gradients in total, taking the serum of the unimmunized mouse as a control, and incubating for 1 h at 37 ℃ after adding the serum; after incubation, the incubation solution was discarded and the washing was repeated 3 times as described above;

(5) diluting a secondary HRP enzyme-labeled anti-mouse antibody to 1:5000 by using a confining liquid, adding 100 mu L of the HRP enzyme-labeled antibody to each hole, and incubating for 1 h at 37 ℃; discard the incubation and repeat 5 washes as described above;

(6) color development: adding 100 mul TMB into each hole, and incubating for 15 min at room temperature in a dark place;

(7) and (4) terminating: adding 100 mul of 0.5mol/L sulfuric acid into each hole to stop color development;

(8) after termination, the absorbance of each well OD450 was measured using a microplate spectrophotometer. And (3) measuring the light absorption value at 450nm, and determining the highest dilution multiple of the OD value of the serum sample/the OD value of the normal saline control group sample to be more than or equal to 2.1.

As shown in FIG. 6, the antibody titer was calculated as the geometric mean of the highest dilution factor of 8 mice, and the antibody titer was 3X 10⁶More than 100 times of VEGF antigen alone.

4.2 detection of neutralizing antibodies against VEGF in sera of immunized mice after immunization with recombinant fusion protein vaccine:

taking ELISA to detect 96-well plate, and adding Na to VEGF165 protein₂CO₃-NaHCO₃And diluting the coating buffer solution with the pH of 9.6 to 80ng/ml, respectively taking 100 mu L of diluted VEGF165 protein, adding the diluted VEGF165 protein into each hole of a 96-hole plate, and incubating overnight at the temperature of 2-8 ℃. The next day, discarding the coating solution, adding 150 μ L PBS blocking solution containing 0.05% (v/v) Tween-20 and 1% BSA into each well, and incubating and blocking for 1 hour at 37 ℃; washing: after the blocking solution is discarded, each well is repeatedly washed for 3 times by 200 microliter PBS containing 0.05% (v/v) Tween-20, and the solution in the well is sucked dryA body; diluting the collected serum of the mice by an initial dilution multiple of 3 times, adding 100 mu L of diluted serum into the leftmost hole of a 96-well plate, wherein each mouse is 1 row, diluting the serum of a blank group of mice (non-immune mice) by taking the serum of the blank group of mice as a control from left to right in a multiple ratio manner, diluting the serum by 12 gradients in total, and incubating the diluted serum for 1 h at 37 ℃; after incubation, the incubation solution was discarded and the washing was repeated 3 times as described above; taking VEGF receptor VEGFR2 Kinase Domain Receptor (KDR) and Fc fusion protein, diluting to 160ng/ml with a blocking solution, adding 100 mu L of the diluted solution into each well, and incubating and blocking for 1 hour at 37 ℃; discard the incubation solution and repeat 3 washes as described above; diluting an HRP enzyme-labeled anti-VEGFR 2 antibody to 1:5000 by using a confining liquid, adding 100 muL of the HRP enzyme-labeled antibody to each hole, and incubating for 1 h at 37 ℃; discard the incubation and repeat 5 washes as described above; color development: adding 100 mul TMB into each hole, and incubating for 15 min at room temperature in a dark place; and (4) terminating: adding 100 mul of 0.5mol/L sulfuric acid into each hole; after termination, the absorbance of each well OD450 was measured using a microplate spectrophotometer.

The results are shown in fig. 7, the higher the OD450 value indicates that the VEGFR2 kinase domain receptor binds to the VEGF165 in higher content, and in the chromogenic results, in the control serogroup, the different concentrations of serum do not affect the chromogenic results, while in the recombinant fusion protein vaccine immunization group, after the low dilution multiple serum is incubated with the VEGF-coated well plate, the binding of the VEGFR2 kinase domain receptor to the VEGF165 is significantly inhibited, and strong neutralizing antibody activity is shown.

4.3 inhibition of vascular endothelial cell proliferation assay:

test materials:

complete medium 1: ECM culture medium was supplemented with 5% FBS (V/V), 1% ECGS and 1% P/S. Placing in glass or plastic bottle, storing at 4 deg.C, and keeping the service life not exceeding the product indication and effective period.

