CN109942709B

CN109942709B - anti-BCMA single domain antibody and application thereof

Info

Publication number: CN109942709B
Application number: CN201910324452.8A
Authority: CN
Inventors: 罗敏; 李光超; 郭锦涛; 莫文俊
Original assignee: Guangzhou Bio Gene Technology Co Ltd
Current assignee: Guangzhou Bio Gene Technology Co Ltd
Priority date: 2019-04-22
Filing date: 2019-04-22
Publication date: 2019-12-27
Anticipated expiration: 2039-04-22
Also published as: CN109942709A

Abstract

The invention relates to an anti-BCMA single domain antibody and application thereof, wherein the anti-BCMA single domain antibody comprises a CDR with an amino acid sequence of any one group of 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 NO.19-21, or a CDR with at least 90% identity with any one group of 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 NO. 19-21. In the single-domain antibody provided by the invention, the killing efficiency of QL197G8 on target cells is high, the release amount of a cytokine IFN-gamma is large, the affinity is strong, and the single-domain antibody has a wide application prospect and a huge market value.

Description

anti-BCMA single domain antibody and application thereof

Technical Field

The invention relates to the field of tumor cellular immunotherapy, in particular to a single domain antibody for resisting BCMA and application thereof.

Background

B-cell maturation antigen (BCMA), which comprises 184 amino acid residues, belongs to a type I transmembrane signal protein lacking a signal peptide, is a member of the tumor necrosis factor receptor family (TNFR), and can bind to both B-cell activating factor BAFF or proliferation-induced ligand (APRIL). In normal tissues, BCMA is expressed on the surfaces of mature B cells and plasma cells and plays an important role in maintaining the survival of the plasma cells, and the mechanism mainly comprises the combination of BCMA and BAFF protein, the up-regulation of anti-apoptosis genes Bcl-2, Mcl-1, Bclw and the like and the maintenance of cell growth. BCMA is generally expressed in multiple myeloma cell lines and plays an important role in promoting malignant proliferation of myeloma cells, and the BCMA can be used as one of the targets of CAR-T cells for cellular immunotherapy of multiple myeloma.

Multiple myeloma is a malignant plasma cell disease, which is characterized by malignant clonal proliferation of bone marrow plasma cells, secretion of monoclonal immunoglobulin or a fragment thereof (M protein) causes damage to related target organs or tissues such as bones and kidneys, and is clinically manifested by bone pain, anemia, renal insufficiency, infection and the like. At present, multiple myeloma is the second most malignant tumor of the blood system, accounts for 10% of the malignant tumors of the blood system, is frequently attacked in men, has the incidence rate increasing year by year with the increase of age, and has the trend of being younger in recent years.

Chimeric Antigen Receptor T cells (CAR-T) refer to T cells that are genetically modified to recognize a specific Antigen of interest in an MHC-unrestricted manner and to continuously activate expanded T cells. Biological immune cell therapy has become a fourth means for treating tumors except for surgery, radiotherapy and chemotherapy, and will become a necessary means for treating tumors in the future. CAR-T cell back-infusion therapy is the most clearly effective form of immunotherapy in current tumor therapy. A large number of studies show that the CAR-T cells can effectively recognize tumor antigens, cause specific anti-tumor immune response and remarkably improve the survival condition of patients.

Therefore, the novel single-domain antibody for resisting BCMA is provided and applied to preparing the chimeric antigen receptor, and has wide application prospect and great market value in the field of preparing medicaments for preventing and/or treating and/or diagnosing tumors.

Disclosure of Invention

The invention provides a BCMA-resistant single-domain antibody and application thereof aiming at the defects of the prior art and the market demand, seven single-domain antibodies are obtained through experimental screening, and a BCMA-targeted chimeric antigen receptor is prepared, wherein the antibody QL197G8 has high killing efficiency on target cells, large release amount of cytokine IFN-gamma, strong affinity, good specificity, wide application prospect and great market value.

In order to achieve the purpose, the invention adopts the following technical scheme:

an anti-BCMA single domain antibody or antigen binding fragment thereof, comprising a CDR having the amino acid sequence of any one of 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 No.19-21 or a CDR having at least 90% identity to any one of 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 No. 19-21.

