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CN104418952B - A kind of anti-ErbB bispecific antibody for being used to treat breast cancer - Google Patents

A kind of anti-ErbB bispecific antibody for being used to treat breast cancer Download PDF

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CN104418952B
CN104418952B CN201310396323.2A CN201310396323A CN104418952B CN 104418952 B CN104418952 B CN 104418952B CN 201310396323 A CN201310396323 A CN 201310396323A CN 104418952 B CN104418952 B CN 104418952B
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CN104418952A (en
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钱卫珠
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Taizhou Mai BARTEC Pharmaceutical Co. Ltd.
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SHANGHAI HAISI TAIKE PHARMACEUTICAL Co Ltd
SHANGHAI ZHANGJIANG BIO-TECH Co Ltd
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Abstract

Bispecific antibody the invention belongs to biological technical field there is provided a class for treating breast cancer.One class is used for the anti-ErbB bispecific antibody for treating breast cancer, its antigen binding domain is made up of the antigen binding domain of Trastuzumab antibody and Pertuzumab antibody, and the present invention builds the bispecific antibody that a class targets ErbB2 molecule different epitopes using biotechnology.Present invention also offers the DNA sequence dna for encoding this antibody-like, the expression vector containing this DNA sequence dna and the host cell containing this expression vector.The class that the present invention is provided is used for the bispecific antibody for treating breast cancer, specific can be combined with the outer II areas of ErbB2 molecular cells and IV areas, and the effect with notable in vitro and in vivo anti-breast cancer.

Description

A kind of anti-ErbB bispecific antibody for being used to treat breast cancer
Technical field
The invention belongs to biological technical field, more specifically, the invention discloses the anti-human ErbB2 molecules different epitopes of a class Bispecific antibody, its preparation method and its application in antineoplastic is prepared.
Background technology
Tumour especially malignant tumour is the disease that the world today seriously endangers human health, dead caused by various diseases In be in second.And in recent years, its incidence of disease is in obvious ascendant trend.Therapeutic effect of malignant tumour is poor, the late period rate of transform Height, it is not good more than prognosis.Although the conventional treatments such as Radiotherapy chemotherapy and operative treatment that are clinically used at present are largely On alleviate slight illness, extend life span, but these methods have significant limitation, its curative effect is difficult to further carry It is high.
Breast cancer is there is ErbB2 in the most common malignant tumour of women, 20%-30% primary breast cancer patients (HER2) amplification of gene and the overexpression of albumen.Herceptin（Trastuzumab, trade name Herceptin）It is beautiful The Humanized monoclonal antibodies for ErbB2 acceptors that Genentech companies of state develop, obtained U.S. FDA batch in 1998 The mutatis mutandis treatment in ErbB2 height expression metastatic breast cancers.But Trastuzumab is to the metastatic breast cancer of ErbB2 low expressions Antitumor activity can not be shown, even if to the metastatic breast cancer of ErbB2 height expression, the mono- medicine first-line treatments of Trastuzumab Objective reactivity in 30%-50%.It is active demand clinically to develop more efficiently ErbB2 targeted therapeutics antibody.
Handkerchief trastuzumab（Pertuzumab, trade name Perjeta）It is being directed to for Genentech companies of U.S. development The Humanized monoclonal antibodies of ErbB2 acceptors, are a kind of HER dimerisation inhibitors, by combining ErbB2 molecules, block ErbB2 Molecule is heterodimeric with other HER receptors, so as to slow down the growth of tumour.Handkerchief trastuzumab passed through the U.S. in 2012 FDA certifications, the metastatic breast cancer positive for treating HER2.Pertuzumab is that Roche group is developed for treatment pair In the novel drugs of the nearly unavailable HER-2 positive breast cancers of hormonotherapy.
Pertuzumab is combined with the extracellular II areas of ErbB2 molecules, and epitope targeted Trastuzumab Positioned at the extracellular IV areas of ErbB2 acceptors.
Human epidermal growth factor acceptor（Human epidermal growth factor receptor, HER/erbB） There are 4 members in family：EGFR/HER1, ErbB2/HER2, ErbB3/HER3 and ErbB4/HER4.ErbB2 is with tyrosine-kinase The transmembrane protein of enzymatic activity, it does not have native ligand, inactive under free state.ErbB2 can be with HER families other 3 Member (EGFR, ErbB3, ErbB4) forms the heterodimer of part non-dependent or ligand-dependent and activated, and causes downstream signal Path MAPK and PI3K/Akt are activated, and ultimately result in tumor cell proliferation and survival.Research shows that Trastuzumab can not be complete Suppress ErbB2 heterodimer activated pathway, this is probably the major reason that Trastuzumab resistances are produced.
The content of the invention
In order to solve problem above, the present inventor is studied for a long period of time, through lot of experiments, utilizes genetic engineering One class of technique construction targets the bispecific antibody of ErbB2 molecule different epitopes, and this kind of bispecific antibody contains simultaneously Trastuzumab（Herceptin）And Pertuzumab（Handkerchief trastuzumab）Epitope land.
The invention discloses：
1. a class bispecific antibody, to include Trastuzumab and Pertuzumab antigen binding domain antibody egg In vain, the extracellular IV areas of people's ErbB2 acceptors can be both combined, the extracellular II areas of ErbB2 acceptors can be combined again.
2. the anti-human ErbB2 molecules different epitopes bispecific antibody of a class, is TPL, with SEQ ID NO:10 and SEQ ID NO:Amino acid sequence shown in 12.
3. the anti-human ErbB2 molecules different epitopes bispecific antibody of a class, is TPS, with SEQ ID NO:14 and SEQ ID NO:Amino acid sequence shown in 16.
4. the anti-human ErbB2 molecules different epitopes bispecific antibody of a class, is PTL, with SEQ ID NO:18 and SEQ ID NO:Amino acid sequence shown in 20.
5. the anti-human ErbB2 molecules different epitopes bispecific antibody of a class, is PTS, with SEQ ID NO:22 and SEQ ID NO:Amino acid sequence shown in 24.
6. a kind of nucleic acid molecules of separation, the bispecific antibody described in coding above-mentioned 1.
7. a kind of nucleic acid molecules of separation, the bispecific antibody TP described in coding above-mentioned 2L, with SEQ ID NO:9 Hes SEQ ID NO:Nucleotide sequence shown in 11.
8. a kind of nucleic acid molecules of separation, the bispecific antibody TP described in coding above-mentioned 3S, with SEQ ID NO:13 With SEQ ID NO:Nucleotide sequence shown in 15.
9. a kind of nucleic acid molecules of separation, the bispecific antibody PT described in coding above-mentioned 4L, with SEQ ID NO:17 With SEQ ID NO:Nucleotide sequence shown in 19.
10. a kind of nucleic acid molecules of separation, the bispecific antibody PT described in coding above-mentioned 5S, with SEQ ID NO:21 With SEQ ID NO:Nucleotide sequence shown in 23.
11. a kind of carrier, the series of operations containing any described nucleic acid molecules of above-mentioned 6-10 and the nucleic acid molecules Connected expression regulation sequence, wherein carrier can be pDR1, pcDNA3.1（+）, pcDNA3.1/ZEO（+）, pDHFR it One.
12. the carrier described in above-mentioned 11, is pcDNA3.1（+）Or pcDNA3.1/ZEO（+）.
13. a kind of host cell, is eukaryotic containing the carrier described in above-mentioned 12.
14. the host cell described in above-mentioned 13, is mammalian cell.
15. the host cell described in above-mentioned 14, is Chinese hamster ovary celI.
16. a kind of method for preparing any described bispecific antibodies of above-mentioned 1-5, this method includes：
A) Trastuzumab weights chain variable region gene is connected to Pertuzumab with the different catenation sequence of length On antibody weight chain gene, TPs and TP are constructed respectivelyLWeight chain gene, by Pertuzumab weight chain variable region genes It is connected to the different catenation sequence of length on Trastuzumab antibody weight chain genes, PTs and PT is constructed respectivelyLIt is light Heavy chain gene,
B) by bispecific antibody TPs, TPL, PTs and PTLWeight chain gene be cloned into carrier for expression of eukaryon respectively pcDNA3.1（+）, any described host cells of transfection claim 13-15, screening high-expression clone,
C) under expression condition, any described host cells of the above-mentioned 13-15 of culture, expression bispecific antibody TPs, TPL, PTs and PTL,
D) bispecific antibody described in isolated or purified.
17. a kind of composition, contains any described bispecific antibodies of above-mentioned 1-5 and pharmaceutically acceptable carrier.
18. above-mentioned 1,2,3,4,5,17 any described bispecific antibodies or preparation are in the medicine of anti-breast cancer is prepared Purposes.
19. the purposes described in above-mentioned 18, in addition to be used in combination with other antineoplastics.
In the present invention, any suitable carrier can be used, and can be pDR1, pcDNA3.1（+）, pcDNA3.1/ ZEO（+）One of, pDHFR, expression vector includes being connected with the fusion dna sequence of suitable transcription and translation regulatory sequence.
The expression of mammal or insect host cell culture systems available for the bispecific antibody of the present invention, COS, The cells such as CHO, NS0, sf9 and sf21 may be applicable to the present invention.
Available host cell is the prokaryotic containing above-mentioned carrier, can be DH5a, BL21 (DE3), one of TG1.
The preparation method that being used for disclosed in the present invention treats the anti-EebB2 bispecific antibodies of breast cancer is in expression bar Under part, above-mentioned host cell is cultivated, so that bispecific antibody is expressed, the bispecific antibody described in isolated or purified.
