CN109942709A - The single domain antibody of anti-BCMA a kind of and its application - Google Patents

The single domain antibody of anti-BCMA a kind of and its application Download PDF

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CN109942709A
CN109942709A CN201910324452.8A CN201910324452A CN109942709A CN 109942709 A CN109942709 A CN 109942709A CN 201910324452 A CN201910324452 A CN 201910324452A CN 109942709 A CN109942709 A CN 109942709A
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CN109942709B (en
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罗敏
李光超
郭锦涛
莫文俊
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Guangzhou Bio Gene Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Abstract

The present invention relates to the single domain antibody of anti-BCMA a kind of and its applications, the anti-BCMA single domain antibody include amino acid sequence be SEQ ID NO.1-3, SEQ ID NO.4-6, SEQ ID NO.7-9, SEQ ID NO.10-12, SEQ ID NO.13-15, any one group of CDR in SEQ ID NO.16-18 or SEQ ID NO.19-21, or with SEQ ID NO.1-3, SEQ ID NO.4-6, SEQ ID NO.7-9, SEQ ID NO.10-12, SEQ ID NO.13-15, SEQ ID NO.16-18 or SEQ ID N O.19-21 any one group of CDR at least 90% identity in.In single domain antibody provided by the invention, QL197G8 is high to target cell killing-efficiency, and the burst size of cell factor IFN-γ is big, and affinity is strong, has broad application prospects and huge market value.

Description

The single domain antibody of anti-BCMA a kind of and its application
Technical field
The present invention relates to the single domain antibody of the cellular immunotherapy field of tumour more particularly to a kind of anti-BCMA and its answer With.
Background technique
B cell maturation antigen (B-cell maturation antigen, BCMA), including 184 amino acid residues, BCMA belongs to the I type transmembrane signaling proteins for lacking signal peptide, is the member of Tumor Necrosis Factor Receptors family (TNFR), Neng Gouyu Two kinds of ligands of B cell activity factor BAFF or proliferation-inducing ligand (A proliferation induced ligand, APRIL) It combines.In the normal tissue, BCMA is expressed in mature B cell and thick liquid cell surface, rises in the survival for maintaining thick liquid cell Important role, mechanism mainly includes BCMA and BAFF protein binding, and raises anti-apoptotic genes expression Bcl-2, Mcl-1 and Bclw Deng maintenance cell growth.BCMA is generally expressed in multiple myeloma cell line, has played weight to the neoplasm of myeloma cell The facilitation wanted prompts BCMA to can be used as cellular immunity of one of the target spot of CAR-T cell for Huppert's disease and controls It treats.
Huppert's disease is a kind of malignant plasma cell disease, shows as bone marrow plasma cells malignant clone hyperplasia, secretion Monoclonal immunoglobulin or its segment (M albumen) lead to the related target organ such as bone, kidney or tissue damage, common clinical Show as ostalgia, anaemia, renal insufficiency, infection etc..Huppert's disease is the second largest malignant tumour of hematological system at present, is accounted for The 10% of Malignancy, frequently-occurring disease increase year by year with advancing age in male, disease incidence, are even more in recent years Has the tendency that rejuvenation.
Chimeric antigen receptor T cell (Chimeric Antigen Receptor-T cell, CAR-T) refers to and repairs through gene After decorations, specific purpose antigen, and the T cell of continuous activation amplification can be identified with MHC non-limiting way.Biological immune cell Treatment have become operation, radiotherapy, the 4th kind for the treatment of tumour outside chemotherapy means, and future tumors will be become and treat essential hand Section.CAR-T cell adoptive therapy is most clear effective immunotherapeutic form in current cancer therapies.A large number of studies show that CAR-T cell can effectively identify tumour antigen, cause the anti-tumor immune response of specificity, significantly improve the existence of patient Situation.
Therefore it provides the single domain antibody of novel anti-BCMA a kind of and it is applied to preparation Chimeric antigen receptor, it is pre- in preparation The drug field of anti-and/or treatment and/or diagnosing tumour has broad application prospects and huge market value.
Summary of the invention
In view of the deficiencies of the prior art with the demand in market, the present invention provides the single domain antibody of anti-BCMA a kind of and its answers With the present invention obtains seven single domain antibodies by experiment screening, the Chimeric antigen receptor of targeting BCMA is prepared, wherein antibody QL197G8 is high to target cell killing-efficiency, and the burst size of cell factor IFN-γ is big, and affinity is strong, and specificity is good, has wide Application prospect and huge market value.
To achieve this purpose, the present invention adopts the following technical scheme:
A kind of single domain antibody or its antigen-binding fragment of anti-BCMA, which is characterized in that the anti-BCMA single domain antibody packet It is SEQ ID NO.1-3, SEQ ID NO.4-6, SEQ ID NO.7-9, SEQ ID NO.10-12, SEQ containing amino acid sequence Any one group of CDR in ID NO.13-15, SEQ ID NO.16-18 or SEQ ID NO.19-21, or with SEQ ID NO.1- 3、SEQ ID NO.4-6、SEQ ID NO.7-9、SEQ ID NO.10-12、SEQ ID NO.13-15、SEQ ID NO.16-18 Or any one group of CDR at least 90% identity in SEQ ID NO.19-21.
In the present invention, anti-BCMA single domain antibody includes that amino acid sequence can be and SEQ ID NO.1-3, SEQ ID NO.4-6, SEQ ID NO.7-9, SEQ ID NO.10-12, SEQ ID NO.13-15, SEQ ID NO.16-18 or SEQ ID Any one group of CDR has the CDR of at least 90% identity in NO.19-21, for example, can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity does not change the CDR region structure and active identity CDR is within that scope of the present invention.
Preferably, which is characterized in that the amino acid sequence of the antibody of the anti-BCMA is as shown in SEQ ID NO.22-28.
In the present invention, yamma (llama) is immunized with BCMA antigen extracellular region polypeptide in inventor, by anti-to a large amount of BCMA Body carries out screening analysis, and screening obtains seven single domain antibodies, and wherein antibody QL197G8 affinity is strong, and specificity is good, and they The small advantage of head, the binding site that tumor cell surface is smaller, more hidden can also be played.Targeting BCMA is prepared Chimeric antigen receptor, it is high to target cell killing-efficiency, have broad application prospects and huge market value.
Sequence is shown in Tables 1 and 2；
Table 1
Antibody Designation Amino acid sequence
QL197A11CDR1SEQ ID NO.1 RTVSTATM
QL197A11CDR2SEQ ID NO.2 AALNWSGNKSYYADS
QL197A11CDR3SEQ ID NO.3 AGPDLNYYTNYDARRYDH
QL197C9CDR1SEQ ID NO.4 HTTAINAA
QL197C9CDR2SEQ ID NO.5 LVGLIHNDGSTQYAQFA
QL197C9CDR3SEQ ID NO.6 NIDSRGVGPVWA
QL197D11CDR1SEQ ID NO.7 NIYSINRMA
QL197D11CDR2SEQ ID NO.8 VVATSLVDGTTNYGDSVKD
QL197D11CDR3SEQ ID NO.9 NVEGNRIDYAPGSRYPTHSYVEL
QL197E2CDR1SEQ ID NO.10 ASFNDYHM
QL197E2CDR2SEQ ID NO.11 FVAQIARYGAATYYARAVQ
QL197E2CDR3SEQ ID NO.12 TADRSNYYIDNALPD
QL197F4CDR1SEQ ID NO.13 RNFNSYAM
QL197F4CDR2SEQ ID NO.14 FLATISRAAGSTYYADSAK
QL197F4CDR3SEQ ID NO.15 AESWTPTTGWPPTKADEFD
QL197F7CDR1SEQ ID NO.16 FNLDDYAI
QL197F7CDR2SEQ ID NO.17 RVSCISSDGRTSHTGSAK
QL197F7CDR3SEQ ID NO.18 ERTSRLCSFLSYD
QL197G8CDR1SEQ ID NO.19 RTFSDHTL
QL197G8CDR2SEQ ID NO.20 GAISWSGGSTYYADSVS
QL197G8CDR3SEQ ID NO.21 ADDRYSDYR
Table 2
Second aspect, the present invention provide a kind of coding single domain antibody of anti-BCMA or its antigen binding as described in relation to the first aspect The nucleic acid fragment of segment.The nucleic acid fragment is as shown in SEQ ID NO.29-35, table 3 specific as follows；
Table 3
Affinity matured antibody is the conventional means in antibody technique, can be mutated by single-point completely random and combine hot spot Combination approach (PM) and section completely random mutation (BM) approach change the affinity of antibody.The former is commonly used in small size affinity Raising/minimum sequence alterations, the latter are used for significant affinity maturation.The amino of claimed 7 nano antibodies Acid lays special stress on protecting the CDR1 of each antibody, 2,3 sequences, and the sequence with above-mentioned CDR region sequence identity greater than 90%.
The third aspect, the present invention provide a kind of carrier, and the carrier includes at least one copy as described in second aspect Nucleic acid fragment.
Fourth aspect, the present invention provide a kind of host cell, and the host cell includes carrier described in the third aspect.
5th aspect, the present invention provide a kind of Chimeric antigen receptor, and the extracellular domain is to resist as described in relation to the first aspect The antibody of BCMA.
Preferably, the amino acid sequence of the Chimeric antigen receptor is as shown in SEQ ID NO.36-42.
The sequence is shown in Table 4；
Table 4
The nucleic acid sequence of the Chimeric antigen receptor is as shown in table 5 below；
Table 5
6th aspect, the present invention provides a kind of T cell, passes through its volume using the Chimeric antigen receptor as described in terms of the 5th The nucleic acid sequence of code, which is transfected into T cell, to be expressed.
Preferably, the mode of the transfection is by viral vectors and/or eukaryotic expression plasmids to T cell, preferably To be transfected into T cell by viral vectors.
In the present invention, the T cell has good targeting killing effect, while can discharge low dosage immune factor, Has the high immunologic cytotoxicity reaction property of hypotoxicity.
