阅读说明：本技术 抗cd56蛋白单克隆抗体及其细胞株、制备方法和应用 (anti-CD 56 protein monoclonal antibody and cell strain, preparation method and application thereof ) 是由 林洋 杨清海 陈惠玲 王小亚 周洪辉 程本亮 陈昌星 于 2021-08-12 设计创作，主要内容包括：本发明涉及一种可以识别人CD56抗原的单克隆抗体,分泌细胞株、其制备方法以及其在免疫检测中的用途。上述技术方案选取CD56蛋白C末端的476-608位氨基酸为抗原肽,进行优化,成为适合在大肠杆菌BL21表达的基因片段,最后得到的重组蛋白包含TRX蛋白标签、CD56蛋白片段及组氨酸蛋白标签。所述重组蛋白对小鼠进行免疫,经细胞融合、筛选和亚克隆,获得高效分泌抗CD56蛋白单克隆抗体的小鼠杂交瘤细胞株33A2,以及由该细胞株所分泌的抗CD56蛋白单克隆抗体。本方案得到的抗体具有高特异性、敏感性,可以特异性识别表达CD56蛋白的细胞,适用于免疫学检测,特别是免疫组化检测。(The invention relates to a monoclonal antibody capable of recognizing human CD56 antigen, a secretory cell strain, a preparation method thereof and application thereof in immunoassay. The technical scheme selects 476-608 amino acids at the C terminal of the CD56 protein as antigen peptides for optimization to form a gene fragment suitable for expression in Escherichia coli BL21, and the finally obtained recombinant protein comprises a TRX protein tag, a CD56 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 33A2 capable of efficiently secreting the anti-CD 56 protein monoclonal antibody and the anti-CD 56 protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing the CD56 protein, and is suitable for immunological detection, particularly immunohistochemical detection.)

1. The monoclonal antibody for resisting the CD56 protein is characterized in that the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown as SEQ ID NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

2. The monoclonal antibody according to claim 1, wherein the coding DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No.2, and the coding DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No. 3.

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes the CD56 protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG2a kappa subtype monoclonal antibody.

5. An anti-CD 56 monoclonal antibody is produced by hybridoma cell strain with preservation number of CGMCC NO 21978.

6. The preparation method of the anti-CD 56 protein monoclonal antibody is characterized in that an antigen used for immunizing a mouse is recombinant protein, and the recombinant protein is expressed by escherichia coli in a recombinant mode and comprises a TRX protein tag, a CD56 protein fragment and a His protein tag.

7. The preparation method of claim 6, wherein the CD56 protein fragment is the 476 th to 608 th amino acid fragment of the CD56 protein, and the amino acid sequence of the fragment is the amino acid sequence shown in SEQ ID NO. 1.

8. A hybridoma cell strain capable of secreting anti-CD 56 protein molecules is a mouse hybridoma cell strain 33A2, is preserved in the China general microbiological culture Collection center (CGMCC), and has the preservation number: CGMCC NO 21978.

9. Use of the monoclonal antibody of any one of claims 1-5 in an immunoassay for CD56 protein.

10. An immunohistochemical detection reagent for CD56 protein, comprising the anti-CD 56 monoclonal antibody according to claim 1 as an active ingredient.

Technical Field

The invention relates to the field of biomedical engineering, in particular to a CD56 protein resistant monoclonal antibody, a cell strain, a preparation method and application thereof.

Background

The CD56 antigen is a glycoprotein expressed on the cell surface with 140KD, is a member of immunoglobulin (Ig) superfamily, belongs to NCAM subtype, and is involved in the growth and migration of cells.

CD56 is thought to originate in NK/T cells and is a hallmark of isoforms of neuronal cell adhesion molecules and natural killer lymphocytes (NK). In the hematopoietic system, CD56 is typically expressed on the surface of NK cells and CD4+/CD8+ T cells, but not on peripheral lymphocytes, monocytes, granulocytes, and CD34+ hematopoietic stem cells. CD56 is hardly expressed on the surface of normal plasma cells, the positive rate of CD56 on the surface of multiple myeloma tumor cells is obviously increased to 70-80%, and the juvenile plasma cells are more likely to express CD56 than mature plasma cells, so that the expression of CD56 is often indicative of malignant plasma cells.