Complete medium 2: ECM culture medium was supplemented with 0.5% FBS (V/V), 1% P/S. Placing in glass or plastic bottle, storing at 4 deg.C, and keeping the service life not exceeding the product indication and effective period.

Complete medium 3: to complete medium 2 was added 10% CCK-8 (ready to use).

The test steps are as follows:

(1) plate paving: after digestion of the cells, the cells were counted and diluted to 3X 10 with complete medium 1⁴And (4) the cells per ml are inoculated in a 96-well cell culture plate, and each well is 100 microliter. 37 ℃ and 5% CO₂Culturing for 18-24 h under the condition.

(2) The medium in the 96 well cell culture plate was changed to complete medium 2 and the cells were starved for 16-24 h.

(3) Sample preparation: blank control group: complete medium 2 without VEGF165, 120 μ l per well of 96-well plates, totaling 20 wells. ② comparison group: serum of a blank group of mice is diluted by 2 times by using complete culture medium 2 containing 6.25ng/ml VEGF165, the concentration of 10 gradients is totally obtained, each gradient is made into 2 holes, and the final volume of each hole is 120 mu l. Positive control group: avastin (commercial VEGF monoclonal antibody) was diluted 2-fold with complete medium 2 containing 6.25ng/ml VEGF165 at a final concentration of 800ng/ml in the first well for a total of 10 concentration gradients, 2 wells per gradient and a final volume of 120 μ l per well. Fourthly, a test sample group: the recombinant fusion protein vaccine immunization group serum is diluted by 2 times with complete culture medium 2 containing 6.25ng/ml VEGF165, 10 gradient concentrations are calculated, each gradient is 2 holes, and the final volume of each hole is 120 mu l.

All samples to be tested were incubated at 37 ℃ with 5% CO₂Incubated for 2h under the same conditions.

(4) Sample adding and co-incubation: and (3) taking the cell culture plate prepared in the step (2), discarding supernatant of each hole, and adding 100 mu l of the sample to be detected with different concentration gradients incubated in the step (3), wherein the total volume of each hole is 100 mu l. 37 ℃ and 5% CO₂Culturing for 72h under the condition.

(5) And (3) detection: 10. mu.l of CCK-8 was added to each well of the culture of step (4) and incubated at 37 ℃ for 5 hours in an incubator, and then the absorbance at 450nm was measured using a microplate reader.

The detection result is shown in fig. 8, and similar to avastin monoclonal antibody, the recombinant fusion protein vaccine immunization group can obviously inhibit the proliferation of vascular endothelial cells, while the serum of the control group is not obviously inhibited.

Example 5: the recombinant fusion protein (comprising the sequence shown in SEQ ID NO: 2) is emulsified with liquid adjuvant Montanide ISA 51 VG and used for immunizing mice

The design, expression and preparation process of the recombinant fusion protein (shown as SEQ ID NO: 2) are as described in examples 2 and 3, except that the DNA coding sequence (shown as SEQ ID NO: 4) of VEGF107-CRM197 is completely synthesized and constructed onto the expression plasmid pCDFDuet-1-DsbC by means of gene synthesis. The sequence alignment of SEQ ID NO 1 and SEQ ID NO 2 is shown in FIG. 10.

The emulsification method and the antibody titer detection method were as described in example 4.

As shown in FIG. 9, the recombinant fusion protein comprising the amino acid sequence of SEQ ID NO. 2 also induced high titers of anti-VEGF 165 antibody in mice.

Example 6: recombinant fusion protein vaccine for immunizing rhesus monkey

The recombinant VEGF fusion protein (shown as SEQ ID NO: 1) and adjuvant Montanide ISA 51 VG are emulsified, 0.8ml of emulsified liquid is injected into the position of biceps brachii of rhesus monkey after emulsification, 1 time per week, 4 times in total and 1 week after the 4 th injection for blood collection, and anti-VEGF 165 antibody titer detection, anti-VEGF 165 neutralizing antibody detection and vascular endothelial cell proliferation inhibition detection are respectively carried out as described in example 4.