7th aspect, the present invention provides a kind of recombinant slow virus, by comprising as described in the third aspect carrier and packaging it is auxiliary The recombinant slow virus for helping plasmid co-transfection mammalian cell to obtain.
Preferably, the mammalian cell is any one in 293 cells, 293T cell or 293F cell or at least two Combination.
Eighth aspect, the present invention provide a kind of pharmaceutical composition, and described pharmaceutical composition includes as described in relation to the first aspect Nucleic acid described in single domain antibody or its antigen-binding fragment, second aspect, the carrier as described in the third aspect, such as fourth aspect institute The host cell stated, the Chimeric antigen receptor as described in terms of the 5th, the T cell as described in terms of the 6th or such as institute in terms of the 7th In the recombinant slow virus stated any one or at least two combination.
It optionally, further include pharmaceutically acceptable carrier and/or excipient.
It preferably, also include a kind of or at least two chemotherapeutics.
9th aspect, single domain antibody or its antigen-binding fragment, the core as described in second aspect as described in relation to the first aspect Acid, the carrier as described in the third aspect, the host cell as described in fourth aspect, the chimeric antigen as described in terms of the 5th by Body, the T cell as described in terms of the 6th, the recombinant slow virus as described in terms of the 7th or the pharmaceutical composition as described in eighth aspect Application of the object in the drug of preparation prevention and/or treatment and/or diagnosing tumour.
According to the present invention, the tumour is disease relevant to the expression of B cell maturation antigen protein, such as multiple bone Myeloma, Hodgkin lymphoma, leukaemia or glioblastoma.
Compared with prior art, the invention has the following beneficial effects:
In the present invention, yamma (llama) is immunized with BCMA antigen extracellular region polypeptide in inventor, by anti-to a large amount of BCMA Body carries out screening analysis, and screening obtains seven single domain antibodies, and wherein antibody QL197G8 affinity is strong, and specificity is good, and they The small advantage of head, the binding site that tumor cell surface is smaller, more hidden can also be played.Targeting BCMA is prepared Chimeric antigen receptor, high to target cell killing-efficiency, the burst size of cell factor IFN-γ is big, has broad application prospects With huge market value.
Detailed description of the invention
Fig. 1 is CAR structural schematic diagram of the invention；
Fig. 2 is pLVX-EF1-MCS Vector map of the invention of the invention；
Fig. 3 is the killing-efficiency figure of CAR-T cell of the invention to target cell；
Fig. 4 is the release conditions of cell factor IFN-γ after CAR-T cell and target cell of the invention are incubated for altogether；
Fig. 5 (A)-Fig. 5 (B) is SDS-PAGE electrophoresis result figure of the invention；
Fig. 6 (A)-Fig. 6 (F) is anti-human BCMA nano antibody affinity analysis result of the invention；
Fig. 7 is the ELISA testing result of the binding ability of 7 anti-BCMA nano antibodies and BCMA of the invention.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below in conjunction with attached drawing and by specific real Mode to further illustrate the technical scheme of the present invention is applied, but the present invention is not limited in scope of embodiments.
In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art, Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from The conventional products of acquisition.
1 antibody screening of embodiment
Two yammas (llama) are immune with BCMA extracellular region Antigenic Peptide, and 1 is immune with free peptide (QL197), and another 1 It is immune with hemocyanin (KLH) coupling peptide (QL198).QL197llama generates high titre antibody, obtains the single core of its peripheral blood Cell (PBMC) is separated for VHH library construction and antibody cloning, and QL198 alpaca does not generate good antibody and is terminated；
(1) QL197 and QL198 titre is measured by BCMA-free peptide, BSA and protein:
It is diluted to 1:10 000-1:100 ten thousand, is exempted from antibody titer, envelope antigen, serum in ELISA method measurement antiserum Serum before epidemic disease inoculation starts is used as control, is detected with the anti-alpaca antibody of goat (heavy chain+light chain) and tmb substrate of HRP coupling The alpaca antibody of capture.Since titre is bad, the experiment of QL198 is terminated；
(2) the PBMC cell quantity in QL197VHH single domain antibody library: 3.2 × 10e8；
(3) total serum IgE purifies: purifying PBMC total serum IgE, silica gel spin column method purifying using phenol/chloroform extraction；With nothing The H of RNA enzyme2O elutes total serum IgE；Using the matter of (non denatured) the evaluation RNA of OD260/280 ratio (2.0) and agarose gel electrophoresis Amount；RNA concentration is estimated with the formula of 1.0OD260=40 μ g/mL；
(4) cDNA is synthesized: according to the scheme of QoolAbs company, the U.S., using the special reverse primer of llama IgG heavy chain Start total serum IgE, using the quality of heavy chain specific primers PCR detection cDNA；
(5) the VHH product of expected size library construction: is amplified from cDNA；
Ago-Gel separates VH and VHH, and VHH product is further purified, and is modified with the site sfil, is cloned into pADL20c In phage display vector, the DNA after ligation is converted into TG1 cell, which obtains 1.3 × 10e9 independent cloning altogether.
Note: due to the great diversity of frame 4 and hinge, VHH insertion piece is divided into two parts: one end on FR4, generate~ 420bp product；The other end is after hinge, generation~580bp product.
Library size: 1.3 × 10e9 independent cloning；
Specific colony screening: Qoolabs phage selection scheme is used, screens phage display technology on people's BCMA coated panel Show antibody library, screened by 4 wheels, 95 clones of every screening select positive colony individual at random.
The culture pyrolysis product of primary ELISA method detection R4 clone, ELISA envelope antigen behaviour BCMA are shown in Table 6；
Table 6
Verification mark clone culture pyrolysis product ELISA as a result, ELISA envelope antigen with BCMA free peptide, people BCMA, HSA is negative control, is shown in Table 7；
Table 7
In flat lining out, each clone takes 2 single colonies to carry out ELISA verifying, is then sequenced all clones.
Positive colony culture pyrolysis product ELISA is verified before sequencing as a result, ELISA envelope antigen is with BCMA free peptide, people BCMA, HSA are negative control, are shown in Table 8；
Table 8
7 different positive colonies of sequence are obtained, DNA sequence dna is shown in SEQ ID NO.29-35；
Embodiment 2: the design of Chimeric antigen receptor
The present embodiment constructs the Chimeric antigen receptor of anti-BCMA, and structural schematic diagram is shown in Fig. 1, and Chimeric antigen receptor includes one The signal peptide sequence (Leader) of section CD8 α, the single domain antibody sequence (Anti-BCMA VHH) in conjunction with BCMA antigentic specificity, The hinge area (Hinge) and transmembrane domain (Transmembrane) of CD8 α, 4-1BB costimulation domain sequence and CD3 ζ signal pass Domain sequence is led, specific each section sequence is as follows:
The amino acid sequence (SEQ ID NO.50) of CD8 alpha signal peptide (leader): MALPVTALLLPLALLLHAARP；
The nucleotide sequence (SEQ ID NO.51) of CD8 alpha signal peptide (leader): ATGGCACTGCCAGTGACAGCCCT GCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCT；
The amino acid sequence (SEQ ID NO.52) of CD8 α hinge area (hinge): TTTPAPRPPTPAPTIASQPLSLRP EACRPAAGGAVHTRGLDFACD；
The nucleotide sequence (SEQ ID NO.53) of CD8 α hinge area (hinge): ACCACGACGCCAGCGCCGCGACCA CCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCG CAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT；
The amino acid sequence (SEQ ID NO.54) of CD8 α transmembrane region (TM): IYIWAPLAGTCGVLLLSLVITLYC；
The nucleotide sequence (SEQ ID NO.55) of CD8 α transmembrane region (TM): ATCTACATCTGGGCGCCCTTGGCCGGG ACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC；
The amino acid sequence (SEQ ID NO.56) of 4-1BB costimulation domain (ICD) intracellular: KRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCEL；
The nucleotide sequence (SEQ ID NO.57) of 4-1BB costimulation domain (ICD) intracellular: AAGAGAGGCAGGAAGAAGC TGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTT CCCAGAGGAGGAGGAGGGAGGATGTGAGCTG；
The amino acid sequence (SEQ ID NO.58) in CD3 ζ signal transduction domain: RVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR；
The nucleotide sequence (SEQ ID NO.59) in CD3 ζ signal transduction domain: AGGGTGAAGTTTTCTCGGAGCGCCGAT GCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGC TGGATAAGCGGAGAGGCAGAGATCCCGAGATGGGAGGCAAGCCAAGGAGGAAGAACCCTCAGGAGGGCCTGTATAA TGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGA CACGATGGCCTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCCCTGCCAC CTAGG.
Embodiment 3: the Chimeric antigen receptor expression vector of anti-BCMA is constructed
(1) according to the nucleic acid sequence of CAR gene, in Guangzhou, Ai Ji Bioisystech Co., Ltd carries out full genome synthesis；
(2) the CAR gene and empty carrier pLVX-EF1-MCS synthesized with EcoRI and BamHI double digestion full genome, in 37 DEG C In water-bath after digestion 30min, DNA electrophoresis is carried out using 1.5% Ago-Gel, is then tried using the Ago-Gel of Tiangeng The processing of agent box purification and recovery；
(3) connection of pLVX-EF1-MCS carrier and CAR genetic fragment:
Linked system is as shown in table 9:
Table 9
Component Additive amount (μ l)
PLVX-EF1-MCS carrier 2(50ng)
CAR gene 10(150ng)
T4DNA connection buffer 2
T4DNA ligase (NEB) 1
dd H2O 5
In total 20
In 22 DEG C of connection 1h, connection product directly converts Stbl3 competent escherichia coli cell, takes 200 μ l converted products It is coated with the LB plate of ammonia benzyl resistance, LB plate is inverted overnight incubation in 37 DEG C of incubator.Select 3 lists the next morning at random Clone carries out bacterium colony PCR identification, and positive colony sample presentation is sequenced.
Wherein, the Vector map of the Chimeric antigen receptor Lentiviral pLVX-EF1 α-G8CAR-Km of anti-BCMA is shown in Fig. 2.