In the present invention, the anti-BCMA single domain antibody comprises CDRs whose amino acid sequences may be at least 90% identical, e.g. may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, to the CDRs of any one of SEQ ID nos. 1 to 3, 4 to 6, 7 to 9, 10 to 12, 13 to 15, 16 to 18 or 19 to 21, and the identical CDRs without altering the structure and activity of the CDR regions are within the scope of the present invention.

Preferably, the amino acid sequence of the anti-BCMA antibody is represented by SEQ ID No. 22-28.

In the invention, the inventor uses BCMA antigen extracellular region polypeptide to immunize llama (llama), and screens and analyzes a large amount of BCMA antibodies to obtain seven single-domain antibodies, wherein the antibody QL197G8 has strong affinity and good specificity, and the antibodies can also play the advantage of small size and recognize smaller and more hidden binding sites on the surface of tumor cells. The chimeric antigen receptor targeting BCMA is prepared, has high killing efficiency on target cells, and has wide application prospect and great market value.

The sequences are shown in table 1 and table 2;

TABLE 1

Name of antibody	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

In a second aspect, the present invention provides a nucleic acid fragment encoding the anti-BCMA single domain antibody or antigen-binding fragment thereof according to the first aspect. The nucleic acid fragment is shown as SEQ ID NO.29-35, and is specifically shown as the following table 3;

TABLE 3

Antibody affinity maturation is a common approach in antibody technology and can alter the affinity of antibodies by single point fully random mutagenesis in combination with the hotspot combination Pathway (PM) and the block fully random mutagenesis (BM) pathways. The former is typically used for small amplitude affinity enhancement/minimal sequence engineering and the latter for significant affinity maturation. The invention claims 7 nano-antibody amino acids, and mainly protects the CDR1, 2, 3 sequence of each antibody and the sequence with more than 90% of sequence identity with the CDR region.

In a third aspect, the present invention provides a vector comprising at least one copy of a nucleic acid fragment according to the second aspect.

In a fourth aspect, the present invention provides a host cell comprising the vector of the third aspect.

In a fifth aspect, the invention provides a chimeric antigen receptor, the extracellular domain being an antibody against BCMA as described in the first aspect.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 36-42.

The sequences are shown in Table 4;

TABLE 4

The nucleic acid sequence of the chimeric antigen receptor is shown in table 5 below;

TABLE 5

In a sixth aspect, the invention provides a T cell which is transfected with a chimeric antigen receptor according to the fifth aspect via its encoded nucleic acid sequence for expression in the T cell.

Preferably, the transfection is by transfection into T cells by viral vectors and/or eukaryotic expression plasmids, preferably by viral vectors.

In the invention, the T cell has good target killing effect, can release low-dose immune factors, and has the properties of low toxicity and high immune killing reaction.

In a seventh aspect, the present invention provides a recombinant lentivirus, which is obtained by co-transfecting a mammalian cell with a vector according to the third aspect and a packaging helper plasmid.

Preferably, the mammalian cell is any one of 293 cell, 293T cell or 293F cell or a combination of at least two thereof.

In an eighth aspect, the present invention provides a pharmaceutical composition comprising any one of or a combination of at least two of the single domain antibody or antigen-binding fragment thereof according to the first aspect, the nucleic acid according to the second aspect, the vector according to the third aspect, the host cell according to the fourth aspect, the chimeric antigen receptor according to the fifth aspect, the T cell according to the sixth aspect or the recombinant lentivirus according to the seventh aspect.

Optionally, it further comprises a pharmaceutically acceptable carrier and/or excipient.

Preferably, it further comprises one or at least two chemotherapeutic drugs.

In a ninth aspect, the use of a single domain antibody or antigen binding fragment thereof according to the first aspect, a nucleic acid according to the second aspect, a vector according to the third aspect, a host cell according to the fourth aspect, a chimeric antigen receptor according to the fifth aspect, a T cell according to the sixth aspect, a recombinant lentivirus according to the seventh aspect or a pharmaceutical composition according to the eighth aspect for the manufacture of a medicament for the prevention and/or treatment and/or diagnosis of a tumor.

According to the invention, the tumor is a disease associated with expression of B cell maturation antigen protein, such as multiple myeloma, hodgkin's lymphoma, leukemia or glioblastoma.