Monkey serum anti-VEGF 165 antibody titres the assay was similar to the anti-VEGF 165 antibody titres of example 4, with the only difference being that the secondary antibody was replaced with an HRP enzyme labelled anti-monkey antibody.

The results of the monkey serum anti-VEGF 165 antibody titer detection are shown in FIG. 11, which shows that the antibody titer exceeded 10⁶The antibody titer of the reported VEGF vaccine in monkeys has been significantly exceeded (no more than 8000).

The anti-VEGF 165 neutralizing antibody detection and the vascular endothelial cell proliferation inhibition assay were similar to those described in example 4. The results are shown in fig. 12, which shows that the recombinant VEGF fusion protein vaccine can stimulate monkeys to produce antibodies that inhibit binding of VEGF165 to its receptor and inhibit proliferation of vascular endothelial cells, i.e., break immune tolerance, induce production of anti-VEGF antibodies, inhibit proliferation of vascular endothelial cells, thereby inhibiting angiogenesis and inhibiting tumor growth.

Example 7: antibody purified after immunization of recombinant fusion protein vaccine for inhibiting tumor growth

Recombinant VEGF fusion proteins were prepared as described in examples 2 and 3, and the prepared rabbits were immunized with adjuvant 1mg each time, four times, and rabbit sera were collected 1 month after the four immunizations, and the vaccine and adjuvant combination method was as described in example 4.

Preparing rabbit serum antibodies after immunization: the rabbit serum is subjected to the steps of centrifugation, ammonium sulfate precipitation, collection of precipitate, re-melting and the like to obtain an antibody crude extract, the antibody is captured by protein A filler, and the purified antibody is eluted and collected by a citric acid buffer solution with the pH value of 3.0.

Rhabdomyosarcoma A673 cells were selected and cultured in an environment of 5% by volume CO2 at 37 deg.C, and the growth medium was 90% DMEM +10% FBS. The tumor-bearing link comprises washing cells with PBS for 3 times, adding pancreatin for digestion, adding growth culture solution after the cells become round, blowing and resuspending the cells, counting, and controlling cell density to 4 × 10⁷One per ml.

Selecting nude mice with the weight of 18-22 mu g, and injecting each nude mouse with the weight of 4 multiplied by 10⁶One cell, injection volume 100. mu.l/mouse. 5 days after cell injection, 1mg of purified antibody (the activity of VEGF antibody is equivalent to the activity of 40 μ g of commercial VEGF monoclonal antibody drug avastin) was injected into each mouse 1 time per week, and the purified antibody from the serum of the non-immunized rabbit and the commercial VEGF monoclonal antibody drug avastin were used as controls. Tumor size was measured twice weekly in mice after tumor loading.

The results are shown in fig. 13, the antibody after the VEGF fusion protein vaccine immunization can significantly inhibit tumor growth and significantly prolong the survival time of mice (fig. 14).

Comparative example 1

Comparison of VEGF121-CRM197 and VEGF107-CRM197 immunized mice

VEGF121-CRM197 recombinant fusion protein and VEGF107-CRM197 recombinant fusion protein were expressed and prepared by the methods of example 2 and example 3, respectively. Mice were immunized with the prepared VEGF121-CRM197 and VEGF107-CRM197 recombinant fusion protein vaccines, respectively, in the manner described in example 4.

Comparing the antibody titers after mice immunized with VEGF121-CRM197 and VEGF107-CRM197, the results at 1 month after four immunizations are shown in FIG. 15, and the anti-VEGF specific antibody titer generated after mice immunized with VEGF107-CRM197 in which the C-terminal 14 amino acid residues were truncated can be increased 2-3 times compared to the mice immunized with VEGF121-CRM 197.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Huidong Thai Biotech Co., Ltd

<120> VEGF-CRM197 recombinant fusion protein vaccine, and preparation method and application thereof