Embodiment 4: slow virus packaging
Slow virus packaging is carried out to the Lentiviral in embodiment respectively, using four pUC pUCs, specific steps It is as follows:
Gag/pol, Rev, VSV-G and structure of the present invention needed for (1) four pUC pUC expresses slow virus carrier packaging respectively The CAR expression vector built: four plasmids are carried out to transiently transfect 293T cell, DNA content is 2 μ g/mL；
(2) above-mentioned plasmid is mixed with PEI transfection reagent, is added into the DMEM of the serum-free of certain volume, after mixing Place 15 minutes, above-mentioned mixed liquor is added into the T75 culture bottle for the cell for being covered with 293T cell, is mixed gently, in 37 DEG C, 5%CO2Cell incubator culture 6h；
(3) fresh culture is replaced after 6h, continues to cultivate, and the butyric acid sodium solution of 10mM is added, after 72 hours The culture supernatant for collecting slow virus carries out purification assays.
The amplification of embodiment 5:CAR-T cell
Every volunteer adopts the whole blood of 30ml, and peripheral blood and physiological saline 1:1 are diluted, are added in centrifuge tube Ficoll, the peripheral blood after being slowly added into dilution, 1500rpm are centrifuged PBMC layers of 30min gentle aspiration and move into another centrifuge tube；
With brine PBMC more times, it is transferred to the X-VIVO culture medium (IL- of the OKT3 containing 50ng/mL, 300IU/mL 2) it is cultivated in, after PBMC separation, needs to be activated with the OKT3 containing 50ng/mL, the X-VIVO of the IL-2 of 300IU/ml, Culture medium is replaced with the X-VIVO containing 300IU/mL after 2 days to expand culture, carries out within then every two days primary count and more The X-VIVO containing 300IU/mL is changed, and cell concentration is maintained 0.5 × 106-1×106/ mL is observed continuously 10 days.
Embodiment 6: slow-virus infection T cell
It improves slow virus using RetroNectin the RetroNectin of 30 μ g is coated in the efficiency of infection of T cell In 6 orifice plates, it is put in 37 DEG C of cell incubator 2h；RetroNectin is drawn, closes packet using Hank ' the s solution containing 2.5%BSA 6 orifice plates by after are put in 37 DEG C of cell incubator 0.5h；Confining liquid is drawn, Hank ' the s solution washing 6 containing 2%Hepes is utilized X-VIVO culture medium is added in orifice plate, and suitable slow virus solution is added, and 2000g is centrifuged 2h；Supernatant is abandoned, is added 1 × 106T Cell (the CD3 positive > 90%), 1000g is centrifuged 10min, in 37 DEG C, 5%CO2It is cultivated in the cell incubator of certain humidity, Second day repeats the above process.
Embodiment 7: killing-efficiency detection
Infection 7-10 days after progress cytotoxicity test experience, and use blank control NC (T cell of untransfected) as pair According to, the specific steps are as follows:
Using LDH detection CAR-T cell to the toxicity of K562 and K562-BCMA (the K562 cell for stablizing expression BCMA)；
After the centrifugation of various cells, with serum-free without phenol red, RPMI1640 culture medium counts after repeatedly washing, take 1 × 106Each 50 μ L of K562 and K562-BCMA cell, be plated in 96 orifice plates, as target cell, by target cell: effector cell= 1:1 be separately added into untransfected T cell and each CAR-T cell, in 37 DEG C, 5%CO2It is trained in the cell incubator of certain humidity Support 12h；Lysate is added as positive control, then 250g is centrifuged 5min, and every hole takes 100 μ L culture supernatants, new 96 are added In orifice plate, 20 μ L reaction solutions are added, is put in darkroom and reacts 20-30min.
Microplate reader 590nm measurement calculates dissolution percentage according to formula:
Cytotoxicity (%)=[(experimental port-culture medium background hole)-(spontaneous LDH relief hole-culture medium of effector cell Background hole)-(the spontaneous LDH relief hole of target cell-culture medium background hole)]/[(target cell maximum LDH relief hole-volume correction Hole)-(the spontaneous LDH relief hole of target cell-culture medium background hole)] × 100%.
Specific step referring to application No. is 201510362935.5 patent " CD33 specific chimeric antigen receptor and its Using ".
CAR-T cell is shown in Fig. 3 to the killing-efficiency of target cell；
From the figure 3, it may be seen that the T cell (T mock) of untransfected CAR is to K562 and K562-BCMA all without significant killing；It is each CAR-T cell can kill the positive cell K562-BCMA of BCMA expression to some extent, and several to BCMA negative cells K562 There is no killing ability；The wherein killing rate highest of G8CAR-T.
The release of embodiment 8:IFN- γ detects
Respectively by K562 and K562-BCMA cell according to 5 × 105Cells/well is inoculated with 24 orifice plates.By every hole 5 × 105Cell It is separately added into T cell (T mock) cell of CAR-T, untransfected, it is small to co-culture 12 in incubator for supplement culture solution to 1.5mL Shi Hou；Using people's IFN-γ ELISA detection kit (Xin Bosheng biology), detected that (specific steps are shown in supernatant is co-cultured ELISA detection kit specification).The release conditions of cell factor IFN-γ are shown in figure after CAR-T cell and target cell are incubated for altogether 4。
As shown in Figure 4: the T cell (T mock) of untransfected CAR is hardly secreted after co-culturing with K562 and K562-BCMA IFN-γ；After each CAR-T cell and K562-BCMA are co-cultured, the IFN-γ of various concentration can be detected in supernatant, and with The release of few cell factor IFN-γ after BCMA negative cells K562 is co-cultured.
The test of 9 affinity of embodiment
By 7 alpaca antibody sequences of table 2 together with IgG leader peptide sequence (MAVVLAALLQSVQA), the IgG2b of llama Hinge legion sequence (epktpkpqpqpqpqpnptteskcpkcpapellggps) and His sequence label (HHHHHH), carry out After codon optimization on building to pcDNA4/His B carrier, plasmid is obtained.
Sequence explanation:
>QL197A11SEQ ID NO.60
MAVVLAALLQSVQAqvllvesggglvqaggslrlscavsgrtvstatmgwfrqapgkerefvaalnws gnksyyadsvkgrfaisrdeakntvylqmnslkpedtavyhcaagpdlnyytnydarrydhwgqgtqvtvssepkt pkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH
>QL197C9SEQ ID NO.61
MAVVLAALLQSVQAqgqyvesgggsvqageslrlscigshttainaagwyrqtpgkqrelvglihndg stqyaqfakgrftisrddakdavylqmnslkvedtgvyycnidsrgvgpvwahwgqgtqvtvssepktpkpqpqpq pqpnptteskcpkcpapellggpshhhhhh
>QL197D11SEQ ID NO.62
MAVVLAALLQSVQAqaqlkesgggsvrtgeslrlsceasgniysinrmawyrqvsgmqrevvatslvd gttnygdsvkdrftvsrdnakkmvflqmnslepadtgvyycnvegnridyapgsrypthsyvelwgqgtqvtvsse pktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH
>QL197E2SEQ ID NO.63
MAVVLAALLQSVQAqvqlvenggglvqpggslrlscaasgasfndyhmgwfrqapgqerkfvaqiary gaatyyaravqgrftisvddakntvylqmngltpddtgvyyctadrsnyyidnalpdywgqgtqvtvssepktpkp qpqpqpqpnptteskcpkcpapellggpsHHHHHH
>QL197F4SEQ ID NO.64
MAVVLAALLQSVQAqvqliesggglvqagtsltlscassgrnfnsyamgwfrqapgkereflatisra agstyyadsakgrftisrdnrkefaylqihdlkpddtavyycaaeswtpttgwpptkadefdywgqgtqvtvssep ktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH
>QL197F7SEQ ID NO.65
MAVVLAALLQSVQAqdrlvesgggsvqaggslklscrtsgfnlddyaigwfrqapgkerervscissd grtshtgsakgrftirsanarntvylqlnrltpedagvyfcaaertsrlcsflsydywgqgtqvtvssepktpkpq pqpqpqpnptteskcpkcpapellggpsHHHHHH
>QL197G8SEQ ID NO.66
MAVVLAALLQSVQAqvqlvesggglvqaggslrlscaasgrtfsdhtlgwfrqapgkerefvgaisws ggstyyadsvsgrftisrdkakntgylqmnslkpedtavyycaaaddrysdyrywgqgtqvtvssepktpkpqpqp qpqpnptteskcpkcpapellggpsHHHHHH
Control antibodies BBHL-Fab-VL SEQ ID NO.67:
DIVLTQSPASLAVSLGERATINCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGS GSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIK
Control antibodies BBHL-Fab-VH SEQ ID NO.68:
QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSINWVRQAPGQGLEWMGWINTETREPAYAYDFRGRFV FSLDTSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVSS
After above-mentioned plasmid order-checking is correct, 293F transient transfection is carried out, cell is harvested after 2 days, and (there may be the feelings of degradation Condition), after Ni-His affinity column affinity purification nano antibody, as a result SDS-PAGE electrophoresis detection is shown in Fig. 5 (A) and Fig. 5 (B), wherein Fig. 5 (A) be 15% reproducibility SDS-PAGE analyze 7 anti-BCMA nano antibodies molecular weight results figure, 5 (B) be 15% it is non-also Originality SDS-PAGE analyzes the molecular weight results figure of 7 anti-BCMA nano antibodies；
By Fig. 5 (A) and Fig. 5 (B) it is found that 7 antibody molecules all realize expression, there is degradation situation in C-terminal.