Compared with the prior art, the invention has the following beneficial effects:

in the invention, the inventor uses BCMA antigen extracellular region polypeptide to immunize llama (llama), and screens and analyzes a large amount of BCMA antibodies to obtain seven single-domain antibodies, wherein the antibody QL197G8 has strong affinity and good specificity, and the antibodies can also play the advantage of small size and recognize smaller and more hidden binding sites on the surface of tumor cells. The chimeric antigen receptor targeting BCMA is prepared, has high killing efficiency on target cells, large release amount of cell factor IFN-gamma, wide application prospect and great market value.

Drawings

FIG. 1 is a schematic structural diagram of a CAR of the present invention;

FIG. 2 is a vector map of pLVX-EF1-MCS of the present invention;

FIG. 3 is a graph of the killing efficiency of CAR-T cells of the invention against target cells;

FIG. 4 shows the release of the cytokine IFN- γ after co-incubation of the CAR-T cells of the invention with target cells;

FIGS. 5(A) -5 (B) are SDS-PAGE result charts of the present invention;

FIGS. 6(A) -6 (F) are the results of the anti-human BCMA nanobody affinity analysis of the present invention;

FIG. 7 shows the results of ELISA detection of the binding ability of 7 anti-BCMA nanobodies of the present invention to BCMA.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 antibody screening

Two llamas (llama) were immunized with BCMA extracellular domain antigenic peptide, 1 with free peptide (QL197) and 1 with hemocyanin (KLH) conjugated peptide (QL 198). QL197llama produces high titer antibodies, and Peripheral Blood Mononuclear Cells (PBMCs) thereof are obtained for VHH library construction and antibody clone isolation, and QL198 alpacas are terminated without producing good antibodies;

(1) QL197 and QL198 titers were determined from BCMA-free peptide, BSA and protein:

antibody titer in antiserum was determined by ELISA, antigen was coated, the serum was diluted to 1: 10000-1: 100 million, the serum before initiation of immunization was used as a control, and the captured alpaca antibodies were detected with HRP-conjugated goat anti-alpaca antibodies (heavy chain + light chain) and TMB substrate. The QL198 experiment was terminated due to poor titer;

(2) PBMC cell number of QL197VHH single domain antibody library: 3.2 × 10e 8;

(3) total RNA purification: purifying PBMC total RNA by a phenol/chloroform extraction method and purifying by a silica gel spin column method; with RNase-free H₂O eluting the total RNA; the quality of RNA was assessed by OD260/280 ratio (2.0) and agarose gel electrophoresis (non-denaturing); RNA concentration was estimated using the formula 1.0OD260 ═ 40 μ g/mL;

(4) cDNA synthesis, according to the scheme of Qoolabs company in USA, using the llama IgG heavy chain specific reverse primer to start the total RNA, adopting the heavy chain specific primer PCR to detect the quality of cDNA;

(5) library construction: amplifying VHH products of the expected size from the cDNA;

VH and VHH were separated on agarose gel, VHH product was further purified, modified with sfil sites, cloned into pADL20c phage display vector, ligated DNA was transformed into TG1 cells, and the library yielded a total of 1.3 × 10e9 independent clones.

Note that due to the high diversity of the frame 4 and hinges, the VHH insert was divided into two parts, one end on FR4, resulting in a-420 bp product; the other end after the hinge, yields a 580bp product.

Library size 1.3 × 10e9 independent clones;

screening specific clones, namely screening a phage display antibody library on a human BCMA coated plate by adopting a Qolabs phage screening scheme, and randomly selecting positive clone individuals after screening 95 clones every time through 4 rounds of screening.

Detecting a culture lysate of the R4 clone by a primary ELISA method, wherein an ELISA coated antigen is human BCMA (cell-binding antigen), and the ELISA coated antigen is shown in Table 6;

TABLE 6

The ELISA result of the labeled clone culture lysate was verified, and the ELISA-coated antigen was negative control with BCMA free peptide, human BCMA, HSA, as shown in Table 7;

TABLE 7

All clones were streaked on plates, and 2 single colonies per clone were subjected to ELISA validation and then sequenced.