Bispecific antibody disclosed by the invention can be isolated and purified using the method for affinity chromatography, according to institute's profit The characteristic of affinity column, can use conventional method method elution of bound such as high-salt buffer, change PH affine Bispecific antibody on post.
Can be substantially uniform material by bispecific antibody purifying, such as in SDS-PAGE electricity using the above method It is single band in swimming.
According to a preferred embodiment of the present invention, a class bispecific antibody comprising Trastuzumab and Four kinds of the antibody protein of Pertuzumab antigen binding domain, respectively TPL、TPS、PTL、PTS 。
One class bispecific antibody comprising Trastuzumab and Pertuzumab antigen binding domain antibody be by with What lower method was obtained：
TPs/LIt is that Trastuzumab weight chain variable district (VH) and light chain variable district (VL) are passed through into overlap PCR Method be connected in series to Pertuzumab heavy chain and the N-terminal of light chain respectively via Linker.
PTs/LStructure and TPs/LIt is similar, Pertuzumab weight chain variable district (VH) and light chain variable district (VL) is logical The method for crossing overlap PCR is connected in series to Trastuzumab heavy chain and the N-terminal of light chain via Linker respectively.
Wherein above-mentioned linker points are short Linker (SL) and long Linker (LL), the interval SL and LL ammonia of weight chain variable Base acid is respectively AST and ASTKGPSVF, and the interval SL and LL amino acid of light chain variable is respectively TVA and TVAAPSVFI.
By the above-mentioned weight built, chain variable region gene by overlap PCR methods respectively with human IgG1's antibody Weight constant region of light chain connection, constructs complete heavy and light chain gene.
Above-mentioned complete weight, the light chain gene built is respectively charged into eukaryotic expression vector pcDNA3.1（+） (Invitrogen Products).Above-mentioned plasmid one reinstates liposome transfection CHO-K1 cells（ATCC）, and with containing 600 μ g/ The cell clone of the ml G418 stable expression bispecific antibody of Selective agar medium screening.Using Protein A chromatographic columns, lead to Cross affinity chromatography and purified from the supernatant of cell culture and obtain bispecific antibody.
According to a preferred embodiment of the present invention, a class bispecific antibody comprising Trastuzumab and The antibody protein of Pertuzumab antigen binding domain is TPL、TPS、PTL、PTS , competion experiment shows TPLCan be effectively competing Strive the combination for suppressing Trastuzumab and Pertuzumab and ErbB2 molecules, i.e. TPLWith Trastuzumab and Pertuzumab double specific binding activities.We determine TP using ELISA methodLAffinity, find its with Trastuzumab affinity is similar but affinity apparently higher than Pertuzumab.Pharmacokinetic studies show TPLMedicine It is similar to tradition IgG molecules for kinetic parameter, point out it that there is highly stable structure.With Trastuzumab and Pertuzumab combinations are compared, TPLThe ErbB2 heterodimers of part non-dependent and ligand-dependent can more effectively be blocked Formation.TPLAlso show than the Trastuzumab and Pertuzumab suppression ErbB2 molecules that are remarkably reinforced of combination and its under Swim the effect of signal path activation.
Utilize ErbB2 height expression breast cancer cell lines（BT-474, SK-BR-3 and HCC-1954）It is thin with ErbB2 low expressions Born of the same parents are (MDA-MB-468, MDA-MB-231 and MCF-7), and we are to Trastuzumab, Pertuzumab, Trastuzumab+ Pertuzumab and TPLGrowth in vitro inhibitory activity to tumour cell under the conditions of part non-dependent and ligand-dependent is carried out Evaluate.Test result indicates that, compared with other ErbB2 antibody, TPLEmbody the extracorporeal anti-tumor function significantly increased.Utilize BT-474, HCC-1954, MDA-MB-231 and MCF-7 breast cancer bearing mouse model of foundation, we are further to these The Anticancer effect in vivo of ErbB2 antibody is studied, test result indicates that TPLShow much stronger than other ErbB2 antibody Tumor growth inhibiting activity.
Therefore, the antibody egg of antigen binding domain of the class bispecific antibody comprising Trastuzumab and Pertuzumab It is preferably TP in vainL 。
Above-mentioned bispecific antibody or preparation are applied in the medicine for prepare anti-breast cancer, in addition to antitumor with others Drug combination.
The present invention discloses above-mentioned bispecific antibody, can constitute pharmaceutical preparation together with pharmaceutically acceptable auxiliary material Composition is so as to more stably play curative effect, and these preparations can ensure bispecific antibody amino acid core disclosed by the invention The conformation integrality of sequence, prevents it from degrading while also wanting the polyfunctional group of protected protein matter（Including but not limited to cohesion, deamination Or oxidation）.Under normal circumstances, for liquid preparation, it can generally preserve and at least stablize 1 year under the conditions of 2 °C -8 °C, for Lyophilized formulations, keep stable at least six months at 30 °C.Preparation can be the conventional suspension of pharmaceutical field, liquid drugs injection, freeze herein Deng preparation, preferably liquid drugs injection or lyophilized formulations, for the liquid drugs injection or lyophilized formulations of above-mentioned bispecific antibody disclosed by the invention, medicine Acceptable auxiliary material includes one or a combination set of surfactant, solution stabilizer, isotonic regulator and buffer solution on, its Middle surfactant includes nonionic surface active agent such as Polyoxyethylene Sorbitol Fatty Acid Esters（Polysorbas20 or 80）； poloxamer（Such as poloxamer 188）；Triton；Lauryl sodium sulfate（SDS）；Sodium Laurylsulfate；Myristyl, Asia Oil base or octadecyl methyl amimoacetic acid；Pluronics；MONAQUATTMDeng its addition should make the granulating of bispecific antibody become Gesture is minimum, and solution stabilizer can be carbohydrate, including reducing sugar and nonreducing sugar, amino acids include monosodium glutamate or Histidine, alcohols includes one or a combination set of trihydroxylic alcohol, senior sugar alcohol, propane diols, polyethylene glycol, the addition of solution stabilizer The preparation those skilled in the art eventually formed should be made to think to reach holding stable state, isotonic tune in the stable time It can be one of sodium chloride, mannitol to save agent, and buffer solution can be one of TRIS, histidine buffering liquid, phosphate buffer.
Above-mentioned preparation is the composition comprising bispecific antibody, antitumor after to animal administration including people Effect is obvious.Specifically, to tumour prevention and/or treatment is effective, can be used as antineoplastic.
Bispecific antibody and combinations thereof is being that dosage is because of disease to animal administration including people in the present invention The age of people and body weight, disease traits and seriousness, and method of administration and it is different, may be referred to the result of zoopery and a variety of Situation, total dosage is no more than certain limit.The dosage being specifically injected intravenously is 0.1~3000mg/ days.
Breast cancer of the invention signified, refers to the positive breast cancer of erbB2, including high expression erbB2 breast cancer and low Express erbB2 breast cancer.
Antineoplastic alleged by the present invention, refers to the medicine for suppressing and/or treating tumour, can include with tumour The delay of related symptoms development and/or the reduction of these severity of symptom are grown, it further comprises already present tumour Grow the mitigation of simultaneous phenomenon and prevent the appearance of other symptoms, still reduce or prevent transfer.
Bispecific antibody disclosed by the invention and combinations thereof can also be used for other antineoplastic administering drug combinations The treatment of tumour, these antineoplastics include 1, cytotoxic drug（1）Act on the medicine of DNA chemical constitutions：Alkylating agent Such as nitrogen mustards, nitrous urine class, pyrovinic acid esters；Platinum-like compounds such as cis-platinum, carboplatin and oxalic acid platinum etc.；Mitomycin （MMC）；（2）Influence the medicine of nucleic acid synthesis：Dihydrofolate reductase inhibitor such as methopterin（MTX）With Alimta etc.； Thymus nucleoside synthetase inhibitors such as fluorouracil（5FU, FT-207, capecitabine）Deng；Purine nucleosides synthetase inhibitors Such as Ismipur（6-MP）With 6-TG etc.；Ribonucleotide reductase inhibitor such as hydroxycarbamide（HU）Deng；DNA poly enzyme levels Agent such as cytarabine（Ara-C）And gemzar（Gemz）Deng；（3）Act on the medicine of transcribed nucleic acid：Selectively acting is in DNA moulds Plate, suppresses DNA dependenc RNA polymerases, so as to suppress the medicine of RNA synthesis such as：Actinomycin D, daunorubicin, adriamycin, table Adriamycin, aclacinomycin, mithramycin etc.；（4）Mainly act on the medicine of tubulin synthesis：Taxol, taxotere, length Spring flower alkali, vinorelbine, Podophyllum emodi var chinense alkali class, homoharringtonine；（5）Other Cytotoxic drugs：L-Asparaginasum mainly suppresses albumen The synthesis of matter；2nd, steroids antiestrogenic：TAM, Droloxifene, Exemestane etc.；Arimedex：Ammonia Shandong Meter Te, Lactel are grand, Letrozole, auspicious Ningde etc.；Antiandrogen：Its ammonia RH-LH agonist/antagonist of fluorine：Zoladex, enatone Deng；3rd, BRM：It is main that tumour interferon is suppressed by body's immunity；Interleukin 2；Thymus gland Peptides；4th, monoclonal antibody：Mabthera (MabThera)； Cetuximab (C225) ；Trastuzumab (Trastuzumab) Bevacizumab (Avastin) ；5th, other to include some current mechanism not clear and need the medicine further studied；Cell Differentiating inducer such as retinoids；Cell death inducer.Bispecific antibody disclosed by the invention and combinations thereof can with it is upper One or a combination set of antineoplastic stated drug combination.
