CN117886941B - TRPA1 antibody, preparation method and application thereof - Google Patents
TRPA1 antibody, preparation method and application thereof Download PDFInfo
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- CN117886941B CN117886941B CN202410084491.6A CN202410084491A CN117886941B CN 117886941 B CN117886941 B CN 117886941B CN 202410084491 A CN202410084491 A CN 202410084491A CN 117886941 B CN117886941 B CN 117886941B
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Classifications
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/04—Centrally acting analgesics, e.g. opioids
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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Abstract
The invention discloses a TRPA1 antibody, a method for preparing the same and application thereof, wherein the TRPA1 antibody comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID NO. 56-58 respectively; and/or the light chain variable region comprises the amino acid sequences shown as SEQ ID NO. 59, SEQ ID NO.9 and SEQ ID NO.10 as LCDR1, LCDR2 and LCDR3 respectively. The TRPA1 antibody provided by the invention has relatively high affinity to TRPA1, and the screened TRPA1 antibody has obvious inhibition effect on AITC activated TRPA1 channel, and the IC50 is lower than 1.54 mu M and can reach 0.56 mu M at the lowest.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a TRPA1 antibody, a preparation method and application thereof.
Background
Transient receptor potential ankyrin subtype 1protein (trp a 1) is a non-selective cation channel permeable to Ca 2+、Na+ and K +. TRPA1 is present in a subset of aδ and C fiber nociceptive sensory neurons and other sensory cells including epithelial cells. In primary sensory neurons, ca 2+ and Na + flow into cells through TRPA1, causing membrane depolarization, action potential discharge, and neurotransmitter release at peripheral and central nerve projections.
In addition to activation by cysteine and lysine reactive electrophiles and oxidants, TRPA1 may also be indirectly activated by pro-inflammatory agents through the phospholipase C signaling pathway, with cytosolic Ca 2+ being an important regulator of channel gating.
Various evidences suggest that TRPA1 is associated with pain sensation. TRPA1 is expressed in sensory neurons and co-localized with pain markers such as TRPV1 and bradykinin receptors. Its expression is increased in DRG neurons of animal models of inflammation and neuropathic pain and in human avulsion lesions. TRPA1 agonists cause neurotransmitter release, pain and inflammation in rodents and humans, whereas endogenous agonists such as 4-HNE are elevated in human pathological conditions. In several animal models, gene knockout reduces agonist sensitivity, antagonist treatment reduces pain, TRPA1 is believed to play a role in a number of sensory modes, including chemonociception, mechanociception, and cold injury.
In recent years, opioid addiction and overdose have grown at a striking rate and have become a serious national crisis in the united states, leading to death in many people every day. There is a need to develop new non-opioid pain medications without addictive potential at any time today than ever before. Because TRPA1 is involved in chronic and acute pain, TRPA1 antagonists have the potential to provide next generation pain medications, which can help alleviate the current opioid crisis.
The studies on indications for TRPA1 for the treatment of respiratory diseases such as pain, skin diseases and asthma are relatively hot. The TRPA1 small molecule antagonist mainly comprises xanthine, sulfanilamide, oxadiazolone, oxadiazole, carboxamide and other structures. Five small molecules have entered the clinic by 2019 but all have been stopped by poor pharmacokinetic properties. Compared to small molecule drugs, large molecule antibody drugs have significant advantages in that they limit Central Nervous System (CNS) permeability (when targeted for peripheral therapy), low immunogenicity, high selectivity, and favorable half-life, making antibody development of TRPA1 an attractive alternative to biological agents. However, screening for functionally active antibodies remains a well-known challenge due to the dynamic nature of TRPA1 conformation and the minimal extracellular region. The existing commercial TRPA1 antibody is mainly a polyclonal antibody applied to TRPA1 immunological detection experiments, and has poor performances such as affinity, specificity, functionality and the like.
Disclosure of Invention
In order to solve the problem that a functional active antibody targeting TRPA1 is lacking in the prior art, the invention provides a TRPA1 antibody, a preparation method and application thereof, wherein immunogen used in the preparation process is nanodisc packed and purified protein, the preparation of the antibody adopts hybridoma technology, affinity screening is mainly ELISA and FACS, and fluorescent intensity change caused by calcium ions flowing into cells through ion channels is read by combining Flexstation3, so that the antibody with both affinity and functionality is screened. Several therapeutic antibodies with TRPA1 antagonistic function are obtained by the work of affinity test, functional test, sequence acquisition, etc.
In order to solve the technical problem described above, the first aspect of the present invention provides a TRPA1 antibody comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOS 56-58, respectively; and/or the light chain variable region comprises an LCDR1, an LCDR2 and an LCDR3 having amino acid sequences as shown in SEQ ID NO. 59, SEQ ID NO. 9 and SEQ ID NO. 10, respectively; or alternatively, the first and second heat exchangers may be,
The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO 26-28 respectively; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NOS 29-31, respectively; or alternatively, the first and second heat exchangers may be,
The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO. 36-38 respectively; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NOS: 39-41, respectively; or alternatively, the first and second heat exchangers may be,
The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO. 51-53 respectively; and/or the light chain variable region comprises the amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:54, SEQ ID NO:30 and SEQ ID NO:55, respectively.