Before sequencing, ELISA results of positive clone culture lysate are verified, and ELISA envelope antigens take BCMA free peptide, human BCMA and HSA as negative controls, and are shown in Table 8;

TABLE 8

Obtaining 7 positive clones with different sequences, wherein the DNA sequences of the positive clones are shown in SEQ ID NO. 29-35;

example 2: design of chimeric antigen receptors

In this example, a chimeric antigen receptor against BCMA was constructed, the schematic structure of which is shown in fig. 1, and which comprises a signal peptide sequence (Leader) of CD8 α, a single domain antibody sequence (Anti-BCMA VHH) that specifically binds to BCMA antigen, a Hinge region (Hinge) and Transmembrane region sequence (Transmembrane) of CD8 α, a 4-1BB co-stimulatory domain sequence and a CD3 zeta signaling domain sequence, the following specific sequences of parts:

amino acid sequence of CD8 α signal peptide (leader) (SEQ ID NO. 50): MALPVTALLLPLALLLHAARP, respectively;

nucleotide sequence of CD8 alpha signal peptide (leader) (SEQ ID NO. 51): ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCT, respectively;

amino acid sequence of the CD8 α hinge region (hinge) (SEQ ID No. 52): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD, respectively;

nucleotide sequence of CD8 alpha hinge region (hinge) (SEQ ID NO. 53): ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT, respectively;

amino acid sequence of CD8 α transmembrane region (TM) (SEQ ID NO. 54): IYIWAPLAGTCGVLLLSLVITLYC, respectively;

nucleotide sequence of CD8 α transmembrane region (TM) (SEQ ID NO. 55): ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC, respectively;

amino acid sequence of 4-1BB intracellular co-stimulatory domain (ICD) (SEQ ID NO. 56): KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, respectively;

4-1BB Intracellular Costimulatory Domain (ICD) (SEQ ID NO. 57): AAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCAGAGGAGGAGGAGGGAGGATGTGAGCTG, respectively;

amino acid sequence of the zeta-signaling domain of CD3 (SEQ ID NO. 58): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR, respectively;

nucleotide sequence of the CD3 zeta signaling domain (SEQ ID No. 59): AGGGTGAAGTTTTCTCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGATCCCGAGATGGGAGGCAAGCCAAGGAGGAAGAACCCTCAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCCCTGCCACCTAGG are provided.

Example 3: construction of chimeric antigen receptor expression vectors against BCMA

(1) According to the nucleic acid sequence of the CAR gene, the whole gene synthesis is carried out in Egyptian biotechnology, Inc., Guangzhou;

(2) carrying out double enzyme digestion on the CAR gene synthesized by the whole gene and an empty vector pLVX-EF1-MCS by using EcoRI and BamHI, carrying out enzyme digestion for 30min in water bath at 37 ℃, carrying out DNA electrophoresis by using 1.5% agarose gel, and then purifying and recycling by using an agarose gel kit of Tiangen;

(3) ligation of pLVX-EF1-MCS vector to CAR gene fragment:

the attachment system is shown in table 9:

TABLE 9

Assembly	Addition amount (μ l)
		pLVX-EF1-MCS vector	2(50ng)
CAR gene	10(150ng)
		T4DNA ligation buffer	2
T4DNA ligase (NEB)	1
		dd H₂O	5
In all	20

Ligation was performed at 22 ℃ for 1h, the ligation products were directly transformed into Stbl3 E.coli competent cells, 200. mu.l of the transformation products were spread on ampicillin-resistant LB plates, and the LB plates were cultured in an incubator at 37 ℃ overnight in an inverted manner. Randomly selecting 3 monoclonals the next morning for colony PCR identification, and sending positive clones to sequence.

Among them, the vector map of the anti-BCMA chimeric antigen receptor lentiviral expression vector pLVX-EF1 alpha-G8 CAR-Km is shown in FIG. 2.

Example 4: lentiviral packaging

The lentiviral expression vectors in the examples were each subjected to lentiviral packaging using a four plasmid system, the specific steps of which were as follows:

(1) four plasmid systems respectively express gag/pol, Rev, VSV-G required by packaging of lentiviral vectors and the CAR expression vector constructed by the invention: transient transfection is carried out on the four plasmids to 293T cells, and the DNA content is 2 mug/mL;

(2) mixing the plasmid and PEI transfection reagent, adding into serum-free DMEM with a certain volume, standing for 15 minutes after mixing uniformly, adding the mixed solution into a T75 culture bottle paved with 293T cell cells, mixing uniformly and gently, and carrying out 5% CO treatment at 37 DEG C₂Culturing for 6h in a cell culture box;

(3) after 6h, the culture medium was replaced with fresh medium and the culture was continued, and 10mM sodium butyrate solution was added, and culture supernatant of lentivirus was collected 72 hours later for purification assay.