Since it mediates cell-to-cell interactions, it may regulate cell homing leading to the spread of acute myeloid leukemia cells. In recent domestic analysis on CD56+ AML, it was suggested that CD56 expression reduces OS and DFS in patients, and that CRR is reduced and recurrence rate is high, further suggesting that CD56 may be a bad factor affecting AML prognosis.

Therefore, the development of monoclonal antibodies for the immunohistochemical detection of CD56 is of great importance.

Disclosure of Invention

The invention provides a monoclonal antibody for resisting CD56 protein, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

Furthermore, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.2, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 3.

Further, the monoclonal antibody specifically recognizes the CD56 protein.

Further, the monoclonal antibody is a mouse IgG2a kappa subtype monoclonal antibody.

Furthermore, the monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 21978.

The inventor also provides a preparation method of the anti-CD 56 protein monoclonal antibody, wherein the antigen used for immunizing mice is recombinant protein, and the recombinant protein is expressed by escherichia coli in a recombinant mode and comprises a TRX protein tag, a CD56 protein fragment and a His protein tag.

Further, the CD56 protein fragment is the amino acid fragment from 476 th position to 608 th position of the CD56 protein, and the amino acid sequence of the CD56 protein fragment is the amino acid sequence shown in SEQ ID NO. 1.

The inventor also provides a hybridoma cell strain secreting anti-CD 56 protein molecules, wherein the cell strain is a mouse hybridoma cell strain 33A2 which has been preserved in China general microbiological culture Collection center (CGMCC NO 21978) at 24 months 3 in 2021, with the preservation number of CGMCC NO 21978 and the address of China academy of sciences microbial research institute No.3 of North China western street 1 institute in the morning, Chaoyang, Beijing city.

The inventor also provides the application of the monoclonal antibody in immunodetection of the CD56 protein.

Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

The inventor finally provides a CD56 protein immunohistochemical detection reagent, wherein the immunohistochemical detection reagent contains an amino acid sequence of which the amino acid sequence of a heavy chain variable region is shown as SEQ ID NO. 4; the monoclonal antibody of anti-CD 56 protein with the amino acid sequence of the light chain variable region as shown in SEQ ID No.5 as the effective component.

Different from the prior art, the invention has the beneficial technical effects that: according to the technical scheme, amino acids 476 to 608 from the C terminal of the CD56 protein are selected as antigen peptides, the antigen peptides are optimized into a gene fragment suitable for being expressed in escherichia coli BL21, and finally the obtained recombinant protein comprises a TRX protein tag, a CD56 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 33A2 capable of efficiently secreting the anti-CD 56 protein monoclonal antibody and the anti-CD 56 protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing the CD56 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a comparison of immunohistochemical staining results for small cell lung cancer; the left is CD56 secreted by 33A2, and the right is commercial CD 56.

FIG. 2 is a comparison of immunohistochemical staining of the brain; the left is CD56 secreted by 33A2, and the right is commercial CD 56.

Detailed Description

To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

EXAMPLE 1 preparation of recombinant CD56 protein fragment

First, gene optimization and synthesis

CD56 protein fragment with amino acids 476-608 was selected according to the protein sequence with accession number P13591 in the Uniprot database, and was directly optimized into a gene fragment suitable for expression in E.coli BL 21. EcoR I and XhoI cleavage sites were added at the 5 'and 3' ends of the gene, respectively, during PCR.

And separating and recovering the PCR product through agarose gel electrophoresis, carrying out EcoR I and XhoI enzyme digestion on the recovered fusion protein gene and a plasmid vector Pet32a for expression respectively, carrying out electrophoresis recovery again, and connecting with T4 DNA ligase. And transforming the connecting product into escherichia coli competent cells BL21, selecting clones on a plate for inoculation, and performing PCR identification on bacterial liquid. And selecting clones with positive PCR results for sequencing analysis, and using the clones with completely correct sequences.