Using the fixed sensor of Anti-hFc, loadding human BCMA-hFc antigen, 7 kinds of antibody proteins are initially dense Degree concentration is set as 200nM, carries out affinity analysis, as a result sees Fig. 6 (A)-Fig. 6 (B) and table 10；
By Fig. 6 (A), Fig. 6 (B) and table 10 it is found that in 7 kinds of antibody QL197E2 and QL197G8 affinity can achieve with it is right According to the comparable level of BBHL-Fab, but both also need to verify whether be and Fc sections of human of combination；
Table 10
Using the fixed sensor of Anti-hFc, loadding human BCMA-hFc antigen and irrelevant antigen human PD1-hFc, according to affinity analysis above as a result, QL197E2 is set concentration as 100nm, by QL197G8 set concentration as BBHL-Fab (positive control antibodies) is set concentration as 50nm by 200nm, analyzes whether three kinds of albumen are non-specific bindings, knot Fruit sees Fig. 6 (C)-Fig. 6 (F) and table 11；
Table 11
By Fig. 6 (C)-Fig. 6 (F) and table 11 it is found that QL197E2 is the antibody combined with Fc sections of human, and QL197G8 is not It is combined with humanFc sections, subsequent research can be carried out.
We further with the binding ability (Fig. 7) of ELISA detection 7 anti-BCMA nano antibodies and BCMA, are coated with 7 respectively Kind antibody and control antibodies BBHL-Fab, concentration 0.5ug/mL, by human BCMA-hFc nothing to do with the anti-of human Fc Diluted concentration is 20ng/ml and 100ng/ml to original respectively, and Anti-human HRP colour developing is added.
As shown in Figure 7: QL197E2 is the antibody combined with Fc sections of human, and QL197G8 is specific binding, be can be used as Follow-up study.
In conclusion the present invention provides the single domain antibody of anti-BCMA a kind of and its applications, wherein QL197G8 is thin to target Born of the same parents' killing-efficiency is high, and the burst size of cell factor IFN-γ is big, and is not combined with Fc sections of human, mentions for B cell related disease For a more effective therapeutic choice.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
SEQUENCE LISTING
<110>hundred caip gene Science and Technology Ltd. of Guangzhou
<120>single domain antibody of anti-BCMA a kind of and its application
<130> 2019
<160> 68
<170> PatentIn version 3.3
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Arg Thr Val Ser Thr Ala Thr Met
1 5
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Ala Ala Leu Asn Trp Ser Gly Asn Lys Ser Tyr Tyr Ala Asp Ser
1 5 10 15
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<212> PRT
<213>artificial synthesized sequence
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Ala Gly Pro Asp Leu Asn Tyr Tyr Thr Asn Tyr Asp Ala Arg Arg Tyr
1 5 10 15
Asp His
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<212> PRT
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His Thr Thr Ala Ile Asn Ala Ala
1 5
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<212> PRT
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Leu Val Gly Leu Ile His Asn Asp Gly Ser Thr Gln Tyr Ala Gln Phe
1 5 10 15
Ala
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<212> PRT
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Asn Ile Asp Ser Arg Gly Val Gly Pro Val Trp Ala
1 5 10
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Asn Ile Tyr Ser Ile Asn Arg Met Ala
1 5
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Val Val Ala Thr Ser Leu Val Asp Gly Thr Thr Asn Tyr Gly Asp Ser
1 5 10 15
Val Lys Asp
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Asn Val Glu Gly Asn Arg Ile Asp Tyr Ala Pro Gly Ser Arg Tyr Pro
1 5 10 15
Thr His Ser Tyr Val Glu Leu
20
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Ala Ser Phe Asn Asp Tyr His Met
1 5
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Phe Val Ala Gln Ile Ala Arg Tyr Gly Ala Ala Thr Tyr Tyr Ala Arg
1 5 10 15
Ala Val Gln
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Thr Ala Asp Arg Ser Asn Tyr Tyr Ile Asp Asn Ala Leu Pro Asp
1 5 10 15
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Arg Asn Phe Asn Ser Tyr Ala Met
1 5
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Phe Leu Ala Thr Ile Ser Arg Ala Ala Gly Ser Thr Tyr Tyr Ala Asp
1 5 10 15
Ser Ala Lys
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Ala Glu Ser Trp Thr Pro Thr Thr Gly Trp Pro Pro Thr Lys Ala Asp
1 5 10 15
Glu Phe Asp
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Phe Asn Leu Asp Asp Tyr Ala Ile
1 5
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Arg Val Ser Cys Ile Ser Ser Asp Gly Arg Thr Ser His Thr Gly Ser
1 5 10 15
Ala Lys
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Glu Arg Thr Ser Arg Leu Cys Ser Phe Leu Ser Tyr Asp
1 5 10
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<400> 19
Arg Thr Phe Ser Asp His Thr Leu
1 5
<210> 20
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Gly Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
1 5 10 15
Ser
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Ala Asp Asp Arg Tyr Ser Asp Tyr Arg
1 5
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Gln Val Leu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Thr Val Ser Thr Ala
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Leu Asn Trp Ser Gly Asn Lys Ser Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Glu Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr His Cys
85 90 95
Ala Ala Gly Pro Asp Leu Asn Tyr Tyr Thr Asn Tyr Asp Ala Arg Arg
100 105 110
Tyr Asp His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 23
<211> 118
<212> PRT
<213>artificial synthesized sequence
<400> 23
Gln Gly Gln Tyr Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Ile Gly Ser His Thr Thr Ala Ile Asn Ala
20 25 30
Ala Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Leu Val Gly
35 40 45
Leu Ile His Asn Asp Gly Ser Thr Gln Tyr Ala Gln Phe Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asp Ala Val Tyr Leu Gln
65 70 75 80
Met Asn Ser Leu Lys Val Glu Asp Thr Gly Val Tyr Tyr Cys Asn Ile
85 90 95
Asp Ser Arg Gly Val Gly Pro Val Trp Ala His Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 24
<211> 129
<212> PRT
<213>artificial synthesized sequence
<400> 24
Gln Ala Gln Leu Lys Glu Ser Gly Gly Gly Ser Val Arg Thr Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Asn Ile Tyr Ser Ile Asn
20 25 30
Arg Met Ala Trp Tyr Arg Gln Val Ser Gly Met Gln Arg Glu Val Val
35 40 45
Ala Thr Ser Leu Val Asp Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys
50 55 60
Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Lys Met Val Phe Leu
65 70 75 80
Gln Met Asn Ser Leu Glu Pro Ala Asp Thr Gly Val Tyr Tyr Cys Asn
85 90 95
Val Glu Gly Asn Arg Ile Asp Tyr Ala Pro Gly Ser Arg Tyr Pro Thr
100 105 110
His Ser Tyr Val Glu Leu Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 25
<211> 123
<212> PRT
<213>artificial synthesized sequence
<400> 25
Gln Val Gln Leu Val Glu Asn Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Ser Phe Asn Asp Tyr
20 25 30
His Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Lys Phe Val
35 40 45
Ala Gln Ile Ala Arg Tyr Gly Ala Ala Thr Tyr Tyr Ala Arg Ala Val
50 55 60
Gln Gly Arg Phe Thr Ile Ser Val Asp Asp Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Gly Leu Thr Pro Asp Asp Thr Gly Val Tyr Tyr Cys
85 90 95
Thr Ala Asp Arg Ser Asn Tyr Tyr Ile Asp Asn Ala Leu Pro Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 26
<211> 128
<212> PRT
<213>artificial synthesized sequence
<400> 26
Gln Val Gln Leu Ile Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Thr
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ser Ser Gly Arg Asn Phe Asn Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu
35 40 45
Ala Thr Ile Ser Arg Ala Ala Gly Ser Thr Tyr Tyr Ala Asp Ser Ala
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Arg Lys Glu Phe Ala Tyr
65 70 75 80
Leu Gln Ile His Asp Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Glu Ser Trp Thr Pro Thr Thr Gly Trp Pro Pro Thr Lys Ala
100 105 110
Asp Glu Phe Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
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<212> PRT
<213>artificial synthesized sequence
<400> 27
Gln Asp Arg Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Arg Thr Ser Gly Phe Asn Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val
35 40 45
Ser Cys Ile Ser Ser Asp Gly Arg Thr Ser His Thr Gly Ser Ala Lys
50 55 60
Gly Arg Phe Thr Ile Arg Ser Ala Asn Ala Arg Asn Thr Val Tyr Leu
65 70 75 80
Gln Leu Asn Arg Leu Thr Pro Glu Asp Ala Gly Val Tyr Phe Cys Ala
85 90 95
Ala Glu Arg Thr Ser Arg Leu Cys Ser Phe Leu Ser Tyr Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 28
<211> 119
<212> PRT
<213>artificial synthesized sequence
<400> 28
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His
20 25 30
Thr Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Gly Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Ser Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys Asn Thr Gly Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asp Asp Arg Tyr Ser Asp Tyr Arg Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 29
<211> 378
<212> PRT
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Cys Ala Gly Gly Thr Gly Cys Thr Gly Cys Thr Gly Gly Thr Ala Gly
1 5 10 15
Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Ala Thr Thr
20 25 30
Gly Gly Thr Gly Cys Ala Gly Gly Cys Thr Gly Gly Gly Gly Gly Cys
35 40 45
Thr Cys Thr Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Thr Gly Thr Cys Thr Gly Gly Ala Cys Gly
65 70 75 80
Cys Ala Cys Cys Gly Thr Cys Ala Gly Thr Ala Cys Cys Gly Cys Ala
85 90 95
Ala Cys Cys Ala Thr Gly Gly Gly Cys Thr Gly Gly Thr Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala
115 120 125
Gly Gly Ala Gly Cys Gly Thr Gly Ala Gly Thr Thr Thr Gly Thr Cys
130 135 140
Gly Cys Ala Gly Cys Gly Cys Thr Thr Ala Ala Cys Thr Gly Gly Ala
145 150 155 160
Gly Thr Gly Gly Cys Ala Ala Thr Ala Ala Ala Thr Cys Ala Thr Ala
165 170 175
Cys Thr Ala Thr Gly Cys Thr Gly Ala Thr Thr Cys Cys Gly Thr Gly
180 185 190
Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Gly Cys Cys Ala
195 200 205
Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Gly Ala Ala Gly Cys
210 215 220
Cys Ala Ala Gly Ala Ala Cys Ala Cys Gly Gly Thr Gly Thr Ala Thr
225 230 235 240
Cys Thr Ala Cys Ala Gly Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys
245 250 255
Thr Gly Ala Ala Ala Cys Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys
260 265 270
Gly Gly Cys Cys Gly Thr Thr Thr Ala Thr Cys Ala Cys Thr Gly Thr
275 280 285
Gly Cys Ala Gly Cys Ala Gly Gly Ala Cys Cys Gly Gly Ala Thr Thr
290 295 300
Thr Ala Ala Ala Thr Thr Ala Cys Thr Ala Thr Ala Cys Cys Ala Ala
305 310 315 320
Thr Thr Ala Thr Gly Ala Cys Gly Cys Cys Cys Gly Ala Cys Gly Gly
325 330 335
Thr Ala Thr Gly Ala Cys Cys Ala Thr Thr Gly Gly Gly Gly Gly Cys
340 345 350
Ala Gly Gly Gly Gly Ala Cys Cys Cys Ala Gly Gly Thr Cys Ala Cys
355 360 365
Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala
370 375
<210> 30
<211> 354
<212> DNA
<213>artificial synthesized sequence
<400> 30
caggggcagt acgtggagtc tgggggcgga tcggtccagg ctggggagtc tctaagactc 60
tcgtgcatcg ggtctcacac tactgccatc aacgccgcgg gctggtatcg tcagactcca 120
gggaagcagc gcgaactggt cggcctgatt cacaatgatg gcagtacgca atatgcccaa 180
ttcgcgaagg gccgattcac tatttccagg gacgacgcca aggacgcggt gtacctgcaa 240
atgaacagcc taaaagttga agacacgggc gtctattatt gcaatatcga ctcgcgcgga 300
gtggggccgg tctgggccca ctggggcccg gggactcagg tcaccgtctc ctca 354
<210> 31
<211> 387
<212> DNA
<213>artificial synthesized sequence
<400> 31
caggcgcagc tgaaggagtc tgggggaggc tcggtgcgga ctggggagtc tctgcggctc 60
tcctgtgaag cctccggaaa tatctacagt atcaatagaa tggcctggta ccgccaggtt 120
tcagggatgc agcgcgaggt ggtcgcgacc agtctcgttg atggtacaac gaactatgga 180
gactccgtga aggaccgatt caccgtttcc agagacaacg ccaaaaaaat ggtgtttctg 240
cagatgaata gcctagaacc cgcggacacg ggcgtctatt attgtaatgt cgagggaaat 300
cggatcgact atgcacctgg tagtcgctac ccgacacaca gttatgtcga actttggggc 360
caggggctcg cggtcactgt ctcttca 387
<210> 32
<211> 369
<212> DNA
<213>artificial synthesized sequence
<400> 32
caggtgcaac tggtagaaaa tgggggagga ttggtgcagc ctgggggctc tctgagactc 60
tcctgtgcag cctctggtgc ctccttcaat gactatcaca tgggctggtt ccgccaggct 120
ccggggcagg agcgaaagtt tgtcgctcag attgcacggt atggtgctgc cacgtattat 180
gcgcgggccg ttcagggccg gttcaccatc tccgtcgacg acgccaagaa tacggtgtat 240
ctgcaaatga acggtttgac acctgacgac acgggcgttt attactgtac agcagatcgc 300
agcaattatt atatcgacaa tgccctgcct gattattggg gccaggggac ccaggtgacc 360
gtctcctca 369
<210> 33
<211> 384
<212> DNA
<213>artificial synthesized sequence
<400> 33
caggtgcagt tgatcgagtc tggaggagga ttggtgcagg cgggaacgtc tctgacactc 60
tcctgtgcgt cctccggacg caacttcaat agttacgcca tgggctggtt tcgccaggct 120
ccaggaaagg agcgtgaatt tctggctact attagccggg ctgctggcag cacatattat 180
gctgactccg cgaagggccg attcaccatt tctcgagaca accgcaagga attcgcgtat 240
ctacaaatac acgacctgaa acctgacgac acggccgttt attactgtgc agcagagtca 300
tggaccccga ctacgggctg gcctccgacg aaggcagatg agtttgacta ctggggccag 360
ggtacccagg tcaccgtctc ctca 384
<210> 34
<211> 366
<212> DNA
<213>artificial synthesized sequence
<400> 34
caggaccggc tcgtggaatc cgggggaggc tcggtgcagg ctggggggtc cctgaaactc 60
tcctgtcgaa cctctggatt caatctcgat gattatgcca tcggctggtt ccgccaggcc 120
ccaggaaagg agcgtgaaag ggtctcttgt attagtagtg atggccggac gtcacatacc 180
ggctccgcga agggccgatt taccatcagg agcgccaacg cccggaatac ggtgtatctc 240
caactgaatc gcctgacccc tgaagacgcg ggcgtttatt tttgtgctgc agagcggacg 300
agtcgattgt gttccttcct atcgtatgat tactggggcc aggggaccca ggtcaccgtc 360
tcctca 366
<210> 35
<211> 357
<212> DNA
<213>artificial synthesized sequence
<400> 35
caggtgcagc tggtagagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg taccttcagt gaccataccc tgggctggtt ccgccaggct 120
cccgggaagg agcgtgagtt tgtaggagct atttcctgga gtggtggtag cacatactat 180
gcagactccg tgagcggccg attcaccatc tctcgagaca aggccaagaa cacgggctat 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agcagccgac 300
gatcgctata gtgactatcg ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 36
<211> 370
<212> PRT
<213>artificial synthesized sequence
<400> 36
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Leu Leu Val Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg
35 40 45
Thr Val Ser Thr Ala Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Phe Val Ala Ala Leu Asn Trp Ser Gly Asn Lys Ser Tyr
65 70 75 80
Tyr Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Glu Ala
85 90 95
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
100 105 110
Ala Val Tyr His Cys Ala Ala Gly Pro Asp Leu Asn Tyr Tyr Thr Asn
115 120 125
Tyr Asp Ala Arg Arg Tyr Asp His Trp Gly Gln Gly Thr Gln Val Thr
130 135 140
Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
145 150 155 160
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
165 170 175
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
180 185 190
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
195 200 205
Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
210 215 220
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
225 230 235 240
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
245 250 255
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
260 265 270
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
275 280 285
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
290 295 300
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
305 310 315 320
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
325 330 335
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
340 345 350
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
355 360 365
Pro Arg
370
<210> 37
<211> 362
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<213>artificial synthesized sequence
<400> 37
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Gly Gln Tyr Val Glu Ser Gly Gly Gly Ser
20 25 30
Val Gln Ala Gly Glu Ser Leu Arg Leu Ser Cys Ile Gly Ser His Thr
35 40 45
Thr Ala Ile Asn Ala Ala Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln
50 55 60
Arg Glu Leu Val Gly Leu Ile His Asn Asp Gly Ser Thr Gln Tyr Ala
65 70 75 80
Gln Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asp
85 90 95
Ala Val Tyr Leu Gln Met Asn Ser Leu Lys Val Glu Asp Thr Gly Val
100 105 110
Tyr Tyr Cys Asn Ile Asp Ser Arg Gly Val Gly Pro Val Trp Ala His
115 120 125
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr Thr Thr Pro Ala
130 135 140
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
145 150 155 160
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
165 170 175
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
180 185 190
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
195 200 205
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
210 215 220
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
225 230 235 240
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
245 250 255
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
260 265 270
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
275 280 285
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
290 295 300
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
305 310 315 320
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
325 330 335
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
340 345 350
Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 38
<211> 373
<212> PRT
<213>artificial synthesized sequence
<400> 38
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Ala Gln Leu Lys Glu Ser Gly Gly Gly Ser
20 25 30
Val Arg Thr Gly Glu Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Asn
35 40 45
Ile Tyr Ser Ile Asn Arg Met Ala Trp Tyr Arg Gln Val Ser Gly Met
50 55 60
Gln Arg Glu Val Val Ala Thr Ser Leu Val Asp Gly Thr Thr Asn Tyr
65 70 75 80
Gly Asp Ser Val Lys Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys
85 90 95
Lys Met Val Phe Leu Gln Met Asn Ser Leu Glu Pro Ala Asp Thr Gly
100 105 110
Val Tyr Tyr Cys Asn Val Glu Gly Asn Arg Ile Asp Tyr Ala Pro Gly
115 120 125
Ser Arg Tyr Pro Thr His Ser Tyr Val Glu Leu Trp Gly Gln Gly Thr
130 135 140
Gln Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
145 150 155 160
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
165 170 175
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
180 185 190
Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val
195 200 205
Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys
210 215 220
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
225 230 235 240
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
245 250 255
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
260 265 270
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
275 280 285
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
290 295 300
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
305 310 315 320
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
325 330 335
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
340 345 350
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
355 360 365
Ala Leu Pro Pro Arg
370
<210> 39
<211> 367
<212> PRT
<213>artificial synthesized sequence
<400> 39
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Asn Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala
35 40 45
Ser Phe Asn Asp Tyr His Met Gly Trp Phe Arg Gln Ala Pro Gly Gln
50 55 60
Glu Arg Lys Phe Val Ala Gln Ile Ala Arg Tyr Gly Ala Ala Thr Tyr