Example 5: expansion of CAR-T cells

Collecting 30ml of whole blood from each volunteer, diluting peripheral blood with physiological saline 1:1, adding Ficoll into a centrifugal tube, slowly adding diluted peripheral blood, centrifuging at 1500rpm for 30min, gently sucking PBMC layer, and transferring into another centrifugal tube;

PBMC are washed with physiological saline for multiple times, transferred into X-VIVO culture medium (containing 50ng/mL OKT3, 300IU/mL IL-2) for culture, after PBMC is separated, X-VIVO containing 50ng/mL OKT3, 300IU/mL IL-2 is needed for activation, after 2 days, the culture medium is changed into X-VIVO containing 300IU/mL for amplification culture, then counting and changing the X-VIVO containing 300IU/mL are carried out every two days, and the cell concentration is maintained at 0.5X 10⁶-1×10⁶mL, for 10 consecutive days.

Example 6: lentiviral infection of T cells

The infection efficiency of lentivirus to T cells is improved by using retroNectin, and 30 mu g of retroNectin is coated in a 6-hole plate and placed in a cell culture box at 37 ℃ for 2 h; absorbing RetroNectin, sealing the coated 6-well plate by using Hank's solution containing 2.5% BSA, and placing the plate in a 37 ℃ cell culture box for 0.5 h; sucking the confining liquid, washing a 6-well plate by using a Hank's solution containing 2% Hepes, adding an X-VIVO culture medium, adding a proper amount of a lentivirus solution, adding 2000g of the lentivirus solution, and centrifuging for 2 hours;discard the supernatant and add 1X 10⁶T cells (CD3 positive)>90%), 1000g, centrifugation for 10min at 37 deg.C and 5% CO₂And culturing in a cell culture box with certain humidity, and repeating the process the next day.

Example 7: killing efficiency detection

Cytotoxicity assays were performed 7-10 days after infection, and blank control NC (untransfected T cells) was used as a control, as follows:

detecting toxicity of CAR-T cells to K562 and K562-BCMA (K562 cells stably expressing BCMA) using LDH;

centrifuging various cells, washing with serum-free phenol red-free RPMI1640 medium for several times, counting, and collecting 1 × 10⁶And K562-BCMA cells, 50 μ L each, were plated in 96-well plates as target cells, as per target cell: effector cells 1:1 untransfected T cells and CAR-T cells were added separately at 37 ℃ with 5% CO₂Culturing in a cell culture box with certain humidity for 12 h; adding lysis solution as positive control, centrifuging for 5min at 250g, collecting 100 μ L culture supernatant per well, adding into new 96-well plate, adding 20 μ L reaction solution, and placing in dark room for reaction for 20-30 min.

And (3) measuring by a microplate reader at 590nm, and calculating the dissolution percentage according to the formula:

cytotoxicity (%) - [ (experimental well-medium background well) - (effector cell spontaneous LDH release well-medium background well) - (target cell spontaneous LDH release well-medium background well) ]/[ (target cell maximum LDH release well-volume corrected well) - (target cell spontaneous LDH release well-medium background well) ] × 100%.

The specific steps refer to the patent with application number 201510362935.5, CD33 specific chimeric antigen receptor and application thereof.

The killing efficiency of CAR-T cells against target cells is shown in figure 3;

as can be seen in figure 3, T cells that were not transfected with CAR (T mock) did not significantly kill both K562 and K562-BCMA; each CAR-T cell can kill BCMA positive cells K562-BCMA to different degrees, and has almost no killing capacity on BCMA negative cells K562; among them, G8CAR-T has the highest killing rate.

Example 8: IFN-gamma release assay

K562 and K562-BCMA cells were plated at 5X 10⁵Cells/well were seeded in 24-well plates. At a rate of 5X 10 per hole⁵Adding CAR-T and untransfected T cell (T mock) into the cells respectively, supplementing a culture solution to 1.5mL, and co-culturing in an incubator for 12 hours; the co-culture supernatant was assayed using human IFN-. gamma.ELISA assay kit (Xinbo Sheng Bio) (see ELISA assay kit for details). The release of the cytokine IFN-. gamma.after incubation of CAR-T cells with target cells is shown in FIG. 4.