Brief description of the drawings
Fig. 1 bispecific antibody structural representations.
Fig. 2 bispecific antibodies SDS-PAGE schemes.
Fig. 3 bispecific antibody competion experiments.
Fig. 4 bispecific antibodies block heterodimer.
Fig. 5 bispecific antibody disabling signal paths.
Fig. 6 bispecific antibodies in-vitro multiplication suppresses.
Fig. 7 bifunctional antibodies suppress breast cancer cell tumor formation curve.
Embodiment
The present invention is further detailed for following examples, experimental example, should not be construed as limiting the invention.It is real Applying example does not include detailed descriptions of conventional methods, and such as those are used for the method for carrier construction and plasmid, by the base of encoding proteins Method as the method for host cell is introduced for this area because of the method for carrier as being inserted into and plasmid or by plasmid In have ordinary skill personnel be it is well known that and be all described in many publications, including Sambrook, J., Fritsch, E.F. and Maniais, T.（1989） Molecular Cloning： A Laboratory Manual, 2ndEdition, Cold spring Harbor Laboratory Press.
The clone of the human antibody light and heavy chain constant region of embodiment 1. and Fc areas gene
With lymphocyte separation medium separating health human lymphocyte, carried with Trizol reagents (Invitrogen Products) Total serum IgE is taken, according to document（Cell,1980,22：197-207）And document（Nucleic Acids Research, 1982,10： 4071-4079）The sequence of report separately designs primer amplification heavy chain of antibody and light chain constant region gene, and PCR reactions are using heat Start, reaction condition：94oC 3 minutes；94oC 45 seconds, 60oC 45 seconds, 72oC 1 points 10 seconds, 30 circulation；72oC 10 minutes. PCR primer purifies through agarose gel electrophoresis and reclaims and be cloned into pGEM-T carriers（Promega Products）In, sequence verification Confirm to obtain correct clone afterwards.SEQ ID NO:1 and SEQ ID NO:2 respectively illustrate heavy chain constant region（CH）Nucleosides Acid and amino acid sequence.SEQ ID NO:3 and SEQ ID NO:4 respectively illustrate the nucleotides and ammonia of constant region of light chain (CL) Base acid sequence.Correct clone in this example is denoted as pGEM-T/CH, pGEM-T/CL.
Embodiment 2:The structure of New-type bifunctional antibody
Specific construction method：We are according to Trastuzumab [Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, et al. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA 1992;89:4285-9] and Pertuzumab [Adams CW, Allison DE, Flagella K, Presta L, Clarke J, Dybdal N, et al. Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab. Cancer Immunol Immunother 2006;55: 717-27] two antibody the series sequence of variable region and the difference of small peptide amino acid number construct respectively following four kinds it is double special Different in nature tetravalent antibody：TPs、TPL, PTs and PTL.In order to verify whether enhancing that we build antibody function is due to tetravalence and is drawn Rise, construct TTLAnd PPLTwo tetravalent antibodies.TPs/LIt is by Trastuzumab weight chain variable district (VH) and light chain variable district (VL) connected respectively Pertuzumab heavy chain and the N-terminal of light chain by the small peptide of different number amino acid, is then anti- The constant region of body.Similarly, PTs/LStructure and TPs/LIt is similar, by Pertuzumab weight chain variable district (VH) and light chain variable Connected respectively Trastuzumab heavy chain and the N-terminal of light chain by the small peptide of different number amino acid in area (VL).TTLAnd PPLFor the N-terminal for the light and weight chain that the variable region of the light and weight chain of itself is connected serially to parental antibody respectively respectively.Briefly we are by X The heavy light chain variable district of antibody is connected in series to Y antibody weight light chain variable district via Linker respectively by overlap PCR method 5 ' end.X, Y antibody represent Trastuzumab and Pertuzumab respectively, wherein linker points for short Linker (SL) and Long Linker (LL), the interval SL and LL amino acid of weight chain variable is respectively AST and ASTKGPSVF, the interval SL of light chain variable And LL amino acid is respectively TVA and TVAAPSVFI.By the weight of the heavy and light chain variable region gene built respectively with human IgG1's antibody Constant region of light chain is connected, and constructs complete heavy and light chain gene.
Above-mentioned heavy, light chain gene is respectively charged into eukaryotic expression vector pcDNA3.1 (Invitrogen Products).On State plasmid one and reinstate liposome transfection CHO-K1 cells（ATCC）, and screened with the Selective agar medium containing 600 μ g/ml G418 The cell clone of stable expression bispecific antibody.Using Protein A posts by affinity chromatography from the supernatant of cell culture Middle purifying bispecific antibody.
Build genetic engineering antibody following six：
TPS (Vtrastuzumab-SL-Vpertuzumab-constant); TPL (Vtrastuzumab-LL-Vpertuzumab- constant);
PTS (Vpertuzumab-SL-Vtrastuzumab-constant); PTL (Vpertuzumab-LL-Vtrastuzumab- constant);
TTL (Vtrastuzumab-LL-Vtrastuzumab-constant); PPL (Vpertuzumab-LL-Vpertuzumab- constant)。
Below with TPLExemplified by describe in detail bispecific antibody construction expression process：
TPLHeavy chain and light chain variable district be TPLVH and TPLVL, purpose band is reclaimed simultaneously after being synthesized through Overlap PCR It is cloned into pGEM-T carriers, screening positive clone sequencing respectively obtains pGEM-T/TPLVH（Nucleotide sequence and amino acid sequence Row such as SEQ ID NO:5 and SEQ ID NO:Shown in 6）And pGEM-T/TPLVL（Nucleotide sequence and amino acid sequence such as SEQ ID NO:7 and SEQ ID NO:Shown in 8）.PCR uses the high-fidelity amplification system of Roche companies, and reaction condition is：95˚C 5 minutes；94 C 45 seconds, 55 C45 seconds, 72 C 55 seconds, 30 circulations；72 C 7 minutes.
Using Overlap PCR methods by pGEM-T/TPLPGEM-T/TP in VHLIn VH genes and plasmid pGEM-T/CH Weight chain constant area gene connection, and be cloned into pGEM-T carriers, select after correct clone with Hind III and the digestions of EcoR I, Purpose fragment is reclaimed through agarose gel electrophoresis purifying, is carried out with the plasmid pcDNA3.1 (+) of same digestion with T4 DNA ligases Connection, is built into eukaryotic expression vector pcDNA3.1 (+) (TPLVHCH)（Nucleotide sequence and amino acid sequence such as SEQ ID NO:9 and SEQ ID NO:Shown in 10）.
Similarly, using Overlap PCR methods by pGEM-T/TPLPGEM-T/TP in VLLChain constant in VL genes Area's gene is connected, and is cloned into pGEM-T carriers, selects with Hind III and the digestions of EcoR I after correct clone, solidifying through agarose Purpose fragment is reclaimed in gel electrophoresis purifying, is connected with plasmid pcDNA3.1 (+) carrier of same digestion, is built into carrier for expression of eukaryon pcDNA3.1 (TPLVLCL)（Nucleotide sequence and amino acid sequence such as SEQ ID NO:11 and SEQ ID NO:Shown in 12）.
Similarly TPS、PTL、PTS、TTL、PPLAccording to mode is structured as described above, its sequence is respectively：
TPSSequence is as follows：
pcDNA3.1(+)(TPSVHCH)（Nucleotide sequence and amino acid sequence such as SEQ ID NO:13 and SEQ ID NO:Shown in 14）.
pcDNA3.1 (TPLVLCL)（Nucleotide sequence and amino acid sequence such as SEQ ID NO:15 and SEQ ID NO: Shown in 16）
PTLSequence is as follows：
pcDNA3.1(+)(PTLVHCH)（Nucleotide sequence and amino acid sequence such as SEQ ID NO:17 and SEQ ID NO:Shown in 18）.
pcDNA3.1 (PTLVLCL)（Nucleotide sequence and amino acid sequence such as SEQ ID NO:19 and SEQ ID NO: Shown in 20）
PTSSequence is as follows：
pcDNA3.1(+)(PTSVHCH)（Nucleotide sequence and amino acid sequence such as SEQ ID NO:21 and SEQ ID NO:Shown in 22）.
pcDNA3.1 (PTSVLCL)（Nucleotide sequence and amino acid sequence such as SEQ ID NO:23 and SEQ ID NO: Shown in 24）
TTLSequence is as follows：
pcDNA3.1(+)(TTLVHCH)（Nucleotide sequence and amino acid sequence such as SEQ ID NO:25 and SEQ ID NO:Shown in 26）.
pcDNA3.1 (TTLVLCL)（Nucleotide sequence and amino acid sequence such as SEQ ID NO:27 and SEQ ID NO: Shown in 28）.
PPLSequence is as follows：
pcDNA3.1(+)(PPLVHCH)（Nucleotide sequence and amino acid sequence such as SEQ ID NO:29 and SEQ ID NO:Shown in 30）.
pcDNA3.1 (PPLVLCL)（Nucleotide sequence and amino acid sequence such as SEQ ID NO:31 and SEQ ID NO: Shown in 32）.