<130> P2021-0031

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 659

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 1

Met Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val

1 5 10 15

Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr

20 25 30

Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe

35 40 45

Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp

50 55 60

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

65 70 75 80

Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser

85 90 95

Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Gly Ala Asp Asp

115 120 125

Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn Phe Ser Ser Tyr

130 135 140

His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln Lys Gly Ile Gln

145 150 155 160

Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp Asp Trp Lys Glu

165 170 175

Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly Tyr Ser Val Asp

180 185 190

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

195 200 205

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

210 215 220

Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu Pro Leu Met Glu

225 230 235 240

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

245 250 255

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

260 265 270

Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser Val Glu Leu Glu

275 280 285

Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp Ala Met Tyr Glu

290 295 300

Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg Arg Ser Val Gly

305 310 315 320

Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val Ile Arg Asp Lys

325 330 335

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

340 345 350

Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu Glu Lys Ala Lys

355 360 365

Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu His Pro Glu Leu

370 375 380

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

385 390 395 400

Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val Ile Asp Ser Glu

405 410 415

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

420 425 430

Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala Val His His Asn

435 440 445

Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser Leu Met Val

450 455 460

Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala

465 470 475 480

Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe Gln Val Val His

485 490 495

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

500 505 510

Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr Val Glu Asp Ser

515 520 525

Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His Asp Ile Lys Ile

530 535 540

Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val Leu Leu Pro Thr

545 550 555 560

Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys Thr His Ile Ser Val

565 570 575

Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala Ile Asp Gly Asp Val

580 585 590

Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val Gly Asn Gly Val His

595 600 605

Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser Glu Lys Ile His

610 615 620

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

625 630 635 640

Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser Leu Phe Phe Glu

645 650 655

Ile Lys Ser

<210> 2

<211> 659

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 2

Met Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val

1 5 10 15

Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr

20 25 30

Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe

35 40 45

Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp

50 55 60

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

65 70 75 80

Ile Met Glu Ile Glu Pro Glu Gln Gly Gln His Ile Gly Glu Met Ser

85 90 95

Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Gly Ala Asp Asp

115 120 125

Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn Phe Ser Ser Tyr

130 135 140

His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln Lys Gly Ile Gln

145 150 155 160

Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp Asp Trp Lys Glu

165 170 175

Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly Tyr Ser Val Asp

180 185 190

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

195 200 205

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

210 215 220

Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu Pro Leu Met Glu

225 230 235 240

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

245 250 255

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

260 265 270

Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser Val Glu Leu Glu

275 280 285

Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp Ala Met Tyr Glu

290 295 300

Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg Arg Ser Val Gly

305 310 315 320

Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val Ile Arg Asp Lys

325 330 335

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

340 345 350

Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu Glu Lys Ala Lys

355 360 365

Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu His Pro Glu Leu

370 375 380

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

385 390 395 400

Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val Ile Asp Ser Glu

405 410 415

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

420 425 430

Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala Val His His Asn

435 440 445

Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser Ser Leu Met Val

450 455 460

Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala

465 470 475 480

Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe Gln Val Val His

485 490 495

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

500 505 510

Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr Val Glu Asp Ser

515 520 525

Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His Asp Ile Lys Ile

530 535 540

Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val Leu Leu Pro Thr

545 550 555 560

Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys Thr His Ile Ser Val

565 570 575

Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala Ile Asp Gly Asp Val

580 585 590

Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val Gly Asn Gly Val His

595 600 605

Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser Glu Lys Ile His

610 615 620

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

625 630 635 640

Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser Leu Phe Phe Glu

645 650 655

Ile Lys Ser

<210> 3

<211> 1977

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

atggcaccaa tggctgaagg cggcggtcaa aatcatcacg aagttgttaa attcatggac 60

gtttaccagc gttcttactg ccatcctatc gaaaccctgg tggatatttt ccaggaatat 120

ccggacgaaa ttgaatatat cttcaagccg tcttgtgtgc cgctgatgcg ttgcggcggt 180

tgttgcaatg acgaaggcct ggaatgtgtt cctaccgaag aatctaacat cactatgcag 240

atcatgcgta tcaaaccgca ccagggtcag cacattggcg aaatgagctt tctgcagcac 300

aataagtgcg aatgccgtcc gaaaggtggt ggcggtagcg gtggtggcgg cagcggtggt 360

ggtggtagca tgggcgctga cgatgtggta gactcttcca aaagcttcgt gatggaaaac 420

ttcagctctt atcacggcac caaaccgggt tatgtggact ccattcagaa aggtatccaa 480

aaaccgaaaa gcggtacgca gggcaattac gatgatgatt ggaaggaatt ttactccacc 540

gacaataaat acgacgcggc gggctatagc gtcgataacg aaaatccgct gtctggcaag 600

gcaggcggtg tggtcaaagt tacctatccg ggcctgacca aggtgctggc cctgaaagtg 660

gataacgctg aaactatcaa aaaggagctg ggcctgtctc tgaccgaacc gctgatggaa 720

caggtaggta ccgaggaatt tatcaaacgt ttcggtgacg gcgcttctcg cgtcgtgctg 780

tctctgccgt tcgcggaagg ttctagctcc gttgaataca ttaacaactg ggagcaggcg 840

aaagcgctgt ccgttgaact ggaaatcaac ttcgagacgc gcggcaaacg tggtcaggat 900

gctatgtacg aatatatggc ccaggcttgt gctggtaacc gtgttcgccg ctctgttggc 960

tcctccctga gctgcatcaa cctggactgg gatgttattc gtgataaaac caaaaccaaa 1020

attgaatctc tgaaggaaca cggtccgatc aagaacaaaa tgtctgaatc tccgaataaa 1080

accgtatctg aagaaaaagc gaaacaatac ctggaagaat ttcaccagac cgcgctggaa 1140

cacccagaac tgtctgaact gaaaaccgta accggcacca acccggtttt cgcgggtgcg 1200

aactacgcag catgggcggt taacgtggca caggtcatcg attccgagac cgctgacaac 1260

ctggaaaaaa ccactgcagc actgtccatc ctgccaggca tcggctccgt gatgggtatt 1320

gccgatggcg cggtgcacca caacaccgag gaaattgtgg cccagagcat cgctctgtcc 1380

tccctgatgg ttgcccaggc gatcccactg gtaggcgaac tggttgacat cggtttcgca 1440

gcctataact tcgtcgaaag cattattaac ctgtttcagg ttgtacataa cagctacaac 1500

cgtccggctt actccccagg ccacaaaacc cagccgttcc tgcacgatgg ctacgcggta 1560

agctggaata ccgtcgaaga ttccattatc cgcacgggct tccagggcga gagcggtcac 1620

gacatcaaga tcaccgctga gaacaccccg ctgccgattg ccggcgttct gctgccgacg 1680

atcccgggca agctggatgt taacaaatcc aaaactcaca tcagcgtaaa cggtcgtaaa 1740

atccgtatgc gttgccgcgc catcgacggc gatgtaacct tctgccgccc gaaatccccg 1800

gtatatgttg gcaacggcgt gcacgcgaac ctgcacgttg ctttccatcg cagcagctcc 1860

gaaaaaattc attccaatga aatcagctct gactctatcg gtgtcctggg ttatcagaaa 1920

accgttgacc acaccaaagt taacagcaaa ctgtccctgt tcttcgaaat taagtcc 1977

<210> 4

<211> 1977

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

atggctccga tggctgaagg cggcggccag aaccaccatg aagtagttaa gttcatggac 60

gtttaccaac gctcttattg tcaccctatt gaaaccctgg tggatatctt ccaggagtac 120

cctgacgaaa ttgaatatat cttcaagcca tcttgcgtgc cgctgatgcg ctgcggtggt 180

tgctgtaacg atgagggtct ggaatgcgtg ccgacggaag agtccaacat caccatgcag 240

atcatggaaa tcgaaccgga acagggccag cacattggcg aaatgtcttt cctgcagcac 300

aacaaatgtg aatgccgtcc gaagggtggt ggtggttccg gtggcggcgg tagcggcggt 360

ggtggctcta tgggtgcgga cgatgtagtc gattcttcca aatctttcgt aatggagaac 420

ttctcctctt accacggcac caaaccgggc tacgtcgatt ctatccagaa aggcatccaa 480

aaaccgaaat ccggcactca gggcaactac gacgatgact ggaaagaatt ttacagcacc 540

gacaacaaat acgatgctgc tggttatagc gtagataacg agaacccgct gagcggcaag 600

gcgggtggtg tggtcaaggt tacgtacccg ggtctgacca aagtgctggc actgaaagta 660

gataacgcag aaactatcaa gaaagaactg ggcctgtctc tgactgagcc gctgatggaa 720

caggttggca ccgaagaatt catcaaacgt ttcggtgatg gcgcttctcg tgtggttctg 780

tccctgccgt tcgcagaagg ctctagcagc gtagaataca ttaacaattg ggaacaagcg 840

aaagcactgt ctgtggaact ggaaatcaat ttcgagactc gtggcaaacg tggccaggat 900

gcgatgtacg aatacatggc acaggcatgt gctggtaacc gtgttcgtcg ttctgtgggc 960

tcttccctgt cctgcatcaa cctggactgg gacgttatcc gtgataaaac taaaactaaa 1020

atcgaatctc tgaaggagca cggtccaatt aagaataaga tgtccgaatc cccgaacaaa 1080

acggtaagcg aagaaaaagc aaaacagtac ctggaagaat tccaccagac cgcactggaa 1140

cacccggagc tgtctgaact gaaaaccgtg acgggtacca acccggtttt cgcaggcgcc 1200