As can be seen from fig. 4: t cells (T mock) not transfected with CAR secreted little IFN-. gamma.after coculture with K562 and K562-BCMA; different concentrations of IFN-gamma could be detected in the supernatant after co-culturing each CAR-T cell with K562-BCMA, whereas very little cytokine IFN-gamma was released after co-culturing with BCMA negative cells K562.

Example 9 affinity assay

The 7 alpaca antibody sequences in table 2 were constructed to pcDNA4/His B vector after codon optimization, together with IgG leader peptide sequence (MAVVLAALLQSVQA), hinge region sequence of IgG2B in llama (epktppqpqpqpqpnptteskpcpcpapelggps) and His tag sequence (hhhhhhhhhhhhhh), to obtain plasmids.

Description of the sequence:

>QL197A11SEQ ID NO.60

MAVVLAALLQSVQAqvllvesggglvqaggslrlscavsgrtvstatmgwfrqapgkerefvaalnwsgnksyyadsvkgrfaisrdeakntvylqmnslkpedtavyhcaagpdlnyytnydarrydhwgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH

>QL197C9SEQ ID NO.61

MAVVLAALLQSVQAqgqyvesgggsvqageslrlscigshttainaagwyrqtpgkqrelvglihndgstqyaqfakgrftisrddakdavylqmnslkvedtgvyycnidsrgvgpvwahwgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpshhhhhh

>QL197D11SEQ ID NO.62

MAVVLAALLQSVQAqaqlkesgggsvrtgeslrlsceasgniysinrmawyrqvsgmqrevvatslvdgttnygdsvkdrftvsrdnakkmvflqmnslepadtgvyycnvegnridyapgsrypthsyvelwgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH

>QL197E2SEQ ID NO.63

MAVVLAALLQSVQAqvqlvenggglvqpggslrlscaasgasfndyhmgwfrqapgqerkfvaqiarygaatyyaravqgrftisvddakntvylqmngltpddtgvyyctadrsnyyidnalpdywgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH

>QL197F4SEQ ID NO.64

MAVVLAALLQSVQAqvqliesggglvqagtsltlscassgrnfnsyamgwfrqapgkereflatisraagstyyadsakgrftisrdnrkefaylqihdlkpddtavyycaaeswtpttgwpptkadefdywgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH

>QL197F7SEQ ID NO.65

MAVVLAALLQSVQAqdrlvesgggsvqaggslklscrtsgfnlddyaigwfrqapgkerervscissdgrtshtgsakgrftirsanarntvylqlnrltpedagvyfcaaertsrlcsflsydywgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH

>QL197G8SEQ ID NO.66

MAVVLAALLQSVQAqvqlvesggglvqaggslrlscaasgrtfsdhtlgwfrqapgkerefvgaiswsggstyyadsvsgrftisrdkakntgylqmnslkpedtavyycaaaddrysdyrywgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH

control antibody BBHL-Fab-VL SEQ ID No. 67:

DIVLTQSPASLAVSLGERATINCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIK

control antibody BBHL-Fab-VH SEQ ID No. 68:

QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSINWVRQAPGQGLEWMGWINTETREPAYAYDFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVSS

after the plasmid is sequenced correctly, 293F transient transfection is carried out, cells are harvested after 2 days (degradation may exist), after the Ni-His affinity column affinity purification of the nano-antibody, SDS-PAGE electrophoresis detection is carried out, and the results are shown in a figure 5(A) and a figure 5(B), wherein the figure 5(A) is a figure of molecular weight results of 7 anti-BCMA nano-antibodies analyzed by 15% reducing SDS-PAGE, and the figure 5(B) is a figure of molecular weight results of 7 anti-BCMA nano-antibodies analyzed by 15% non-reducing SDS-PAGE;

as can be seen from FIGS. 5(A) and 5(B), the expression was achieved for all 7 antibody molecules, and degradation occurred at the C-terminus.