The selection of different antigens for immunization makes it possible to prepare antibodies with different binding characteristics, the molecules present simultaneously a plurality of variants due to variable cleavage, and finally the recognition ability and pattern of different antibodies on antigen-expressing cells are different. The CD56 molecule was analyzed according to published sequences, and based on the structure, antigenicity, hydrophilicity and hydrophobicity of the constituent amino acids and secondary structure on the cell membrane, a region suitable for soluble expression and good immunogenicity was selected for recombinant expression, and amino acid residues 476-608 of CD56 were selected for codon optimization and recombinant expression, with a molecular weight of about 39 kDa. The CD56 protein is obtained by using prokaryotic expression gene sequence through sequence optimization design. The recombinant immunogen consists of a CD56 protein fragment with antigenicity and a protein tag for purifying the recombinant protein, wherein the protein tag is TRX and His.

II, protein expression and purification

The overnight strain cultured by a single colony is transferred to 100mL LB culture medium according to the proportion of 1:100, kanamycin with the final concentration of 10 mug/mL is added, shaking culture is carried out at 37 ℃ until OD600 is 0.6-0.8, 1mmol/L IPTG is added, shaking culture is carried out at 16 ℃ for overnight, and the strain is collected and then is crushed by ultrasound. The recombinant protein is provided with a histidine tag, and affinity purification of the protein is performed by using a nickel column. Elution was carried out with 500mmol/L imidazole and SDS PAGE separation was carried out.

EXAMPLE 2 establishment of hybridoma cell lines

Immunization

The recombinant protein of example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and 4-6 week-old female ICR mice (purchased from Beijing Wintolite laboratory animals technologies, Ltd.) were immunized and injected subcutaneously at 6 o' clock into the abdomen of each mouse at a dose of 60. mu.g/mouse. The booster was administered every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Co., F5506) at a dose of 30. mu.g/mouse. 7 days after 3 times of booster immunization, the titer of multiple antibodies of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength of 450nm), the mice with the highest titer are injected by tail vein for impact immunization, and the antigen is uniformly mixed by normal saline, and the dosage is 50 mu g/mouse.

Second, cell fusion

A mouse spleen cell suspension with qualified immunity is prepared aseptically, mixed with mouse myeloma cell sp2/0(ATCC number CRL-8287) at a ratio of 5:1, and centrifuged at 1500rpm for 5 min. The supernatant was discarded and the tube was placed in a 37 ℃ water bath, 1ml of PEG1500 (Roche) was added slowly over 1 minute, and the cells were agitated. After standing in warm water for 1min, 10ml of serum-free IMDM (Sigma Co.) was added, mixed well, and centrifuged at 1000rpm for 5 min. After discarding the supernatant, 10ml of serum (PAA) was added to the supernatant, the cells were carefully blown up, 5ml of thymocytes mixed with 10XHAT (Sigma) was added, and the mixture was mixed well. Then, 25ml of a semi-solid medium containing 2.1% nitrocellulose (Sigma) was added thereto, mixed well, and poured into 20 cell culture dishes. Placing the cell culture dish into a wet box, and adding 5% CO at 37 deg.C₂Culturing in an incubator.

Cloning and ELISA screening of positive hybridoma cells

The size and density of the clone cell mass are moderate 7 days after fusion, and the round, solid and large clone mass is sucked under a dissecting mirror and is injected into a 96-hole culture plate which is prepared with a culture medium in advance, and the culture is carried out in a 5% CO2 incubator at 37 ℃. After 3 days, the cell mass was approximately 2/3 basal areas, and 100. mu.L of the supernatant was screened by ELISA using the immunogen and the synthetic polypeptide, respectively. Positive clones were completely replaced and 200. mu.L of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, a second ELISA screening was performed and positive clones were transferred to 24-well plates previously prepared in medium (containing feeder cells and HT). After five days, 100 μ l of supernatant was subjected to a third ELISA screening, and the positive clones were transferred to 6-well plates and cell culture flasks successively for expanded culture and frozen. EXAMPLE 3 preparation of monoclonal antibody by ascites Induction method

First, preparation of ascites

Cells in logarithmic growth phase were washed with serum-free medium and suspended, and counted at about 5X 10⁵And 1 ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The ascites fluid taken out was centrifuged at 4000rpm at 4 ℃ for 10 min. The ascites fluid in the middle is carefully aspirated and collected in a centrifuge tube and stored at 4 ℃ or-20 ℃.