aactatgcgg cgtgggccgt taacgtggcg caagttatcg attctgaaac cgctgacaat 1260

ctggaaaaaa cgaccgccgc tctgtctatt ctgccgggta tcggctccgt gatgggcatc 1320

gcggatggtg ccgtgcacca caacaccgaa gaaatcgtag cgcagtccat cgcactgtct 1380

tctctgatgg ttgcacaggc tatcccgctg gtgggcgagc tggtcgacat cggtttcgcg 1440

gcgtacaact tcgtagaaag catcatcaac ctgtttcaag ttgttcacaa ttcctataac 1500

cgcccggctt attctccggg tcacaaaacc caaccttttc tgcacgacgg ctatgccgtt 1560

tcctggaaca ctgtagaaga ctccatcatc cgcaccggct tccagggcga aagcggccac 1620

gatatcaaga tcaccgctga aaatactccg ctgccgatcg cgggcgttct gctgccgact 1680

atcccgggta aactggacgt aaacaagtcc aaaactcaca tcagcgttaa tggtcgcaaa 1740

atccgcatgc gttgtcgtgc catcgacggt gatgtaacct tctgccgccc taaatccccg 1800

gtttacgttg gtaatggcgt tcacgcaaac ctgcatgtcg cttttcaccg ttcctcttcc 1860

gaaaagattc atagcaacga aatctcctct gacagcattg gtgttctggg ttaccagaaa 1920

accgtggacc acaccaaggt aaactctaag ctgagcctgt tcttcgagat caaatct 1977

<210> 5

<211> 107

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 5

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys

1 5 10 15

Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu

20 25 30

Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys

35 40 45

Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu

50 55 60

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

65 70 75 80

Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe

85 90 95

Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys

100 105

<210> 6

<211> 107

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 6

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys

1 5 10 15

Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu

20 25 30

Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys

35 40 45

Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu

50 55 60

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

65 70 75 80

Met Glu Ile Glu Pro Glu Gln Gly Gln His Ile Gly Glu Met Ser Phe

85 90 95

Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys

100 105

<210> 7

<211> 535

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 7

Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn

1 5 10 15

Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln

20 25 30

Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp

35 40 45

Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly

50 55 60

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

65 70 75 80

Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val

85 90 95

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

100 105 110

Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly

115 120 125

Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser

130 135 140

Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser

145 150 155 160

Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp

165 170 175

Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg

180 185 190

Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val

195 200 205

Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly

210 215 220

Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu

225 230 235 240

Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu

245 250 255

His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val

260 265 270

Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val

275 280 285

Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu

290 295 300

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

305 310 315 320

Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser

325 330 335

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

340 345 350

Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe

355 360 365

Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His

370 375 380

Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr

385 390 395 400

Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His

405 410 415

Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val

420 425 430

Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys Thr

435 440 445

His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala Ile

450 455 460

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

465 470 475 480

Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser

485 490 495

Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu

500 505 510

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

515 520 525

Leu Phe Phe Glu Ile Lys Ser

530 535

<210> 8

<211> 121

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 8

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys

1 5 10 15

Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu

20 25 30

Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys

35 40 45

Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu

50 55 60

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

65 70 75 80

Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe

85 90 95

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

100 105 110

Gln Glu Lys Cys Asp Lys Pro Arg Arg

115 120

<210> 9

<211> 779

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 9

Met Gly Ser Ser His His His His His His Gly Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Ser Asp Ser Glu Val Asn Gln Glu