65 70 75 80
Tyr Ala Arg Ala Val Gln Gly Arg Phe Thr Ile Ser Val Asp Asp Ala
85 90 95
Lys Asn Thr Val Tyr Leu Gln Met Asn Gly Leu Thr Pro Asp Asp Thr
100 105 110
Gly Val Tyr Tyr Cys Thr Ala Asp Arg Ser Asn Tyr Tyr Ile Asp Asn
115 120 125
Ala Leu Pro Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
130 135 140
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
145 150 155 160
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
165 170 175
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
180 185 190
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
195 200 205
Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
210 215 220
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
225 230 235 240
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
245 250 255
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
260 265 270
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
275 280 285
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
290 295 300
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
305 310 315 320
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
325 330 335
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
340 345 350
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360 365
<210> 40
<211> 372
<212> PRT
<213>artificial synthesized sequence
<400> 40
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Ile Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Ala Gly Thr Ser Leu Thr Leu Ser Cys Ala Ser Ser Gly Arg
35 40 45
Asn Phe Asn Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Phe Leu Ala Thr Ile Ser Arg Ala Ala Gly Ser Thr Tyr
65 70 75 80
Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Arg
85 90 95
Lys Glu Phe Ala Tyr Leu Gln Ile His Asp Leu Lys Pro Asp Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Ala Glu Ser Trp Thr Pro Thr Thr Gly Trp
115 120 125
Pro Pro Thr Lys Ala Asp Glu Phe Asp Tyr Trp Gly Gln Gly Thr Gln
130 135 140
Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
145 150 155 160
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys
165 170 175
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
180 185 190
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
195 200 205
Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys
210 215 220
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
225 230 235 240
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
245 250 255
Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
260 265 270
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
275 280 285
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
290 295 300
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
305 310 315 320
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
325 330 335
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
340 345 350
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
355 360 365
Leu Pro Pro Arg
370
<210> 41
<211> 366
<212> PRT
<213>artificial synthesized sequence
<400> 41
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Asp Arg Leu Val Glu Ser Gly Gly Gly Ser
20 25 30
Val Gln Ala Gly Gly Ser Leu Lys Leu Ser Cys Arg Thr Ser Gly Phe
35 40 45
Asn Leu Asp Asp Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Arg Val Ser Cys Ile Ser Ser Asp Gly Arg Thr Ser His
65 70 75 80
Thr Gly Ser Ala Lys Gly Arg Phe Thr Ile Arg Ser Ala Asn Ala Arg
85 90 95
Asn Thr Val Tyr Leu Gln Leu Asn Arg Leu Thr Pro Glu Asp Ala Gly
100 105 110
Val Tyr Phe Cys Ala Ala Glu Arg Thr Ser Arg Leu Cys Ser Phe Leu
115 120 125
Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr
130 135 140
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
145 150 155 160
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
165 170 175
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
180 185 190
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
195 200 205
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
210 215 220
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
225 230 235 240
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
245 250 255
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
260 265 270
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
275 280 285
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
290 295 300
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
305 310 315 320
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
325 330 335
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
340 345 350
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360 365
<210> 42
<211> 363
<212> PRT
<213>artificial synthesized sequence
<400> 42
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg
35 40 45
Thr Phe Ser Asp His Thr Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Phe Val Gly Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr
65 70 75 80
Tyr Ala Asp Ser Val Ser Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala
85 90 95
Lys Asn Thr Gly Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Ala Ala Asp Asp Arg Tyr Ser Asp Tyr Arg
115 120 125
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr Thr Thr Pro
130 135 140
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
145 150 155 160
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
165 170 175
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
180 185 190
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
195 200 205
Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
210 215 220
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
225 230 235 240
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
245 250 255
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
260 265 270
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
275 280 285
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
290 295 300
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
305 310 315 320
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
325 330 335
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
340 345 350
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 43
<211> 1086
<212> DNA
<213>artificial synthesized sequence
<400> 43
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cctcaggtgc tgctggtaga gtctggggga ggattggtgc aggctggggg ctctctgaga 120
ctctcctgtg cagtgtctgg acgcaccgtc agtaccgcaa ccatgggctg gttccgccag 180
gctccaggga aggagcgtga gtttgtcgca gcgcttaact ggagtggcaa taaatcatac 240
tatgctgatt ccgtgaaggg ccgattcgcc atctccagag acgaagccaa gaacacggtg 300
tatctacaga tgaacagcct gaaacctgag gacacggccg tttatcactg tgcagcagga 360
ccggatttaa attactatac caattatgac gcccgacggt atgaccattg ggggcagggg 420
acccaggtca ccgtctcctc actgaaacct gaggacacgg ccgtttatta ctgtgcagca 480
gccgacgatc gctatagtga ctatcgctac tggggccagg ggacccaggt caccgtctcc 540
tcaaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 600
ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg 660
ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 720
ctcctgtcac tggttatcac cctttactgc agggtgaagt tttctcggag cgccgatgca 780
ccagcatatc agcagggaca gaatcagctg tacaacgagc tgaatctggg caggcgcgag 840
gagtacgacg tgctggataa gcggagaggc agagatcccg agatgggagg caagccaagg 900
aggaagaacc ctcaggaggg cctgtataat gagctgcaga aggacaagat ggccgaggcc 960
tactctgaga tcggcatgaa gggagagcgg agaaggggca agggacacga tggcctgtat 1020
cagggcctga gcacagccac caaggacacc tacgatgcac tgcacatgca ggccctgcca 1080
cctagg 1086
<210> 44
<211> 1062
<212> DNA
<213>artificial synthesized sequence
<400> 44
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cctcaggggc agtacgtgga gtctgggggc ggatcggtcc aggctgggga gtctctaaga 120
ctctcgtgca tcgggtctca cactactgcc atcaacgccg cgggctggta tcgtcagact 180
ccagggaagc agcgcgaact ggtcggcctg attcacaatg atggcagtac gcaatatgcc 240
caattcgcga agggccgatt cactatttcc agggacgacg ccaaggacgc ggtgtacctg 300
caaatgaaca gcctaaaagt tgaagacacg ggcgtctatt attgcaatat cgactcgcgc 360
ggagtggggc cggtctgggc ccactggggc ccggggactc aggtcaccgt ctcctcactg 420
aaacctgagg acacggccgt ttattactgt gcagcagccg acgatcgcta tagtgactat 480
cgctactggg gccaggggac ccaggtcacc gtctcctcaa ccacgacgcc agcgccgcga 540
ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 600
cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac 660
atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 720
tactgcaggg tgaagttttc tcggagcgcc gatgcaccag catatcagca gggacagaat 780
cagctgtaca acgagctgaa tctgggcagg cgcgaggagt acgacgtgct ggataagcgg 840
agaggcagag atcccgagat gggaggcaag ccaaggagga agaaccctca ggagggcctg 900
tataatgagc tgcagaagga caagatggcc gaggcctact ctgagatcgg catgaaggga 960
gagcggagaa ggggcaaggg acacgatggc ctgtatcagg gcctgagcac agccaccaag 1020
gacacctacg atgcactgca catgcaggcc ctgccaccta gg 1062
<210> 45
<211> 1095
<212> DNA
<213>artificial synthesized sequence
<400> 45
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cctcaggcgc agctgaagga gtctggggga ggctcggtgc ggactgggga gtctctgcgg 120
ctctcctgtg aagcctccgg aaatatctac agtatcaata gaatggcctg gtaccgccag 180
gtttcaggga tgcagcgcga ggtggtcgcg accagtctcg ttgatggtac aacgaactat 240
ggagactccg tgaaggaccg attcaccgtt tccagagaca acgccaaaaa aatggtgttt 300
ctgcagatga atagcctaga acccgcggac acgggcgtct attattgtaa tgtcgaggga 360
aatcggatcg actatgcacc tggtagtcgc tacccgacac acagttatgt cgaactttgg 420
ggccaggggc tcgcggtcac tgtctcttca ctgaaacctg aggacacggc cgtttattac 480
tgtgcagcag ccgacgatcg ctatagtgac tatcgctact ggggccaggg gacccaggtc 540
accgtctcct caaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 600
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 660
acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 720
ggggtccttc tcctgtcact ggttatcacc ctttactgca gggtgaagtt ttctcggagc 780
gccgatgcac cagcatatca gcagggacag aatcagctgt acaacgagct gaatctgggc 840