Secondly, purification of monoclonal antibody

Antibodies were purified from ascites fluid by HiTrap rProtein A FF (GE) affinity chromatography as described. Purity was assessed on SDS-PAGE gels and concentration was determined by Bradford method. Purified antibody was stored at-20 ℃.

EXAMPLE 4 characterization of monoclonal antibodies

First, subclass identification

Coated goat anti-mouse IgG (Beijing China fir Jinqiao Biotechnology Co., Ltd.) was diluted to 0.5. mu.g/ml with 100. mu.l/well at 4 ℃ overnight in 100mM PBS (pH 7.4). The liquid was decanted, washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. mu.l of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well, and incubated at 37 ℃ for 1 h. The liquid was decanted and washed 3 times with PBS-T. 0.1ml of hybridoma supernatant was added to each well and incubated at 37 ℃ for 1 h. The decanted solution was washed 3 times with PBS-T. Using a confining liquid 1: 1000 dilution of HRP-labeled goat anti-mouse (κ, λ) antibody or 1: HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibodies (Southern Biotech) were diluted at 2000 in 0.1ml per well and added to the appropriate wells, followed by incubation at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. Add 50. mu.l of 0.15% ABTS (Southern Biotech) and 0.03% H per well₂O₂The reaction was performed in the citric acid buffer (pH4.0), and the OD value at a wavelength of 405nm was measured within 10 to 20 min.

The results show that the monoclonal antibody of the invention is a murine monoclonal antibody of the IgG2a kappa type.

Second, determination of affinity constant

The recombinant CD56 protein prepared in example 1 was coated at a concentration of 2. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. mu.l of blocking solution to each well and block for 2h at 37 ℃ and wash 3 times with PBS-T. The monoclonal antibody purified in example 3 was prepared from 1: 200 began a 2-fold gradient dilution, and finally 1 well was blank, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody 1: 20000 dilution, 100 μ l per well, incubation at 37 ℃ for 1h, PBS-T wash 3 times. Mu.l of a buffer containing 0.1% TMB (Sigma) and 0.03% H was added to each well₂O₂The reaction mixture was developed in a citric acid-phosphoric acid buffer for 10min, and 50. mu.l of a 0.5M sulfuric acid solution was added thereto to terminate the reaction. And measuring the light absorption value with the wavelength of 450nm by using a microplate reader. Drawing a curve of OD value corresponding to the dilution factor of the antibody, finding the dilution factor A corresponding to half of the maximum binding OD value, and calculating the affinity constant of the antibody to be 3.84 × 10 by using the following formula⁹。

Reaction specificity and application effect of monoclonal antibody

The CD56 recombinant protein prepared in example 1 was used to detect the recognition specificity of the monoclonal antibody of the present invention by immunoblotting, and 12% polyacrylamide gel electrophoresis was performed. The gel protein bands were transferred to PVDF membranes (Millipore) in a Bio-Rad electrotransfer system according to the conventional method. The membrane was placed in TBS-T blocking solution containing 5% skimmed milk powder overnight at 4 ℃. Monoclonal antibodies to the antibody secreted by the 33A2 hybridoma (1: 1000 dilution) were added and incubated overnight at 4 ℃. After washing the membrane with TBS-T, add 1: a5000-diluted goat anti-mouse secondary antibody (Beijing Zhonghua Jinqiao Biotech Co., Ltd.) was incubated at room temperature for 1 hour. Washing the membrane with TBST again, adding ECL (Beijing prilley Gene technology Co., Ltd.), and collecting chemiluminescence image data with ChemiDocMP multicolor fluorescence imaging system (Bio-Rad). Example 5 immunohistochemical tissue chip staining and identification