With TPLExemplified by antibody transfection, 3 × 10 are inoculated with 3.5cm Tissue Culture Dish5CHO-K1 cells, cell Culture is transfected when being merged to 70%-80%：Take the μ g of plasmid 10 (plasmid pcDNA3.1 (+) (TPLVHCH) 4 μ g, plasmid pcDNA3.1 (+) (TPLVLCL) 6 μ g) and 20 μ l Lipofectamine2000 Reagent9 (Invitrogen companies productions Product) 500 μ l plasma-free DMEM mediums are dissolved in respectively, it is stored at room temperature 5 minutes, 2 kinds of liquid will mixes above, and be incubated at room temperature 20 points Clock is so that DNA- liposome complexes are formed, and the serum that contains replaced therebetween with the DMEM culture mediums of 3ml serum-frees in culture dish is trained Base is supported, then the DNA- liposome complexes of formation are added in plate, CO2Incubator culture adds 2ml containing 10% blood after 4 hours Clear DMEM complete mediums, are placed in CO2Continue to cultivate in incubator.Transfection carries out cell after 24h and changed containing 600 μ g/ml G418 Selective agar mediums screen resistance clone.Obtained high-expression clone will be screened and expand culture with serum free medium, used Protein A affinity columns（GE Products）Isolate and purify bispecific antibody TPL.By TPLDialysed with PBS, finally with purple External absorbent method is quantified.TPLStructure it is as shown in Figure 1.TP is also show in Fig. 1S、PTL、PTS、TTL、PPLStructure.
Experimental example 1：SDS-PAGE methods identify bispecific antibody molecular weight and structural intergrity
It is big that SDS-PAGE under the reducing conditions, Trastuzumab antibody and Pertuzumab antibody are rendered as molecular weight Small about 55KDa and 25KDa heavy chain and light chain bands.Double variable regions antibody TTL、PPL、TPs、PTs、TPLAnd PTLThen it is rendered as Molecular size range about 64KDa and 36KDa heavy chain and light chain bands.Under non reducing conditions, a band is presented in all antibody, Trastuzumab antibody and Pertuzumab antibody are 150KDa, Double variable regions antibody TTL、PPL、TPs、PTs、TPLAnd PTLFor 200kDa（Fig. 2）.
Experimental example 2：Bispecific antibody TPLCompetion experiment
Competion experiment shows TPLCan effective competition suppression Trastuzumab and Pertuzumab and ErbB2 molecules With reference to i.e. TPLBispecific with Trastuzumab and Pertuzumab.Though PTs has part suppression to Trastuzumab Effect, but it is unable to the combination of Competitive assays Pertuzumab and ErbB2 molecule,；Though TPs can effective competition suppression Trastuzumab and Pertuzumab combination ErbB2 molecules, but its relative affinity be markedly less than Trastuzumab and Pertuzumab；Similarly PTLAntibody also can effective competition suppress Trastuzumab and Pertuzumab combination, but its Relative affinity is slightly weaker than Trastuzumab and Pertuzumab（Fig. 3）.It is worth noting that, TPLNot only can effective competition suppression Trastuzumab and Pertuzumab processed combination, and its affinity is similar to Trastuzumab and Pertuzumab respectively （Table 1）.Therefore, we will select TPLCarry out next step experiment.
Experimental example 3：Bifunctional antibody TPLAffinity is determined
ErbB2-ECD albumen is the film outskirt of ErbB2 molecules, and the affinity of ErbB2 antibody is targetted with its detection.ELISA Method detects TPLAffinity similar to Trastuzumab affinity but apparently higher than Pertuzumab, shows changing for structure Change does not influence its affinity.The monospecific tetravalent antibody TT that we build in additionLAnd PPLAffinity respectively with its parent Antibody trastuzumab and Pertuzumab are similar（Table 1）
Table 1：Anti-ErbB 2 antibodies affinity is detected
Affinity number ELISA method readings is calculated and obtained, and data are three independent repetition experiment gained averages.
Experimental example 4：Bifunctional antibody TPLInternal pharmacokinetic parameter is determined
The pharmacokinetic parameter of antibody, Trastuzumab-biotin and Pertuzumab- are analyzed in non-compartment model Biotin competitive ELISA method determines the pharmacokinetic parameter in anti-ErbB 2 antibodies body.Experimental result is shown, in serum TPLAntibody can be competed with Trastuzumab and Pertuzumab, illustrate that it can be stabilized and function in serum It is uninfluenced；And TPLPharmacokinetic parameter it is similar to IgG molecules, illustrate the stabilization of its height in vivo（Table 2）.
Table 2：Pharmacokinetic parameter in anti-ErbB 2 antibodies Mice Body
Non- compartment model analyzes the pharmacokinetic parameter of antibody.AUC：TG-AUC, i.e. blood concentration-time curve The scope area i.e. TG-AUC surrounded between the longitudinal axis and transverse axis；t 1/2：Half-life period；CL：Clearance rate；VSS：Apparent steady state is distributed Volume
Experimental example 5：Co-immunoprecipitation experiment detection bifunctional antibody TPLErbB2 heterodimers are blocked to be formed
In order to further verify bispecific antibody TPLFunction, we detected with the mode of co-immunoprecipitation its block The effect of ErbB2 heterodimers formation.We utilize Trastuzumab sensitive cell lines BT-474 and Trastuzumab days Cell line mcf-7 detection Ttrastuzumab, Pertuzumab, the Trastuzumab+Pertuzumab of right resistance, TTL, PPL, TTL + PPLAnd TPLBlocking ErbB2 heterodimers ability.It is thin at two kinds in the case of existing without part In born of the same parents, Ttrastuzumab can more be effectively blocked the formation of ErbB2/ErbB3 heterodimers compared with Pertuzumab, with The above two can become apparent blocking its formation compared to Trastuzumab+Pertuzumab.Situation about existing in ligands, EGF Under, Pertuzumab can more be effectively blocked the formation of ErbB2/EGFR heterodimers compared with Ttrastuzumab. Trastuzumab+Pertuzumab also can preferably block the formation of the heterodimer.But exist in part HRG In the case of, Pertuzumab can more be effectively blocked the formation of ErbB2/ErbB3 heterodimers, and Ttrastuzumab is but It can not play a role, Trastuzumab+Pertuzumab do not block the formation effect of ErbB2/ErbB3 heterodimers also not Effect as performance is used alone in Pertuzumab is obvious.TTL, PPLBlock both the above heterodimer formation no matter The alone effect still shared and the monoclonal antibody of Ttrastuzumab, Pertuzumab two effect it is similar.But it is noticeable It is, in the presence of non-part and part, TPLIt can significantly block the formation (Fig. 4) of ErbB2 heterodimers.
Experimental example 6：TPLSignificantly block ErbB2 intracellular signaling pathways in Trastuzumab sensitivities and mdr cell
Intracellular downstream MAPK and AKT signal paths will be further activated after ErbB2 molecular activations.BT-474 cells are in part In the case of being not present, after antibody processing, ErbB3, MAPK and AKT phosphorylation level decreases, wherein Trastuzumab The effect of antibody is more obvious, and Trastuzumab+Pertuzumab combined effects are better than Trastuzumab antibody, but TPLThen significantly reduce the phosphorylation level of the three.In EGFR phosphorylation level detection, only TPLIt can be significantly reduced Phosphorylation level, other each antibody have not been able to suppress its phosphorylation.In the cell line MDA-MB- of Trastuzumab resistances 231 and HCC-1954, only TPLCan notable disabling signal path, other antibody only show faint effect.
Equally, we detect the ability of antibody blocking signal path activation in the presence of part.BT-474 cells Under ligands, EGF stimulation, EGFR, ErbB2 and MAPK phosphorylation level is significantly improved, and Pertuzumab is compared with Trastuzumab More efficiently its phosphorylation level of reduction.But under part HRG stimulations, Pertuzumab can significantly inhibit ErbB2, ErbB3, MAPK and AKT phosphorylation level, Trastuzumab but fail to show this effect.Trastuzumab + Pertuzumab combinations can play the effect of certain disabling signal path in the presence of part, be deposited in HRG parts The blocking effect that Trastuzumab+Pertuzumab are combined in the case of is alone not as good as Pertuzumab, but TPLBut can The phosphorylation level of each albumen in signal path is enough significantly reduced, the effect of extremely strong disabling signal path is played. The cell line HCC-1954, MDA-MB-468, MDA-MB-231 and MCF-7 of Trastuzumab resistances also obtain similar As a result (Fig. 5).
Experimental example 7：Bifunctional antibody TPLCell suppression test
7.1 TPLSignificantly inhibit the propagation of the sensitive breast cancer cells of Trastuzumabm
MTS methods have detected bispecific TPLAntibody the breast cancer cell line BT-474 and SK- sensitive to Trastuzumabm BR-3 increment inhibitory action.Simultaneously by it with Trastuzumab, Pertuzumab, Trastuzumab+ Pertuzumab, TTL、PPL、TTL+PPLInhibitory action be compared.TPLSignificantly inhibit the increasing of ErbB2 height expression BT-474 and SK-BR-3 cells Grow.It is not added with part group（NONE groups）, 100%, Trastuzumab is set to the proliferation rate that control groups are human IgG function cells And TTLThere is obvious Inhibit proliferaton to act on for cell, and it is 50% or so that inhibiting rate is similar, illustrates Trastuzumab pairs There is obvious inhibitory action in the cell line of ErbB2 height expression.Pertuzumab and PPLTo the proliferation inhibiting effect of cell not Substantially, it is 10% or so that inhibiting rate is close；Trastuzumab+ Pertuzumab are combined and TTL + PPLCombination has obvious Proliferation inhibiting effect, inhibiting rate is about 70%；TPLIn the case of with other ErbB2 antibody same concentrations, play very bright Aobvious proliferation inhibiting effect, inhibiting rate can reach 90% or so.As a result show, TPLShow than Trastuzumab and The breast cancer cell line BT-474 and SK-BR-3 for the suppression Trastuzumabm sensitivities that Pertuzumab combinations are remarkably reinforced rise in value Inhibition.