20 25 30

Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn

35 40 45

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

50 55 60

Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly

65 70 75 80

Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln

85 90 95

Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile

100 105 110

Glu Ala His Arg Glu Gln Ile Gly Gly Ala Pro Met Ala Glu Gly Gly

115 120 125

Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg

130 135 140

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

145 150 155 160

Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met

165 170 175

Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr

180 185 190

Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln

195 200 205

Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu

210 215 220

Cys Arg Pro Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe

245 250 255

Val Met Glu Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val

260 265 270

Asp Ser Ile Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly

275 280 285

Asn Tyr Asp Asp Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr

290 295 300

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

305 310 315 320

Ala Gly Gly Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu

325 330 335

Ala Leu Lys Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu

340 345 350

Ser Leu Thr Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile

355 360 365

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

370 375 380

Ala Glu Gly Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala

385 390 395 400

Lys Ala Leu Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys

405 410 415

Arg Gly Gln Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly

420 425 430

Asn Arg Val Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu

435 440 445

Asp Trp Asp Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu

450 455 460

Lys Glu His Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys

465 470 475 480

Thr Val Ser Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln

485 490 495

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

500 505 510

Thr Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn

515 520 525

Val Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr

530 535 540

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

545 550 555 560

Ala Asp Gly Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser

565 570 575

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

580 585 590

Glu Leu Val Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile

595 600 605

Ile Asn Leu Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr

610 615 620

Ser Pro Gly His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val

625 630 635 640

Ser Trp Asn Thr Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly

645 650 655

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

660 665 670

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

675 680 685

Lys Ser Lys Thr His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg

690 695 700

Cys Arg Ala Ile Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro

705 710 715 720

Val Tyr Val Gly Asn Gly Val His Ala Asn Leu His Val Ala Phe His

725 730 735

Arg Ser Ser Ser Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser

740 745 750

Ile Gly Val Leu Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn

755 760 765

Ser Lys Leu Ser Leu Phe Phe Glu Ile Lys Ser

770 775

<210> 10

<211> 2340

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