Chip preparation process

HE sections were first stained for each sample to identify tumor sites. The tissue chip was produced using a fully automatic tissue chip machine of 3 DHISTECH. And putting the prepared tissue chip wax block into a wax block manufacturing mold, putting the mold into an oven at 68 ℃ for 10 minutes to enable the tissue core and the wax of the receptor wax block to be fused into a whole, then slightly taking the mold out of the oven, cooling the paraffin in a semi-molten state for about 30 minutes at room temperature, putting the mold into a refrigerator at-20 ℃ for freezing for 6 minutes, taking the tissue chip wax block out of the mold, and slicing or putting the tissue chip wax block into the refrigerator at 4 ℃ for storage for later use. And (3) trimming, continuously slicing, setting the thickness to be 3 mu m, floating the continuous slices in 40% alcohol, naturally unfolding, transferring the separated slices into warm water at 50 ℃ for 30 seconds, pasting the slices with a glass slide treated by polylysine, baking the prepared tissue chip in an oven at 68 ℃ for 2 hours, taking out, cooling at room temperature, and storing in a refrigerator at-4 ℃.

IHC staining and analysis

Conventional xylene dewaxing was performed 3 times for 6 minutes each, 100%, 95%, 85% gradient ethanol hydration for 3 minutes each, and finally tap water rinse. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3 min with PBS. Dropwise adding 3% H₂O₂Incubate for 10min and wash with PBS for 3 × 3 min. Spin-drying the slices, dripping primary antibody diluted in a proper proportion (the dilution proportion of the antibody is designed according to the concentration of the antibody in the primary dilution) and incubating for 1 hour at room temperature (25 ℃), washing for 3X 3 minutes by PBS, dripping secondary antibody and incubating for 15-30 minutes at room temperature, washing for 3X 3 minutes by PBS, throwing off the PBS, and developing for 3-10 minutes by using a freshly prepared DAB developing solution. Hematoxylin counterstain for 25 seconds, PBS bluing for 30 seconds. Dehydration was carried out in a gradient of 85% (3 min) -95% (3 min) -100% (3 min) alcohol, finally xylene was transparent for 3 min, and the gel was blocked with neutral gum.

The immunohistochemical staining results were divided into: positive and negative. Positive expression must be at a specific antigenic site in cells and tissues to be considered positive. Under the condition that the tissue staining distribution is clear and the cell positioning is accurate, the staining result is further divided according to the difference of staining intensity, which is as follows:

1. the sample is weakly positive; marked "+";

2. the sample is moderately positive; marked "+";

3. the sample is highly positive; marked as "+ + +".

4. The sample was negative and marked "-".

Data statistics

1. Tumor tissue chip detection results:

the antibody CD56(33A2) and the commercial antibody CD56(56C04) are synchronously detected in 20 cases of small cell lung cancer and the detection results are compared.

Immunohistochemical results for CD56 were counted. The whole experimental process adopts a double-blind design, and the statistical results are as follows:

the result shows that the monoclonal antibody of the anti-CD 56 protein secreted by the 33A2 cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In the immunohistochemical detection, the positive rate is equivalent to that of a commercial antibody, but the partial positive intensity is higher than that of the commercial antibody, which indicates that the CD56 secreted by the 33A2 cell strain is more sensitive and effectively avoids false negative results.

FIG. 1 is a graph comparing the results of immunohistochemical staining of small cell lung cancer (33A 2-secreted CD56 on the left and commercial CD56 on the right).

2. Detection results of the normal tissue chip:

the normal tissue chip comprises 30 normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed operation specimens; each tissue included 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectal, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (young child), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

The antibody (33A2) and the commercial antibody are synchronously detected on a normal tissue chip, and the negative and positive detection results are consistent, which shows that the specificity of the antibody on the normal tissue is equivalent to that of the commercial antibody.

FIG. 2 is a graph comparing the results of immunohistochemical staining of brain tissue (33A2 secreted CD56 on the left and commercial CD56 on the right).

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.

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