In the presence of HRG and EGF parts, control groups is only add part and a mouse IgG, and it is to be not added with part The proliferation rate of group mouse IgG cells（100%）Appreciation rate is tried to achieve for standard, HRG promotes ErbB2 and ErbB3 shapes for ErbB3 part Into heterodimer, EGF promotes ErbB2 and EGFR formation heterodimers for EGFR part, can significantly stimulate BT-474 And the propagation of SK-BR-3 cells.The Trastuzumab and TT in the case of part presenceLThere is Inhibit proliferaton effect for cell There is no non-part group substantially, illustrate that Trastuzumab can not suppress ErbB2 heterodimers very well in the case of part is present Formed.Pertuzumab and PPLTo the proliferation inhibiting effect of cell compared with Trastuzumab and TTLSubstantially, Trastuzumab+ Pertuzumab are combined and TTL + PPLCombination still has obvious proliferation inhibiting effect, but inhibiting rate is bright It is aobvious to be less than bispecific antibody TPL.Result above shows, compared with Trastuzumab and Pertuzumab combinations, TPLCan be more Effectively to suppress T rastuzumabm sensitive breast cancer cell line BT-474 and SK-BR-3 propagation (Fig. 6).
7.2 TPLSignificantly inhibit the breast cancer cell line propagation of Trastuzumabm resistances
MTS methods have detected bispecific TPLAntibody to the breast cancer cell line MDA-MB-468 of Trastuzumabm resistances, MDA-MB-231, MCF-7 and HCC-1954 growth inhibition effect, at the same by it with Trastuzumab, Pertuzumab, Trastuzumab+ Pertuzumab、TTL、PPL、TTL+PPLInhibitory action be compared.
In the case of part is not present Trastuzumab, Pertuzumab, Trastuzumab+ Pertuzumab, TTL、PPL、 TTL+PPLAntibody can not suppress the propagation of three kinds of cells, TPLAntibody remains to suppress their propagation.HRG and In the presence of EGF parts, Pertuzumab, PPL、Trastuzumab+ Pertuzumab、TTL+PPLCan be to a certain degree Suppress the effect of cell propagation, illustrate that they can suppress ErbB2 heterodimers and be formed, so that suppress cell propagation, but its Inhibitory action is significantly smaller than TPL.Compared with Trastuzumab and Pertuzumab combinations, TPLCan more effectively it suppress The breast cancer cell line propagation (Fig. 6) of Trastuzumabm resistances.
Experimental example 8：Bispecific antibody TPLInternal anti-tumor function research
The right armpit inoculation breast cancer cancer cell of each group tumor bearing nude mice has growth.IgG groups are compareed, tumour is given birth in progressive It is long, and the growth of each treatment group tumors is slowed or shut off growth in various degree.To the observation end of term, control IgG group nude mices are One's spirits are drooping, Shrunken skin, becomes thin, and is slow in action and takes off neck execution then.Tumor growth curve is drawn to the time according to tumor size.BT-474 is thin Born of the same parents' tumor-bearing mice group, Trastuzumab being capable of the growth for suppressing knurl body more effective than Pertuzumab；On the contrary, in MDA-MB-231 And in MCF-7 cell tumor-bearing mice groups, Pertuzumab more effective than Trastuzumab can delay the progress of tumour. Trastuzumab+ Pertuzumab can be good at suppress BT-474 cell tumor-bearing mice group tumours growth, but The tumor killing effect of Trastuzumab+Pertuzumab use in conjunction does not have in MDA-MB-231 and MCF-7 cell tumor-bearing mice groups There is Pertuzumab alone substantially.TTL +PPLAssociated with tumor killing effect and Trastuzumab+Pertuzumab use in conjunction Effect it is similar, TPLThe propagation of tumour can significantly be suppressed compared with other treatment group.In natural drug resistance HCC-1954 cells The weaker growth for suppressing tumour of lotus knurl experimental group, Trastuzumab and Pertuzumab, TTL +PPLAssociated with tumor killing effect It is similar to the effect of Trastuzumab+Pertuzumab use in conjunction, suppress the increasing of mouse tumor body to a certain extent It is long, but TPLThe propagation of tumour can significantly be suppressed compared with other treatment group.Result above shows TPLIt can significantly inhibit The breast cancer cell knurl body propagation (Fig. 7) of Trastuzumab resistances and sensitivity.
SEQUENCE LISTING
<110>Shanghai Zhangjiang Biological Technology Co
<120>A kind of anti-ErbB bispecific antibody for being used to treat breast cancer
<160> 32
<170> PatentIn version 3.5
<210> 1
<211> 990
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<213>It is artificial synthesized
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gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc cccgggtaaa 990
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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
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His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
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Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
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Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
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actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60
actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120
aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180
aaggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa 240
cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc 300
ttcaacaggg gagagtgt 318
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Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
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Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
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Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
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<213>It is artificial synthesized
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gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
tcctgtgcag cttctggctt caacattaaa gacacctata tacactgggt gcgtcaggcc 120
ccgggtaagg gcctggaatg ggttgcaagg atttatccta cgaatggtta tactagatat 180
gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240
ctgcagatga acagcctgcg tgctgaggac actgccgtct attattgttc tagatgggga 300
ggggacggct tctatgctat ggactactgg ggtcaaggaa ccctggtcac cgtctcctcg 360
gcctccacca agggcccatc ggtcttcgaa gttcaattag tcgagtctgg tggcggattg 420
gtacagcctg ggggttccct tcgtctctca tgtgctgcct cgggctttac tttcaccgat 480
tatacaatgg actgggtgcg ccaagcaccc ggaaaagggc tagaatgggt tgcggatgtc 540
aatccaaaca gtggtggcag catttacaat cagcgattta agggacggtt caccctgtct 600
gtagacagat ccaaaaacac tttatatttg caaatgaact cacttagggc tgaggatacc 660
gccgtgtact attgcgcacg taacctcggg ccgtcgtttt acttcgacta ttggggtcag 720
ggcacactag ttaccgtcag tagc 744
<210> 6
<211> 248
<212> PRT
<213>It is artificial synthesized
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
130 135 140
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp
145 150 155 160
Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
165 170 175
Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg
180 185 190
Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu
195 200 205
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
210 215 220
Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln
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Gly Thr Leu Val Thr Val Ser Ser
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gacatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggttacc 60
atcacctgcc gtgccagtca ggatgtgaat actgctgtag cctggtatca acagaaacca 120
ggaaaagctc cgaaactact gatttactcg gcatccttcc tctactctgg agtcccttct 180
cgcttctctg gctccagatc tgggacggat ttcactctga ccatcagcag tctgcagccg 240
gaagacttcg caacttatta ctgtcagcaa cattatacta ctcctcccac gttcggacag 300
ggtaccaagg tggagatcaa acgtactgtg gctgcaccat ctgtcttcat cgatattcaa 360
atgactcagt ctccttcctc attatcggct agtgttggtg accgtgtcac catcacatgt 420
aaagccagcc aagatgtatc tataggcgtg gcatggtatc agcaaaagcc cggaaaagcg 480
ccaaagttgc ttatttactc cgcttcatat cgctacacgg gggttccgtc gcgatttagt 540
ggtagcggct ctggaactga cttcaccctc acaatctcct cactacagcc tgaagatttt 600
gccacctatt actgccaaca gtattacata tatccctaca ctttcgggca aggtaccaaa 660
gtcgagatta agcgg 675
<210> 8
<211> 225
<212> PRT
<213>It is artificial synthesized
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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
115 120 125
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
130 135 140
Asp Val Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
145 150 155 160
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro
165 170 175
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
180 185 190
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
195 200 205
Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
210 215 220
Arg
225
<210> 9
<211> 1734
<212> DNA
<213>It is artificial synthesized
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gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
tcctgtgcag cttctggctt caacattaaa gacacctata tacactgggt gcgtcaggcc 120
ccgggtaagg gcctggaatg ggttgcaagg atttatccta cgaatggtta tactagatat 180
gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240
ctgcagatga acagcctgcg tgctgaggac actgccgtct attattgttc tagatgggga 300
ggggacggct tctatgctat ggactactgg ggtcaaggaa ccctggtcac cgtctcctcg 360
gcctccacca agggcccatc ggtcttcgaa gttcaattag tcgagtctgg tggcggattg 420
gtacagcctg ggggttccct tcgtctctca tgtgctgcct cgggctttac tttcaccgat 480
tatacaatgg actgggtgcg ccaagcaccc ggaaaagggc tagaatgggt tgcggatgtc 540
aatccaaaca gtggtggcag catttacaat cagcgattta agggacggtt caccctgtct 600
gtagacagat ccaaaaacac tttatatttg caaatgaact cacttagggc tgaggatacc 660
gccgtgtact attgcgcacg taacctcggg ccgtcgtttt acttcgacta ttggggtcag 720
ggcacactag ttaccgtcag tagcgctagc accaagggcc catcggtctt ccccctggca 780
ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac 840
ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 900
ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 960
tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc cagcaacacc 1020
aaggtggaca agagagttga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 1080
ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 1140
accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1200
gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1260
aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 1320
caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1380
gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1440
accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1500
aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1560
aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctatagcaag 1620
ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1680
gaggctctgc acaaccacta cacgcagaag agcctctccc tgtccccggg taaa 1734
<210> 10
<211> 578
<212> PRT
<213>It is artificial synthesized
<400> 10
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
130 135 140
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp
145 150 155 160
Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
165 170 175
Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg
180 185 190
Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu
195 200 205
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
210 215 220
Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln
225 230 235 240
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
245 250 255
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
260 265 270
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
275 280 285
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
290 295 300
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
305 310 315 320
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
325 330 335
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
340 345 350
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
355 360 365
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
370 375 380
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