Affinity analysis was performed using Anti-hFc immobilized sensor, loading human BCMA-hFc antigen, with 7 antibody proteins initially set at a concentration of 200nM, and the results are shown in fig. 6(a) -6 (B) and table 10;

as can be seen from fig. 6(a), fig. 6(B) and table 10, the affinity of QL197E2 and QL197G8 in the 7 antibodies could reach a level comparable to the control BBHL-Fab, but it was also necessary to verify whether both were binding to the human Fc fragment;

watch 10

Whether or not the three proteins are non-specifically bound was analyzed using Anti-hFc immobilized sensors, the loading human BCMA-hFc antigen and the irrelevant antigen human PD1-hFc, based on the above affinity analysis results, setting QL197E2 to a concentration of 100nm, QL197G8 to a concentration of 200nm, and BBHL-Fab (positive control antibody) to a concentration of 50nm, and the results are shown in fig. 6(C) -fig. 6(F) and table 11;

TABLE 11

As can be seen from fig. 6(C) to fig. 6(F) and table 11, QL197E2 is an antibody that binds to the humanFc fragment, while QL197G8 does not bind to the humanFc fragment, and subsequent studies can be performed.

We further detected the binding ability of 7 Anti-BCMA nanobodies to BCMA by ELISA (FIG. 7), coated 7 antibodies and the control antibody BBHL-Fab at a concentration of 0.5ug/mL, diluted the human BCMA-hFc and the antigen with unrelated human Fc at a concentration of 20ng/mL and 100ng/mL, and added Anti-human HRP for color development.

As can be seen from fig. 7: QL197E2 is an antibody that binds to the human Fc fragment, and QL197G8 is specific binding, and can be used for subsequent studies.

In conclusion, the invention provides a single domain antibody for resisting BCMA and application thereof, wherein QL197G8 has high killing efficiency on target cells, the release amount of cytokine IFN-gamma is large, and the single domain antibody is not combined with human Fc segment, so that a more effective treatment selection is provided for B cell related diseases.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Guangzhou Bai-and-Gen-Tech Co Ltd