aggcgcgagg agtacgacgt gctggataag cggagaggca gagatcccga gatgggaggc 900
aagccaagga ggaagaaccc tcaggagggc ctgtataatg agctgcagaa ggacaagatg 960
gccgaggcct actctgagat cggcatgaag ggagagcgga gaaggggcaa gggacacgat 1020
ggcctgtatc agggcctgag cacagccacc aaggacacct acgatgcact gcacatgcag 1080
gccctgccac ctagg 1095
<210> 46
<211> 1077
<212> DNA
<213>artificial synthesized sequence
<400> 46
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cctcaggtgc aactggtaga aaatggggga ggattggtgc agcctggggg ctctctgaga 120
ctctcctgtg cagcctctgg tgcctccttc aatgactatc acatgggctg gttccgccag 180
gctccggggc aggagcgaaa gtttgtcgct cagattgcac ggtatggtgc tgccacgtat 240
tatgcgcggg ccgttcaggg ccggttcacc atctccgtcg acgacgccaa gaatacggtg 300
tatctgcaaa tgaacggttt gacacctgac gacacgggcg tttattactg tacagcagat 360
cgcagcaatt attatatcga caatgccctg cctgattatt ggggccaggg gacccaggtg 420
accgtctcct cactgaaacc tgaggacacg gccgtttatt actgtgcagc agccgacgat 480
cgctatagtg actatcgcta ctggggccag gggacccagg tcaccgtctc ctcaaccacg 540
acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 600
cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 660
gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 720
ctggttatca ccctttactg cagggtgaag ttttctcgga gcgccgatgc accagcatat 780
cagcagggac agaatcagct gtacaacgag ctgaatctgg gcaggcgcga ggagtacgac 840
gtgctggata agcggagagg cagagatccc gagatgggag gcaagccaag gaggaagaac 900
cctcaggagg gcctgtataa tgagctgcag aaggacaaga tggccgaggc ctactctgag 960
atcggcatga agggagagcg gagaaggggc aagggacacg atggcctgta tcagggcctg 1020
agcacagcca ccaaggacac ctacgatgca ctgcacatgc aggccctgcc acctagg 1077
<210> 47
<211> 1092
<212> DNA
<213>artificial synthesized sequence
<400> 47
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cctcaggtgc agttgatcga gtctggagga ggattggtgc aggcgggaac gtctctgaca 120
ctctcctgtg cgtcctccgg acgcaacttc aatagttacg ccatgggctg gtttcgccag 180
gctccaggaa aggagcgtga atttctggct actattagcc gggctgctgg cagcacatat 240
tatgctgact ccgcgaaggg ccgattcacc atttctcgag acaaccgcaa ggaattcgcg 300
tatctacaaa tacacgacct gaaacctgac gacacggccg tttattactg tgcagcagag 360
tcatggaccc cgactacggg ctggcctccg acgaaggcag atgagtttga ctactggggc 420
cagggtaccc aggtcaccgt ctcctcactg aaacctgagg acacggccgt ttattactgt 480
gcagcagccg acgatcgcta tagtgactat cgctactggg gccaggggac ccaggtcacc 540
gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 600
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 660
agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 720
gtccttctcc tgtcactggt tatcaccctt tactgcaggg tgaagttttc tcggagcgcc 780
gatgcaccag catatcagca gggacagaat cagctgtaca acgagctgaa tctgggcagg 840
cgcgaggagt acgacgtgct ggataagcgg agaggcagag atcccgagat gggaggcaag 900
ccaaggagga agaaccctca ggagggcctg tataatgagc tgcagaagga caagatggcc 960
gaggcctact ctgagatcgg catgaaggga gagcggagaa ggggcaaggg acacgatggc 1020
ctgtatcagg gcctgagcac agccaccaag gacacctacg atgcactgca catgcaggcc 1080
ctgccaccta gg 1092
<210> 48
<211> 1074
<212> DNA
<213>artificial synthesized sequence
<400> 48
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cctcaggacc ggctcgtgga atccggggga ggctcggtgc aggctggggg gtccctgaaa 120
ctctcctgtc gaacctctgg attcaatctc gatgattatg ccatcggctg gttccgccag 180
gccccaggaa aggagcgtga aagggtctct tgtattagta gtgatggccg gacgtcacat 240
accggctccg cgaagggccg atttaccatc aggagcgcca acgcccggaa tacggtgtat 300
ctccaactga atcgcctgac ccctgaagac gcgggcgttt atttttgtgc tgcagagcgg 360
acgagtcgat tgtgttcctt cctatcgtat gattactggg gccaggggac ccaggtcacc 420
gtctcctcac tgaaacctga ggacacggcc gtttattact gtgcagcagc cgacgatcgc 480
tatagtgact atcgctactg gggccagggg acccaggtca ccgtctcctc aaccacgacg 540
ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 600
ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 660
tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 720
gttatcaccc tttactgcag ggtgaagttt tctcggagcg ccgatgcacc agcatatcag 780
cagggacaga atcagctgta caacgagctg aatctgggca ggcgcgagga gtacgacgtg 840
ctggataagc ggagaggcag agatcccgag atgggaggca agccaaggag gaagaaccct 900
caggagggcc tgtataatga gctgcagaag gacaagatgg ccgaggccta ctctgagatc 960
ggcatgaagg gagagcggag aaggggcaag ggacacgatg gcctgtatca gggcctgagc 1020
acagccacca aggacaccta cgatgcactg cacatgcagg ccctgccacc tagg 1074
<210> 49
<211> 1065
<212> DNA
<213>artificial synthesized sequence
<400> 49
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cctcaggtgc agctggtaga gtctggggga ggattggtgc aggctggggg ctctctgaga 120
ctctcctgtg cagcctctgg acgtaccttc agtgaccata ccctgggctg gttccgccag 180
gctcccggga aggagcgtga gtttgtagga gctatttcct ggagtggtgg tagcacatac 240
tatgcagact ccgtgagcgg ccgattcacc atctctcgag acaaggccaa gaacacgggc 300
tatctgcaaa tgaacagcct gaaacctgag gacacggccg tttattactg tgcagcagcc 360
gacgatcgct atagtgacta tcgctactgg ggccagggga cccaggtcac cgtctcctca 420
ctgaaacctg aggacacggc cgtttattac tgtgcagcag ccgacgatcg ctatagtgac 480
tatcgctact ggggccaggg gacccaggtc accgtctcct caaccacgac gccagcgccg 540
cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 600
tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatatc 660
tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 720
ctttactgca gggtgaagtt ttctcggagc gccgatgcac cagcatatca gcagggacag 780
aatcagctgt acaacgagct gaatctgggc aggcgcgagg agtacgacgt gctggataag 840
cggagaggca gagatcccga gatgggaggc aagccaagga ggaagaaccc tcaggagggc 900
ctgtataatg agctgcagaa ggacaagatg gccgaggcct actctgagat cggcatgaag 960
ggagagcgga gaaggggcaa gggacacgat ggcctgtatc agggcctgag cacagccacc 1020
aaggacacct acgatgcact gcacatgcag gccctgccac ctagg 1065
<210> 50
<211> 21
<212> PRT
<213>artificial synthesized sequence
<400> 50
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 51
<211> 63
<212> DNA
<213>artificial synthesized sequence
<400> 51
atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60
cct 63
<210> 52
<211> 45
<212> PRT
<213>artificial synthesized sequence
<400> 52
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 53
<211> 135
<212> DNA
<213>artificial synthesized sequence
<400> 53
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 54
<211> 24
<212> PRT
<213>artificial synthesized sequence
<400> 54
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 55
<211> 72
<212> DNA
<213>artificial synthesized sequence
<400> 55
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 56
<211> 42
<212> PRT
<213>artificial synthesized sequence
<400> 56
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 57
<211> 126
<212> DNA
<213>artificial synthesized sequence
<400> 57
aagagaggca ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg ccccgtgcag 60
acaacccagg aggaggacgg ctgcagctgt cggttcccag aggaggagga gggaggatgt 120
gagctg 126
<210> 58
<211> 112
<212> PRT
<213>artificial synthesized sequence
<400> 58
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 59
<211> 336
<212> DNA
<213>artificial synthesized sequence
<400> 59
agggtgaagt tttctcggag cgccgatgca ccagcatatc agcagggaca gaatcagctg 60
tacaacgagc tgaatctggg caggcgcgag gagtacgacg tgctggataa gcggagaggc 120
agagatcccg agatgggagg caagccaagg aggaagaacc ctcaggaggg cctgtataat 180
gagctgcaga aggacaagat ggccgaggcc tactctgaga tcggcatgaa gggagagcgg 240
agaaggggca agggacacga tggcctgtat cagggcctga gcacagccac caaggacacc 300
tacgatgcac tgcacatgca ggccctgcca cctagg 336
<210> 60
<211> 182
<212> PRT
<213>artificial synthesized sequence
<400> 60
Met Ala Val Val Leu Ala Ala Leu Leu Gln Ser Val Gln Ala Gln Val
1 5 10 15
Leu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu
20 25 30
Arg Leu Ser Cys Ala Val Ser Gly Arg Thr Val Ser Thr Ala Thr Met
35 40 45
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala
50 55 60
Leu Asn Trp Ser Gly Asn Lys Ser Tyr Tyr Ala Asp Ser Val Lys Gly
65 70 75 80
Arg Phe Ala Ile Ser Arg Asp Glu Ala Lys Asn Thr Val Tyr Leu Gln
85 90 95
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr His Cys Ala Ala
100 105 110
Gly Pro Asp Leu Asn Tyr Tyr Thr Asn Tyr Asp Ala Arg Arg Tyr Asp
115 120 125
His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Thr
130 135 140
Pro Lys Pro Gln Pro Gln Pro Gln Pro Gln Pro Asn Pro Thr Thr Glu
145 150 155 160
Ser Lys Cys Pro Lys Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
165 170 175
His His His His His His
180
<210> 61
<211> 174
<212> PRT
<213>artificial synthesized sequence
<400> 61
Met Ala Val Val Leu Ala Ala Leu Leu Gln Ser Val Gln Ala Gln Gly
1 5 10 15
Gln Tyr Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu Ser Leu
20 25 30
Arg Leu Ser Cys Ile Gly Ser His Thr Thr Ala Ile Asn Ala Ala Gly
35 40 45
Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Leu Val Gly Leu Ile
50 55 60
His Asn Asp Gly Ser Thr Gln Tyr Ala Gln Phe Ala Lys Gly Arg Phe
65 70 75 80
Thr Ile Ser Arg Asp Asp Ala Lys Asp Ala Val Tyr Leu Gln Met Asn
85 90 95
Ser Leu Lys Val Glu Asp Thr Gly Val Tyr Tyr Cys Asn Ile Asp Ser
100 105 110
Arg Gly Val Gly Pro Val Trp Ala His Trp Gly Gln Gly Thr Gln Val
115 120 125
Thr Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Gln Pro Gln
130 135 140
Pro Gln Pro Asn Pro Thr Thr Glu Ser Lys Cys Pro Lys Cys Pro Ala
145 150 155 160
Pro Glu Leu Leu Gly Gly Pro Ser His His His His His His
165 170
<210> 62
<211> 185
<212> PRT
<213>artificial synthesized sequence
<400> 62
Met Ala Val Val Leu Ala Ala Leu Leu Gln Ser Val Gln Ala Gln Ala
1 5 10 15
Gln Leu Lys Glu Ser Gly Gly Gly Ser Val Arg Thr Gly Glu Ser Leu
20 25 30
Arg Leu Ser Cys Glu Ala Ser Gly Asn Ile Tyr Ser Ile Asn Arg Met
35 40 45
Ala Trp Tyr Arg Gln Val Ser Gly Met Gln Arg Glu Val Val Ala Thr
50 55 60
Ser Leu Val Asp Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys Asp Arg
65 70 75 80
Phe Thr Val Ser Arg Asp Asn Ala Lys Lys Met Val Phe Leu Gln Met
85 90 95
Asn Ser Leu Glu Pro Ala Asp Thr Gly Val Tyr Tyr Cys Asn Val Glu
100 105 110