385 390 395 400
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
405 410 415
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
420 425 430
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
435 440 445
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
450 455 460
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
465 470 475 480
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
485 490 495
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
500 505 510
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
515 520 525
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
530 535 540
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
545 550 555 560
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
565 570 575
Gly Lys
<210> 11
<211> 993
<212> DNA
<213>It is artificial synthesized
<400> 11
gacatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggttacc 60
atcacctgcc gtgccagtca ggatgtgaat actgctgtag cctggtatca acagaaacca 120
ggaaaagctc cgaaactact gatttactcg gcatccttcc tctactctgg agtcccttct 180
cgcttctctg gctccagatc tgggacggat ttcactctga ccatcagcag tctgcagccg 240
gaagacttcg caacttatta ctgtcagcaa cattatacta ctcctcccac gttcggacag 300
ggtaccaagg tggagatcaa acgtactgtg gctgcaccat ctgtcttcat cgatattcaa 360
atgactcagt ctccttcctc attatcggct agtgttggtg accgtgtcac catcacatgt 420
aaagccagcc aagatgtatc tataggcgtg gcatggtatc agcaaaagcc cggaaaagcg 480
ccaaagttgc ttatttactc cgcttcatat cgctacacgg gggttccgtc gcgatttagt 540
ggtagcggct ctggaactga cttcaccctc acaatctcct cactacagcc tgaagatttt 600
gccacctatt actgccaaca gtattacata tatccctaca ctttcgggca aggtaccaaa 660
gtcgagatta agcggactgt ggctgcacca tctgtcttca tcttcccgcc atctgatgag 720
cagttgaaat ctggaactgc ctctgttgtg tgcctgctga ataacttcta tcccagagag 780
gccaaagtac agtggaaggt ggataacgcc ctccaatcgg gtaactccca ggagagtgtc 840
acagagcagg acagcaagga cagcacctac agcctcagca gcaccctgac gctgagcaaa 900
gcagactacg agaaacacaa agtctacgcc tgcgaagtca cccatcaggg cctgagctcg 960
cccgtcacaa agagcttcaa caggggagag tgt 993
<210> 12
<211> 331
<212> PRT
<213>It is artificial synthesized
<400> 12
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
115 120 125
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
130 135 140
Asp Val Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
145 150 155 160
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro
165 170 175
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
180 185 190
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
195 200 205
Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
210 215 220
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
225 230 235 240
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
245 250 255
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
260 265 270
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
275 280 285
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
290 295 300
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
305 310 315 320
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
325 330
<210> 13
<211> 1716
<212> DNA
<213>It is artificial synthesized
<400> 13
gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
tcctgtgcag cttctggctt caacattaaa gacacctata tacactgggt gcgtcaggcc 120
ccgggtaagg gcctggaatg ggttgcaagg atttatccta cgaatggtta tactagatat 180
gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240
ctgcagatga acagcctgcg tgctgaggac actgccgtct attattgttc tagatgggga 300
ggggacggct tctatgctat ggactactgg ggtcaaggaa ccctggtcac cgtctcctcg 360
gcctccaccg aagttcaatt agtcgagtct ggtggcggat tggtacagcc tgggggttcc 420
cttcgtctct catgtgctgc ctcgggcttt actttcaccg attatacaat ggactgggtg 480
cgccaagcac ccggaaaagg gctagaatgg gttgcggatg tcaatccaaa cagtggtggc 540
agcatttaca atcagcgatt taagggacgg ttcaccctgt ctgtagacag atccaaaaac 600
actttatatt tgcaaatgaa ctcacttagg gctgaggata ccgccgtgta ctattgcgca 660
cgtaacctcg ggccgtcgtt ttacttcgac tattggggtc agggcacact agttaccgtc 720
agtagcgcta gcaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 780
tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 840
gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 900
tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 960
cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagagagtt 1020
gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 1080
gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 1140
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 1200
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1260
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1320
ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1380
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1440
gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1500
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1560
cccgtgctgg actccgacgg ctccttcttc ctctatagca agctcaccgt ggacaagagc 1620
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1680
tacacgcaga agagcctctc cctgtccccg ggtaaa 1716
<210> 14
<211> 572
<212> PRT
<213>It is artificial synthesized
<400> 14
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Glu Val Gln Leu Val
115 120 125
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
130 135 140
Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr Thr Met Asp Trp Val
145 150 155 160
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Asp Val Asn Pro
165 170 175
Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys Gly Arg Phe Thr
180 185 190
Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
195 200 205
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly
210 215 220
Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
245 250 255
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
260 265 270
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
275 280 285
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
290 295 300
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
305 310 315 320
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
325 330 335
Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
340 345 350
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
355 360 365
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
370 375 380
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
385 390 395 400
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
405 410 415
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
420 425 430
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
435 440 445
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
450 455 460
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
465 470 475 480
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
485 490 495
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
500 505 510
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
515 520 525
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
530 535 540
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
545 550 555 560
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 15
<211> 975
<212> DNA
<213>It is artificial synthesized
<400> 15
gacatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggttacc 60
atcacctgcc gtgccagtca ggatgtgaat actgctgtag cctggtatca acagaaacca 120
ggaaaagctc cgaaactact gatttactcg gcatccttcc tctactctgg agtcccttct 180
cgcttctctg gctccagatc tgggacggat ttcactctga ccatcagcag tctgcagccg 240
gaagacttcg caacttatta ctgtcagcaa cattatacta ctcctcccac gttcggacag 300
ggtaccaagg tggagatcaa acgtactgtg gctgatattc aaatgactca gtctccttcc 360
tcattatcgg ctagtgttgg tgaccgtgtc accatcacat gtaaagccag ccaagatgta 420
tctataggcg tggcatggta tcagcaaaag cccggaaaag cgccaaagtt gcttatttac 480
tccgcttcat atcgctacac gggggttccg tcgcgattta gtggtagcgg ctctggaact 540
gacttcaccc tcacaatctc ctcactacag cctgaagatt ttgccaccta ttactgccaa 600
cagtattaca tatatcccta cactttcggg caaggtacca aagtcgagat taagcggact 660
gtggctgcac catctgtctt catcttcccg ccatctgatg agcagttgaa atctggaact 720
gcctctgttg tgtgcctgct gaataacttc tatcccagag aggccaaagt acagtggaag 780
gtggataacg ccctccaatc gggtaactcc caggagagtg tcacagagca ggacagcaag 840
gacagcacct acagcctcag cagcaccctg acgctgagca aagcagacta cgagaaacac 900
aaagtctacg cctgcgaagt cacccatcag ggcctgagct cgcccgtcac aaagagcttc 960
aacaggggag agtgt 975
<210> 16
<211> 325
<212> PRT
<213>It is artificial synthesized
<400> 16
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Asp
100 105 110
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
115 120 125
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly Val
130 135 140
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
145 150 155 160
Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly Ser
165 170 175
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
180 185 190
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr
195 200 205
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
210 215 220
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
225 230 235 240
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
245 250 255
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
260 265 270
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
275 280 285
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
290 295 300
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
305 310 315 320
Asn Arg Gly Glu Cys
325
<210> 17
<211> 1734
<212> DNA
<213>It is artificial synthesized
<400> 17
gaagttcaat tagtcgagtc tggtggcgga ttggtacagc ctgggggttc ccttcgtctc 60
tcatgtgctg cctcgggctt tactttcacc gattatacaa tggactgggt gcgccaagca 120
cccggaaaag ggctagaatg ggttgcggat gtcaatccaa acagtggtgg cagcatttac 180
aatcagcgat ttaagggacg gttcaccctg tctgtagaca gatccaaaaa cactttatat 240
ttgcaaatga actcacttag ggctgaggat accgccgtgt actattgcgc acgtaacctc 300
gggccgtcgt tttacttcga ctattggggt cagggcacac tagttaccgt cagtagcgcc 360
tccaccaagg gcccatcggt cttcgaggtt cagctggtgg agtctggcgg tggcctggtg 420
cagccagggg gctcactccg tttgtcctgt gcagcttctg gcttcaacat taaagacacc 480
tatatacact gggtgcgtca ggccccgggt aagggcctgg aatgggttgc aaggatttat 540
cctacgaatg gttatactag atatgccgat agcgtcaagg gccgtttcac tataagcgca 600
gacacatcca aaaacacagc ctacctgcag atgaacagcc tgcgtgctga ggacactgcc 660
gtctattatt gttctagatg gggaggggac ggcttctatg ctatggacta ctggggtcaa 720
ggaaccctgg tcaccgtctc ctcggctagc accaagggcc catcggtctt ccccctggca 780
ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac 840
ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 900
ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 960
tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc cagcaacacc 1020
aaggtggaca agagagttga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 1080
ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 1140
accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1200
gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1260
aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 1320
caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1380
gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1440
accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1500
aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1560
aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctatagcaag 1620
ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1680
gaggctctgc acaaccacta cacgcagaag agcctctccc tgtccccggg taaa 1734
<210> 18
<211> 578
<212> PRT
<213>It is artificial synthesized
<400> 18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
145 150 155 160
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
165 170 175
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
180 185 190
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
195 200 205
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
225 230 235 240
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
245 250 255
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
260 265 270
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
275 280 285
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
290 295 300
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
305 310 315 320
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
325 330 335
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
340 345 350
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