<120> single domain antibody for anti-BCMA and application thereof

<130> 2019

<160> 68

<170> PatentIn version 3.3

<210> 1

<211> 8

<212> PRT

<213> artificially synthesized sequence

<400> 1

Arg Thr Val Ser Thr Ala Thr Met

1 5

<210> 2

<211> 15

<212> PRT

<213> artificially synthesized sequence

<400> 2

Ala Ala Leu Asn Trp Ser Gly Asn Lys Ser Tyr Tyr Ala Asp Ser

1 5 10 15

<210> 3

<211> 18

<212> PRT

<213> artificially synthesized sequence

<400> 3

Ala Gly Pro Asp Leu Asn Tyr Tyr Thr Asn Tyr Asp Ala Arg Arg Tyr

1 5 10 15

Asp His

<210> 4

<211> 8

<212> PRT

<213> artificially synthesized sequence

<400> 4

His Thr Thr Ala Ile Asn Ala Ala

1 5

<210> 5

<211> 17

<212> PRT

<213> artificially synthesized sequence

<400> 5

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

1 5 10 15

Ala

<210> 6

<211> 12

<212> PRT

<213> artificially synthesized sequence

<400> 6

Asn Ile Asp Ser Arg Gly Val Gly Pro Val Trp Ala

1 5 10

<210> 7

<211> 9

<212> PRT

<213> artificially synthesized sequence

<400> 7

Asn Ile Tyr Ser Ile Asn Arg Met Ala

1 5

<210> 8

<211> 19

<212> PRT

<213> artificially synthesized sequence

<400> 8

Val Val Ala Thr Ser Leu Val Asp Gly Thr Thr Asn Tyr Gly Asp Ser

1 5 10 15

Val Lys Asp

<210> 9

<211> 23

<212> PRT

<213> artificially synthesized sequence

<400> 9

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

<210> 10

<211> 8

<212> PRT

<213> artificially synthesized sequence

<400> 10

Ala Ser Phe Asn Asp Tyr His Met

1 5

<210> 11

<211> 19

<212> PRT

<213> artificially synthesized sequence

<400> 11

Phe Val Ala Gln Ile Ala Arg Tyr Gly Ala Ala Thr Tyr Tyr Ala Arg

1 5 10 15

Ala Val Gln

<210> 12

<211> 15

<212> PRT

<213> artificially synthesized sequence

<400> 12

Thr Ala Asp Arg Ser Asn Tyr Tyr Ile Asp Asn Ala Leu Pro Asp

1 5 10 15

<210> 13

<211> 8

<212> PRT

<213> artificially synthesized sequence

<400> 13

Arg Asn Phe Asn Ser Tyr Ala Met

1 5

<210> 14

<211> 19

<212> PRT

<213> artificially synthesized sequence

<400> 14

Phe Leu Ala Thr Ile Ser Arg Ala Ala Gly Ser Thr Tyr Tyr Ala Asp

1 5 10 15

Ser Ala Lys

<210> 15

<211> 19

<212> PRT

<213> artificially synthesized sequence

<400> 15

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

1 5 10 15

Glu Phe Asp

<210> 16

<211> 8

<212> PRT

<213> artificially synthesized sequence

<400> 16

Phe Asn Leu Asp Asp Tyr Ala Ile

1 5

<210> 17

<211> 18

<212> PRT

<213> artificially synthesized sequence

<400> 17

Arg Val Ser Cys Ile Ser Ser Asp Gly Arg Thr Ser His Thr Gly Ser

1 5 10 15

Ala Lys

<210> 18

<211> 13

<212> PRT

<213> artificially synthesized sequence

<400> 18

Glu Arg Thr Ser Arg Leu Cys Ser Phe Leu Ser Tyr Asp

1 5 10

<210> 19

<211> 8

<212> PRT

<213> artificially synthesized sequence

<400> 19

Arg Thr Phe Ser Asp His Thr Leu

1 5

<210> 20

<211> 17

<212> PRT

<213> artificially synthesized sequence

<400> 20

Gly Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

Ser

<210> 21

<211> 9

<212> PRT

<213> artificially synthesized sequence

<400> 21

Ala Asp Asp Arg Tyr Ser Asp Tyr Arg

1 5

<210> 22

<211> 126

<212> PRT

<213> artificially synthesized sequence

<400> 22

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> artificially 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> artificially 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> artificially 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> artificially 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

<210> 27

<211> 122

<212> PRT

<213> artificially 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> artificially 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

<213> artificially synthesized sequence

<400> 29

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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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

<212> PRT

<213> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially synthesized sequence

<400> 51

atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60

cct 63

<210> 52

<211> 45

<212> PRT

<213> artificially 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> artificially 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> artificially 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> artificially synthesized sequence

<400> 55

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 56

<211> 42

<212> PRT

<213> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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> artificially 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

1. An anti-BCMA single domain antibody, wherein the amino acid sequence of the anti-BCMA single domain antibody is shown in SEQ ID NO. 22-27.

2. A nucleic acid fragment encoding the anti-BCMA single domain antibody of claim 1.

3. The nucleic acid fragment of claim 2, wherein the nucleic acid fragment is represented by SEQ ID No. 29-34.

4. A vector comprising at least one copy of the nucleic acid fragment of claim 2 or 3.

5. A host cell comprising the vector of claim 4.

6. A chimeric antigen receptor, wherein the extracellular domain is the anti-BCMA antibody according to claim 1.

7. The chimeric antigen receptor according to claim 6, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No. 36-41.

8. A T cell produced by transfecting the chimeric antigen receptor of claim 6 or 7 into a T cell by expression of the nucleic acid sequence encoded therein.

9. The T-cell according to claim 8, wherein the transfection is by transfection into the T-cell with a viral vector and/or a eukaryotic expression plasmid.

10. The T-cell of claim 9, wherein the transfection is by transfection into the T-cell with a viral vector.

11. A recombinant lentivirus comprising the vector of claim 4 co-transfected with a packaging helper plasmid into a mammalian cell.

12. The recombinant lentivirus of claim 11, wherein the mammalian cell is any one of 293 cells, 293T cells or 293F cells or a combination of at least two thereof.

13. A pharmaceutical composition comprising any one of the single domain antibody of claim 1, the nucleic acid of claim 2 or 3, the vector of claim 4, the host cell of claim 5, the chimeric antigen receptor of claim 6 or 7, the T cell of any one of claims 8-10, or the recombinant lentivirus of claim 11 or 12, or a combination of at least two thereof.

14. The pharmaceutical composition of claim 13, further comprising a pharmaceutically acceptable carrier and/or excipient.

15. The pharmaceutical composition of claim 14, further comprising one or at least two chemotherapeutic agents.

16. Use of a single domain antibody according to claim 1, a nucleic acid according to claim 2 or 3, a vector according to claim 4, a host cell according to claim 5, a chimeric antigen receptor according to claim 6 or 7, a T cell according to any one of claims 8 to 10, a recombinant lentivirus according to claim 11 or 12 or a pharmaceutical composition according to any one of claims 13 to 15 for the preparation of a medicament for the prevention and/or treatment and/or diagnosis of a tumour.