Gly Asn Arg Ile Asp Tyr Ala Pro Gly Ser Arg Tyr Pro Thr His Ser
115 120 125
Tyr Val Glu Leu Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
130 135 140
Pro Lys Thr Pro Lys Pro Gln Pro Gln Pro Gln Pro Gln Pro Asn Pro
145 150 155 160
Thr Thr Glu Ser Lys Cys Pro Lys Cys Pro Ala Pro Glu Leu Leu Gly
165 170 175
Gly Pro Ser His His His His His His
180 185
<210> 63
<211> 179
<212> PRT
<213>artificial synthesized sequence
<400> 63
Met Ala Val Val Leu Ala Ala Leu Leu Gln Ser Val Gln Ala Gln Val
1 5 10 15
Gln Leu Val Glu Asn Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
20 25 30
Arg Leu Ser Cys Ala Ala Ser Gly Ala Ser Phe Asn Asp Tyr His Met
35 40 45
Gly Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Lys Phe Val Ala Gln
50 55 60
Ile Ala Arg Tyr Gly Ala Ala Thr Tyr Tyr Ala Arg Ala Val Gln Gly
65 70 75 80
Arg Phe Thr Ile Ser Val Asp Asp Ala Lys Asn Thr Val Tyr Leu Gln
85 90 95
Met Asn Gly Leu Thr Pro Asp Asp Thr Gly Val Tyr Tyr Cys Thr Ala
100 105 110
Asp Arg Ser Asn Tyr Tyr Ile Asp Asn Ala Leu Pro Asp Tyr Trp Gly
115 120 125
Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Thr Pro Lys Pro
130 135 140
Gln Pro Gln Pro Gln Pro Gln Pro Asn Pro Thr Thr Glu Ser Lys Cys
145 150 155 160
Pro Lys Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser His His His
165 170 175
His His His
<210> 64
<211> 184
<212> PRT
<213>artificial synthesized sequence
<400> 64
Met Ala Val Val Leu Ala Ala Leu Leu Gln Ser Val Gln Ala Gln Val
1 5 10 15
Gln Leu Ile Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Thr Ser Leu
20 25 30
Thr Leu Ser Cys Ala Ser Ser Gly Arg Asn Phe Asn Ser Tyr Ala Met
35 40 45
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu Ala Thr
50 55 60
Ile Ser Arg Ala Ala Gly Ser Thr Tyr Tyr Ala Asp Ser Ala Lys Gly
65 70 75 80
Arg Phe Thr Ile Ser Arg Asp Asn Arg Lys Glu Phe Ala Tyr Leu Gln
85 90 95
Ile His Asp Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys Ala Ala
100 105 110
Glu Ser Trp Thr Pro Thr Thr Gly Trp Pro Pro Thr Lys Ala Asp Glu
115 120 125
Phe Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
130 135 140
Lys Thr Pro Lys Pro Gln Pro Gln Pro Gln Pro Gln Pro Asn Pro Thr
145 150 155 160
Thr Glu Ser Lys Cys Pro Lys Cys Pro Ala Pro Glu Leu Leu Gly Gly
165 170 175
Pro Ser His His His His His His
180
<210> 65
<211> 178
<212> PRT
<213>artificial synthesized sequence
<400> 65
Met Ala Val Val Leu Ala Ala Leu Leu Gln Ser Val Gln Ala Gln Asp
1 5 10 15
Arg Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu
20 25 30
Lys Leu Ser Cys Arg Thr Ser Gly Phe Asn Leu Asp Asp Tyr Ala Ile
35 40 45
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val Ser Cys
50 55 60
Ile Ser Ser Asp Gly Arg Thr Ser His Thr Gly Ser Ala Lys Gly Arg
65 70 75 80
Phe Thr Ile Arg Ser Ala Asn Ala Arg Asn Thr Val Tyr Leu Gln Leu
85 90 95
Asn Arg Leu Thr Pro Glu Asp Ala Gly Val Tyr Phe Cys Ala Ala Glu
100 105 110
Arg Thr Ser Arg Leu Cys Ser Phe Leu Ser Tyr Asp Tyr Trp Gly Gln
115 120 125
Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln
130 135 140
Pro Gln Pro Gln Pro Gln Pro Asn Pro Thr Thr Glu Ser Lys Cys Pro
145 150 155 160
Lys Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser His His His His
165 170 175
His His
<210> 66
<211> 175
<212> PRT
<213>artificial synthesized sequence
<400> 66
Met Ala Val Val Leu Ala Ala Leu Leu Gln Ser Val Gln Ala Gln Val
1 5 10 15
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu
20 25 30
Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His Thr Leu
35 40 45
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Gly Ala
50 55 60
Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Ser Gly
65 70 75 80
Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys Asn Thr Gly Tyr Leu Gln
85 90 95
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala
100 105 110
Ala Asp Asp Arg Tyr Ser Asp Tyr Arg Tyr Trp Gly Gln Gly Thr Gln
115 120 125
Val Thr Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Gln Pro
130 135 140
Gln Pro Gln Pro Asn Pro Thr Thr Glu Ser Lys Cys Pro Lys Cys Pro
145 150 155 160
Ala Pro Glu Leu Leu Gly Gly Pro Ser His His His His His His
165 170 175
<210> 67
<211> 111
<212> PRT
<213>artificial synthesized sequence
<400> 67
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Ser Val Ile
20 25 30
Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Thr Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ala Glu Asp Ala Ala Ile Tyr Tyr Cys Leu Gln Ser Arg
85 90 95
Ile Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 68
<211> 117
<212> PRT
<213>artificial synthesized sequence
<400> 68
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ser Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp Phe
50 55 60
Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115

Claims (10)

1. the single domain antibody or its antigen-binding fragment of a kind of anti-BCMA, which is characterized in that the anti-BCMA single domain antibody includes Amino acid sequence is SEQ ID NO.1-3, SEQ ID NO.4-6, SEQ ID NO.7-9, SEQ ID NO.10-12, SEQ ID Any one group of CDR in NO.13-15, SEQ ID NO.16-18 or SEQ ID NO.19-21, or with SEQ ID NO.1-3, SEQ ID NO.4-6, SEQ ID NO.7-9, SEQ ID NO.10-12, SEQ ID NO.13-15, SEQ ID NO.16-18 or Any one group of CDR at least 90% identity in SEQ ID NO.19-21.
2. the single domain antibody or its antigen-binding fragment of anti-BCMA according to claim 1, which is characterized in that described anti- The amino acid sequence of the antibody of BCMA is as shown in SEQ ID NO.22-28.
3. a kind of encode the single domain antibody of anti-BCMA or the nucleic acid fragment of its antigen-binding fragment as claimed in claim 1 or 2；
Preferably, the nucleic acid fragment is as shown in SEQ ID NO.29-35.
4. a kind of carrier, which is characterized in that the carrier includes the nucleic acid piece as claimed in claim 3 of at least one copy Section.
5. a kind of host cell, which is characterized in that the host cell includes carrier as claimed in claim 4.
6. a kind of Chimeric antigen receptor, which is characterized in that the extracellular domain is anti-BCMA as claimed in claim 1 or 2 Antibody；
Preferably, the amino acid sequence of the Chimeric antigen receptor is as shown in SEQ ID NO.36-42.
7. a kind of T cell, which is characterized in that the nucleic acid encoded using Chimeric antigen receptor as claimed in claim 6 by it Sequence is transfected into T cell and expresses；
Preferably, the mode of the transfection is by viral vectors and/or eukaryotic expression plasmids to T cell, preferably logical It crosses viral vectors and is transfected into T cell.
8. a kind of recombinant slow virus, which is characterized in that carrier as claimed in claim 4 and packaging helper plasmid corotation will be included The recombinant slow virus that dye mammalian cell obtains；
Preferably, the mammalian cell be 293 cells, 293T cell or 293F cell in any one or at least two group It closes.
9. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes the single domain as described in claims 1 or 2 item Antibody or its antigen-binding fragment, nucleic acid as claimed in claim 3, carrier as claimed in claim 4, such as claim 5 The host cell, Chimeric antigen receptor as claimed in claim 6, T cell as claimed in claim 7 or as right is wanted In recombinant slow virus described in asking 8 any one or at least two combination；
It optionally, further include pharmaceutically acceptable carrier and/or excipient；
It preferably, also include a kind of or at least two chemotherapeutics.
10. single domain antibody or its antigen-binding fragment, nucleic acid as claimed in claim 3 as described in claims 1 or 2 item, As claimed in claim 4 carrier, host cell as claimed in claim 5, chimeric antigen as claimed in claim 6 by Body, T cell as claimed in claim 7, recombinant slow virus as claimed in claim 8 or drug as claimed in claim 9 Application of the composition in the drug of preparation prevention and/or treatment and/or diagnosing tumour.
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CN111139264A (en) * 2020-01-20 2020-05-12 天津达济科技有限公司 Method for constructing single-domain antibody library in mammalian cell line based on linear double-stranded DNA molecules
CN111333730A (en) * 2020-03-11 2020-06-26 南京融捷康生物科技有限公司 Single-domain antibody capable of specifically binding to EpCAM and application thereof
CN111848798A (en) * 2020-07-27 2020-10-30 南京安锐生物科技有限公司 Nano antibody capable of being combined with BCMA (brain cell activating antigen) and application thereof
CN111909271A (en) * 2020-08-12 2020-11-10 深圳市茵冠生物科技有限公司 BCMA chimeric antigen receptor based on single domain antibody and application thereof
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CN111139264B (en) * 2020-01-20 2021-07-06 天津达济科技有限公司 Method for constructing single-domain antibody library in mammalian cell line based on linear double-stranded DNA molecules
CN111139264A (en) * 2020-01-20 2020-05-12 天津达济科技有限公司 Method for constructing single-domain antibody library in mammalian cell line based on linear double-stranded DNA molecules
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CN111848798A (en) * 2020-07-27 2020-10-30 南京安锐生物科技有限公司 Nano antibody capable of being combined with BCMA (brain cell activating antigen) and application thereof
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CN112028996A (en) * 2020-10-30 2020-12-04 南京北恒生物科技有限公司 Single domain antibodies targeting BCMA and uses thereof
WO2022199590A1 (en) * 2021-03-22 2022-09-29 浙江纳米抗体技术中心有限公司 Nanobody targeting bcma and application thereof
WO2022228429A1 (en) * 2021-04-27 2022-11-03 原启生物科技(上海)有限责任公司 Bcma-targeting chimeric antigen receptor and use thereof

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