355 360 365
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
370 375 380
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
385 390 395 400
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
405 410 415
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
420 425 430
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
435 440 445
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
450 455 460
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
465 470 475 480
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
485 490 495
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
500 505 510
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
515 520 525
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
530 535 540
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
545 550 555 560
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
565 570 575
Gly Lys
<210> 19
<211> 993
<212> DNA
<213>It is artificial synthesized
<400> 19
gatattcaaa tgactcagtc tccttcctca ttatcggcta gtgttggtga ccgtgtcacc 60
atcacatgta aagccagcca agatgtatct ataggcgtgg catggtatca gcaaaagccc 120
ggaaaagcgc caaagttgct tatttactcc gcttcatatc gctacacggg ggttccgtcg 180
cgatttagtg gtagcggctc tggaactgac ttcaccctca caatctcctc actacagcct 240
gaagattttg ccacctatta ctgccaacag tattacatat atccctacac tttcgggcaa 300
ggtaccaaag tcgagattaa gcggactgtg gctgcaccat ctgtcttcat cgacatccag 360
atgacccagt ccccgagctc cctgtccgcc tctgtgggcg atagggttac catcacctgc 420
cgtgccagtc aggatgtgaa tactgctgta gcctggtatc aacagaaacc aggaaaagct 480
ccgaaactac tgatttactc ggcatccttc ctctactctg gagtcccttc tcgcttctct 540
ggctccagat ctgggacgga tttcactctg accatcagca gtctgcagcc ggaagacttc 600
gcaacttatt actgtcagca acattatact actcctccca cgttcggaca gggtaccaag 660
gtggagatca aacgtactgt ggctgcacca tctgtcttca tcttcccgcc atctgatgag 720
cagttgaaat ctggaactgc ctctgttgtg tgcctgctga ataacttcta tcccagagag 780
gccaaagtac agtggaaggt ggataacgcc ctccaatcgg gtaactccca ggagagtgtc 840
acagagcagg acagcaagga cagcacctac agcctcagca gcaccctgac gctgagcaaa 900
gcagactacg agaaacacaa agtctacgcc tgcgaagtca cccatcaggg cctgagctcg 960
cccgtcacaa agagcttcaa caggggagag tgt 993
<210> 20
<211> 331
<212> PRT
<213>It is artificial synthesized
<400> 20
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
115 120 125
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
130 135 140
Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
145 150 155 160
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
165 170 175
Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile
180 185 190
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
195 200 205
Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
210 215 220
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
225 230 235 240
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
245 250 255
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
260 265 270
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
275 280 285
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
290 295 300
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
305 310 315 320
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
325 330
<210> 21
<211> 1716
<212> DNA
<213>It is artificial synthesized
<400> 21
gaagttcaat tagtcgagtc tggtggcgga ttggtacagc ctgggggttc ccttcgtctc 60
tcatgtgctg cctcgggctt tactttcacc gattatacaa tggactgggt gcgccaagca 120
cccggaaaag ggctagaatg ggttgcggat gtcaatccaa acagtggtgg cagcatttac 180
aatcagcgat ttaagggacg gttcaccctg tctgtagaca gatccaaaaa cactttatat 240
ttgcaaatga actcacttag ggctgaggat accgccgtgt actattgcgc acgtaacctc 300
gggccgtcgt tttacttcga ctattggggt cagggcacac tagttaccgt cagtagcgcc 360
tccaccgagg ttcagctggt ggagtctggc ggtggcctgg tgcagccagg gggctcactc 420
cgtttgtcct gtgcagcttc tggcttcaac attaaagaca cctatataca ctgggtgcgt 480
caggccccgg gtaagggcct ggaatgggtt gcaaggattt atcctacgaa tggttatact 540
agatatgccg atagcgtcaa gggccgtttc actataagcg cagacacatc caaaaacaca 600
gcctacctgc agatgaacag cctgcgtgct gaggacactg ccgtctatta ttgttctaga 660
tggggagggg acggcttcta tgctatggac tactggggtc aaggaaccct ggtcaccgtc 720
tcctcggcta gcaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 780
tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 840
gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 900
tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 960
cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagagagtt 1020
gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 1080
gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 1140
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 1200
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1260
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1320
ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1380
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1440
gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1500
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1560
cccgtgctgg actccgacgg ctccttcttc ctctatagca agctcaccgt ggacaagagc 1620
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1680
tacacgcaga agagcctctc cctgtccccg ggtaaa 1716
<210> 22
<211> 572
<212> PRT
<213>It is artificial synthesized
<400> 22
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Glu Val Gln Leu Val Glu
115 120 125
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
130 135 140
Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg
145 150 155 160
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr
165 170 175
Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
180 185 190
Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu
195 200 205
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp
210 215 220
Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
245 250 255
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
260 265 270
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
275 280 285
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
290 295 300
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
305 310 315 320
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
325 330 335
Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
340 345 350
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
355 360 365
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
370 375 380
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
385 390 395 400
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
405 410 415
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
420 425 430
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
435 440 445
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
450 455 460
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
465 470 475 480
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
485 490 495
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
500 505 510
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
515 520 525
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
530 535 540
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
545 550 555 560
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 23
<211> 975
<212> DNA
<213>It is artificial synthesized
<400> 23
gatattcaaa tgactcagtc tccttcctca ttatcggcta gtgttggtga ccgtgtcacc 60
atcacatgta aagccagcca agatgtatct ataggcgtgg catggtatca gcaaaagccc 120
ggaaaagcgc caaagttgct tatttactcc gcttcatatc gctacacggg ggttccgtcg 180
cgatttagtg gtagcggctc tggaactgac ttcaccctca caatctcctc actacagcct 240
gaagattttg ccacctatta ctgccaacag tattacatat atccctacac tttcgggcaa 300
ggtaccaaag tcgagattaa gcggactgtg gctgacatcc agatgaccca gtccccgagc 360
tccctgtccg cctctgtggg cgatagggtt accatcacct gccgtgccag tcaggatgtg 420
aatactgctg tagcctggta tcaacagaaa ccaggaaaag ctccgaaact actgatttac 480
tcggcatcct tcctctactc tggagtccct tctcgcttct ctggctccag atctgggacg 540
gatttcactc tgaccatcag cagtctgcag ccggaagact tcgcaactta ttactgtcag 600
caacattata ctactcctcc cacgttcgga cagggtacca aggtggagat caaacgtact 660
gtggctgcac catctgtctt catcttcccg ccatctgatg agcagttgaa atctggaact 720
gcctctgttg tgtgcctgct gaataacttc tatcccagag aggccaaagt acagtggaag 780
gtggataacg ccctccaatc gggtaactcc caggagagtg tcacagagca ggacagcaag 840
gacagcacct acagcctcag cagcaccctg acgctgagca aagcagacta cgagaaacac 900
aaagtctacg cctgcgaagt cacccatcag ggcctgagct cgcccgtcac aaagagcttc 960
aacaggggag agtgt 975
<210> 24
<211> 325
<212> PRT
<213>It is artificial synthesized
<400> 24
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Asp
100 105 110
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
115 120 125
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val
130 135 140
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
145 150 155 160
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
165 170 175
Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
180 185 190
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr
195 200 205
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
210 215 220
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
225 230 235 240
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
245 250 255
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
260 265 270
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
275 280 285
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
290 295 300
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
305 310 315 320
Asn Arg Gly Glu Cys
325
<210> 25
<211> 1737
<212> DNA
<213>It is artificial synthesized
<400> 25
gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
tcctgtgcag cttctggctt caacattaaa gacacctata tacactgggt gcgtcaggcc 120
ccgggtaagg gcctggaatg ggttgcaagg atttatccta cgaatggtta tactagatat 180
gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240
ctgcagatga acagcctgcg tgctgaggac actgccgtct attattgttc tagatgggga 300
ggggacggct tctatgctat ggactactgg ggtcaaggaa ccctggtcac cgtctcctcg 360
gcctccacca agggcccatc ggtcttcgag gttcagctgg tggagtctgg cggtggcctg 420
gtgcagccag ggggctcact ccgtttgtcc tgtgcagctt ctggcttcaa cattaaagac 480
acctatatac actgggtgcg tcaggccccg ggtaagggcc tggaatgggt tgcaaggatt 540
tatcctacga atggttatac tagatatgcc gatagcgtca agggccgttt cactataagc 600
gcagacacat ccaaaaacac agcctacctg cagatgaaca gcctgcgtgc tgaggacact 660
gccgtctatt attgttctag atggggaggg gacggcttct atgctatgga ctactggggt 720
caaggaaccc tggtcaccgt ctcctcggct agcaccaagg gcccatcggt cttccccctg 780
gcaccctcct ccaagagcac ctctgggggc acagcggccc tgggctgcct ggtcaaggac 840
tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 900
accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 960
ccctccagca gcttgggcac ccagacctac atctgcaacg tgaatcacaa gcccagcaac 1020
accaaggtgg acaagagagt tgagcccaaa tcttgtgaca aaactcacac atgcccaccg 1080
tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 1140
gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 1200
gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1260
acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1320
ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1380
ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1440
tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct gacctgcctg 1500
gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1560
aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctatagc 1620
aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1680
catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtcccc gggtaaa 1737
<210> 26
<211> 579
<212> PRT
<213>It is artificial synthesized
<400> 26
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
130 135 140
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp
145 150 155 160
Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
165 170 175
Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser
180 185 190
Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
195 200 205
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
210 215 220
Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly
225 230 235 240
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
245 250 255
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
260 265 270
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
275 280 285
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
290 295 300