atgggcagca gccatcatca tcatcatcac ggcagcggcc tggtgccgcg cggcagccat 60

atggctagca tgtcggactc agaagtcaat caagaagcta agccagaggt caagccagaa 120

gtcaagcctg agactcacat caatttaaag gtgtccgatg gatcttcaga gatcttcttc 180

aagatcaaaa agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggt 240

aaggaaatgg actccttaag attcttgtac gacggtatta gaattcaagc tgatcagacc 300

cctgaagatt tggacatgga ggataacgat attattgagg ctcacagaga acagattggt 360

ggtgcaccca tggcagaagg aggagggcag aatcatcacg aagtggtgaa gttcatggat 420

gtctatcagc gcagctactg ccatccaatc gagaccctgg tggacatctt ccaggagtac 480

cctgatgaga tcgagtacat cttcaagcca tcctgcgtgc ccctgatgcg atgcgggggc 540

tgctgcaatg acgagggcct ggagtgtgtg cccactgagg agtccaacat caccatgcag 600

attatgcgga tcaaacctca ccaaggccag cacataggag agatgagctt cctacagcac 660

aacaaatgtg aatgcagacc aaagggtggc ggtggctctg gtggcggtgg ttccggcggt 720

ggtggctcta tgggcgcaga cgatgttgtt gactcttcta aatcctttgt tatggaaaac 780

ttcagctcct atcacggcac caagccgggt tatgtcgata gcattcagaa aggtatccaa 840

aaaccgaagt ctggcacgca gggtaactac gatgacgatt ggaaggaatt ctacagcacc 900

gacaacaaat atgatgcggc cggttactca gtcgacaacg aaaatccgct gagcggcaag 960

gccggcggtg tggttaaagt gacgtatccg ggcctgacca aggtcctggc cctgaaagtg 1020

gataatgcag aaaccattaa aaaggaactg ggtctgagcc tgacggaacc gctgatggaa 1080

caggttggca ccgaagaatt tatcaaacgc ttcggcgatg gtgccagtcg tgtcgtgctg 1140

tccctgccgt tcgcagaagg tagctctagt gtggaatata ttaacaattg ggaacaagcg 1200

aaggccctgt ccgttgaact ggaaatcaac tttgaaaccc gcggcaaacg tggtcaggat 1260

gcgatgtatg aatacatggc acaagcttgc gcgggtaatc gcgttcgtcg cagcgtcggc 1320

tcctcactgt cttgtatcaa cctggactgg gatgttattc gtgataaaac caagacgaaa 1380

attgaaagtc tgaaggaaca tggcccgatc aagaacaaaa tgagcgaatc tccgaataaa 1440

acggtgtccg aagaaaaggc taaacagtat ctggaagaat tccaccaaac cgcactggaa 1500

catccggaac tgtcagaact gaaaaccgtg acgggtacca acccggtttt tgccggcgca 1560

aattacgcag cttgggctgt gaacgttgcg caagtgattg actcggaaac ggccgataat 1620

ctggaaaaaa ccacggcggc cctgagtatt ctgccgggca tcggttccgt tatgggtatt 1680

gccgacggcg cagtccatca caacaccgaa gaaattgtgg cccagtctat cgcactgtcg 1740

agcctgatgg ttgctcaagc gattccgctg gttggcgaac tggttgatat cggctttgca 1800

gcttacaact tcgtggaaag tatcatcaac ctgtttcagg ttgtccacaa ctcatataat 1860

cgcccggcct actcgccggg tcacaaaacc caaccgttcc tgcatgacgg ctacgcggtt 1920

agctggaata cggtcgaaga ttctattatc cgtaccggct ttcagggtga atctggccac 1980

gacattaaaa tcacggctga aaacaccccg ctgccgattg ccggtgttct gctgccgacc 2040

atcccgggca agctggatgt taataagtca aaaacccata tctcggtcaa cggtcgcaaa 2100

attcgtatgc gctgccgtgc gatcgacggc gatgtgacct tctgtcgtcc gaaaagcccg 2160

gtctatgtgg gcaacggtgt ccatgctaat ctgcacgtgg cgtttcatcg ctctagttcc 2220

gaaaagatcc atagtaacga aatctcatcg gattccattg gtgtgctggg ctaccagaaa 2280

accgtggacc ataccaaagt gaatagtaaa ctgagcctgt tcttcgaaat caaatcctaa 2340

<210> 11

<211> 654

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

atggatgacg cggcaattca acaaacgtta gccaaaatgg gcatcaaaag cagcgatatt 60

cagcccgcgc ccgtagctgg catgaagaca gttctgacta acagcggcgt gttgtacatc 120

accgatgatg gtaaacatat cattcagggg ccaatgtatg acgttagcgg cacggctccg 180

gtcaatgtca ccaataagat gctgttaaag cagttgaatg cgcttgaaaa agagatgatc 240

gtttataaag cgccgcagga aaaacacgtc atcaccgtgt ttactgatat tacctgtggt 300

tactgccaca aactgcatga gcaaatggca gactataacg cgctggggat caccgtgcgt 360

tatcttgctt tcccgcgcca ggggctggac agcgatgcag agaaagaaat gaaagctatc 420

tggtgtgcga aagataaaaa caaagcgttt gatgatgtga tggcaggtaa aagcgtcgca 480

ccagccagct gcgacgtgga tattgccgac cattacgtac ttggcgtcca gcttggcgtt 540

agcggtactc cggcagttgt gctgagcaat ggcacacttg ttccgggtta ccagccgccg 600

aaagagatga aagaattcct cgacgaacac caaaaaatga ccagcggtaa ataa 654