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
305 310 315 320
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
325 330 335
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
340 345 350
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
355 360 365
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
370 375 380
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
385 390 395 400
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
405 410 415
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
420 425 430
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
435 440 445
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
450 455 460
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
465 470 475 480
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
485 490 495
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
500 505 510
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
515 520 525
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
530 535 540
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
545 550 555 560
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
565 570 575
Pro Gly Lys
<210> 27
<211> 993
<212> DNA
<213>It is artificial synthesized
<400> 27
gacatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggttacc 60
atcacctgcc gtgccagtca ggatgtgaat actgctgtag cctggtatca acagaaacca 120
ggaaaagctc cgaaactact gatttactcg gcatccttcc tctactctgg agtcccttct 180
cgcttctctg gctccagatc tgggacggat ttcactctga ccatcagcag tctgcagccg 240
gaagacttcg caacttatta ctgtcagcaa cattatacta ctcctcccac gttcggacag 300
ggtaccaagg tggagatcaa acgtactgtg gctgcaccat ctgtcttcat cgacatccag 360
atgacccagt ccccgagctc cctgtccgcc tctgtgggcg atagggttac catcacctgc 420
cgtgccagtc aggatgtgaa tactgctgta gcctggtatc aacagaaacc aggaaaagct 480
ccgaaactac tgatttactc ggcatccttc ctctactctg gagtcccttc tcgcttctct 540
ggctccagat ctgggacgga tttcactctg accatcagca gtctgcagcc ggaagacttc 600
gcaacttatt actgtcagca acattatact actcctccca cgttcggaca gggtaccaag 660
gtggagatca aacgtactgt ggctgcacca tctgtcttca tcttcccgcc atctgatgag 720
cagttgaaat ctggaactgc ctctgttgtg tgcctgctga ataacttcta tcccagagag 780
gccaaagtac agtggaaggt ggataacgcc ctccaatcgg gtaactccca ggagagtgtc 840
acagagcagg acagcaagga cagcacctac agcctcagca gcaccctgac gctgagcaaa 900
gcagactacg agaaacacaa agtctacgcc tgcgaagtca cccatcaggg cctgagctcg 960
cccgtcacaa agagcttcaa caggggagag tgt 993
<210> 28
<211> 331
<212> PRT
<213>It is artificial synthesized
<400> 28
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
115 120 125
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
130 135 140
Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
145 150 155 160
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
165 170 175
Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile
180 185 190
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
195 200 205
Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
210 215 220
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
225 230 235 240
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
245 250 255
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
260 265 270
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
275 280 285
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
290 295 300
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
305 310 315 320
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
325 330
<210> 29
<211> 1731
<212> DNA
<213>It is artificial synthesized
<400> 29
gaagttcaat tagtcgagtc tggtggcgga ttggtacagc ctgggggttc ccttcgtctc 60
tcatgtgctg cctcgggctt tactttcacc gattatacaa tggactgggt gcgccaagca 120
cccggaaaag ggctagaatg ggttgcggat gtcaatccaa acagtggtgg cagcatttac 180
aatcagcgat ttaagggacg gttcaccctg tctgtagaca gatccaaaaa cactttatat 240
ttgcaaatga actcacttag ggctgaggat accgccgtgt actattgcgc acgtaacctc 300
gggccgtcgt tttacttcga ctattggggt cagggcacac tagttaccgt cagtagcgcc 360
tccaccaagg gcccatcggt cttcgaagtt caattagtcg agtctggtgg cggattggta 420
cagcctgggg gttcccttcg tctctcatgt gctgcctcgg gctttacttt caccgattat 480
acaatggact gggtgcgcca agcacccgga aaagggctag aatgggttgc ggatgtcaat 540
ccaaacagtg gtggcagcat ttacaatcag cgatttaagg gacggttcac cctgtctgta 600
gacagatcca aaaacacttt atatttgcaa atgaactcac ttagggctga ggataccgcc 660
gtgtactatt gcgcacgtaa cctcgggccg tcgttttact tcgactattg gggtcagggc 720
acactagtta ccgtcagtag cgctagcacc aagggcccat cggtcttccc cctggcaccc 780
tcctccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 840
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 900
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 960
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 1020
gtggacaaga gagttgagcc caaatcttgt gacaaaactc acacatgccc accgtgccca 1080
gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 1140
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 1200
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 1260
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 1320
caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 1380
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 1440
ctgcccccat cccgggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 1500
ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1560
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta tagcaagctc 1620
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1680
gctctgcaca accactacac gcagaagagc ctctccctgt ccccgggtaa a 1731
<210> 30
<211> 577
<212> PRT
<213>It is artificial synthesized
<400> 30
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
145 150 155 160
Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
165 170 175
Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
180 185 190
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr
195 200 205
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
245 250 255
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
260 265 270
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
275 280 285
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
290 295 300
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
305 310 315 320
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
325 330 335
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
340 345 350
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
355 360 365
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
370 375 380
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
385 390 395 400
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
405 410 415
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
420 425 430
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
435 440 445
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
450 455 460
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
465 470 475 480
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
485 490 495
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
500 505 510
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
515 520 525
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
530 535 540
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
545 550 555 560
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
565 570 575
Lys
<210> 31
<211> 993
<212> DNA
<213>It is artificial synthesized
<400> 31
gatattcaaa tgactcagtc tccttcctca ttatcggcta gtgttggtga ccgtgtcacc 60
atcacatgta aagccagcca agatgtatct ataggcgtgg catggtatca gcaaaagccc 120
ggaaaagcgc caaagttgct tatttactcc gcttcatatc gctacacggg ggttccgtcg 180
cgatttagtg gtagcggctc tggaactgac ttcaccctca caatctcctc actacagcct 240
gaagattttg ccacctatta ctgccaacag tattacatat atccctacac tttcgggcaa 300
ggtaccaaag tcgagattaa gcggactgtg gctgcaccat ctgtcttcat cgatattcaa 360
atgactcagt ctccttcctc attatcggct agtgttggtg accgtgtcac catcacatgt 420
aaagccagcc aagatgtatc tataggcgtg gcatggtatc agcaaaagcc cggaaaagcg 480
ccaaagttgc ttatttactc cgcttcatat cgctacacgg gggttccgtc gcgatttagt 540
ggtagcggct ctggaactga cttcaccctc acaatctcct cactacagcc tgaagatttt 600
gccacctatt actgccaaca gtattacata tatccctaca ctttcgggca aggtaccaaa 660
gtcgagatta agcggactgt ggctgcacca tctgtcttca tcttcccgcc atctgatgag 720
cagttgaaat ctggaactgc ctctgttgtg tgcctgctga ataacttcta tcccagagag 780
gccaaagtac agtggaaggt ggataacgcc ctccaatcgg gtaactccca ggagagtgtc 840
acagagcagg acagcaagga cagcacctac agcctcagca gcaccctgac gctgagcaaa 900
gcagactacg agaaacacaa agtctacgcc tgcgaagtca cccatcaggg cctgagctcg 960
cccgtcacaa agagcttcaa caggggagag tgt 993
<210> 32
<211> 331
<212> PRT
<213>It is artificial synthesized
<400> 32
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
115 120 125
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
130 135 140
Asp Val Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
145 150 155 160
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro
165 170 175
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
180 185 190
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
195 200 205
Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
210 215 220
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
225 230 235 240
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
245 250 255
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
260 265 270
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
275 280 285
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
290 295 300
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
305 310 315 320
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
325 330

Claims (12)

1. a class bispecific antibody, for the antibody protein comprising Trastuzumab and Pertuzumab antigen binding domain, both The extracellular IV areas of people's ErbB2 acceptors can be combined, the extracellular II areas of ErbB2 acceptors, the anti-human ErbB2 can be combined again Molecule different epitopes bispecific antibody, is TPL, with SEQIDNO:10 and SEQIDNO:Amino acid sequence shown in 12.
2. a kind of nucleic acid molecules of separation, the bispecific antibody described in coding claim 1.
3. a kind of nucleic acid molecules of separation, the bispecific antibody TP described in coding claim 2L, with SEQIDNO:9 Hes SEQIDNO:Nucleotide sequence shown in 11.
4. a kind of carrier, the table that the series of operations containing the nucleic acid molecules described in claim 3 with the nucleic acid molecules is connected Up to regulating and controlling sequence, wherein carrier can be pDR1, pcDNA3.1 (+), pcDNA3.1/ZEO (+), one of pDHFR.
5. the carrier described in claim 4, is pcDNA3.1 (+) or pcDNA3.1/ZEO (+).
6. a kind of host cell, is eukaryotic containing the carrier described in claim 5.
7. the host cell described in claim 6, is mammalian cell.
8. the host cell described in claim 7, is Chinese hamster ovary celI.
9. a kind of method of the bispecific antibody prepared described in claim 1, this method includes：A) it is Trastuzumab is light Heavy chain variable region gene is connected on Pertuzumab antibody weight chain genes with catenation sequence, constructs TPLLight and weight chain base Cause；B) by bispecific antibody TPLWeight chain gene be cloned into eukaryotic expression vector pcDNA3.1 (+) respectively, transfect right It is required that any described host cells of 6-8, screen high-expression clone；C) under expression condition, any institutes of claim 6-8 are cultivated The host cell stated, expression bispecific antibody TPL；D) bispecific antibody described in isolated or purified.
10. a kind of composition, contains the bispecific antibody and pharmaceutically acceptable carrier described in claim 1.
11. the bispecific antibody or right described in claim 1 want the composition described in 10 preparing the medicine of anti-breast cancer In purposes.
12. the purposes described in claim 11, in addition to be used in combination with other antineoplastics.
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CN106554419A (en) * 2015-09-28 2017-04-05 上海抗体药物国家工程研究中心有限公司 Recombinate anti-HER2 bispecific antibodies, its preparation method and application
CN106729743B (en) 2015-11-23 2021-09-21 四川科伦博泰生物医药股份有限公司 anti-ErbB 2 antibody-drug conjugate, and composition, preparation method and application thereof

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