In some embodiments of the invention, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences shown in SEQ ID NOS 5-7, respectively; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NOS 8-10, respectively; or alternatively, the first and second heat exchangers may be,
The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO 17-19 respectively; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NOS 8-10, respectively; or alternatively, the first and second heat exchangers may be,
The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO 17-19 respectively; and/or the light chain variable region comprises the amino acid sequences shown as SEQ ID NO. 46, SEQ ID NO. 9 and SEQ ID NO. 10 of LCDR1, LCDR2 and LCDR3 respectively.
In some embodiments of the invention, the heavy chain variable region further comprises a heavy chain variable region framework region HFWR, and/or the light chain variable region further comprises a light chain variable region framework region LFWR, wherein the HFWR is a heavy chain variable region framework region of human or mouse origin and the LFWR is a light chain variable region framework region of a human or mouse antibody.
In some preferred embodiments of the invention, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 4 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO. 4; and/or the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 3 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown as SEQ ID NO. 3; or alternatively, the first and second heat exchangers may be,
The light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 14 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown as SEQ ID NO. 14; and/or the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 13 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown as SEQ ID NO. 13; or alternatively, the first and second heat exchangers may be,
The light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 14 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown as SEQ ID NO. 14; and/or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID No. 16 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID No. 16; or alternatively, the first and second heat exchangers may be,
The light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 14 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown as SEQ ID NO. 14; and/or the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 21 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown as SEQ ID NO. 21; or alternatively, the first and second heat exchangers may be,
The light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 25 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown as SEQ ID NO. 25; and/or the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 24 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown as SEQ ID NO. 24; or alternatively, the first and second heat exchangers may be,
The light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 35 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown as SEQ ID NO. 35; and/or the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 34 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown as SEQ ID NO. 34; or alternatively, the first and second heat exchangers may be,
The light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 45 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown as SEQ ID NO. 45; and/or the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 44 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown as SEQ ID NO. 44; or alternatively, the first and second heat exchangers may be,
The light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 50 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown as SEQ ID NO. 50; and/or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 49 or has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO. 49.
In some more preferred embodiments of the present invention, the TRPA1 antibody further comprises a heavy chain constant region and a light chain constant region.
In some even more preferred embodiments of the present invention, the TRPA1 antibody heavy chain constant region is a human or mouse-derived antibody heavy chain constant region; the TRPA1 antibody light chain constant region is a human or mouse antibody light chain constant region.
In some embodiments of the invention, the TRPA1 antibody is in the form of any one of the following antibodies:
(a) An intact immunoglobulin molecule;
(b) An scFv;
(c) A fusion protein comprising an scFv;
(d) A Fab fragment;
(e) A Fab' fragment;
(f) F (ab) 2;
Or the TRPA1 antibody is a monoclonal antibody or a polyclonal antibody;
Or the TRPA1 antibody is a humanized antibody or a bispecific antibody.
In order to solve the technical problem described above, the second aspect of the present invention provides a chimeric antigen receptor comprising the TRPA1 antibody according to the first aspect of the present invention.
In order to solve the above technical problem, the third aspect of the present invention provides an isolated nucleic acid encoding the TRPA1 antibody according to the first aspect of the present invention or the chimeric antigen receptor according to the second aspect of the present invention.
In some preferred embodiments of the present invention, the nucleic acid encoding the TRPA1 antibody comprises a polynucleotide sequence as set forth in SEQ ID NO.1 and/or as set forth in SEQ ID NO. 2; or, comprises a polynucleotide sequence as shown in SEQ ID NO. 11 and/or as shown in SEQ ID NO. 12; or, comprises a polynucleotide sequence as shown in SEQ ID NO. 15 and/or as shown in SEQ ID NO. 12; or, comprises the polynucleotide sequence shown in SEQ ID NO. 20 and/or as shown in SEQ ID NO. 12; or, comprises the polynucleotide sequence shown in SEQ ID NO. 22 and/or as shown in SEQ ID NO. 23; or, comprises the polynucleotide sequence shown in SEQ ID NO. 32 and/or as shown in SEQ ID NO. 33; or, comprises a polynucleotide sequence as shown in SEQ ID NO. 42 and/or as shown in SEQ ID NO. 43; or, comprises a polynucleotide sequence as shown in SEQ ID NO. 47 and as shown in SEQ ID NO. 48.
In order to solve the above technical problem, the fourth aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid according to the third aspect of the present invention.
In order to solve the above technical problem, a fifth aspect of the present invention provides a transformant comprising the recombinant expression vector according to the fourth aspect in a host cell.
In order to solve the above technical problem, a sixth aspect of the present application provides a method for producing a TRPA1 antibody, comprising culturing the transformant according to the fifth aspect of the present application, and obtaining the TRPA1 antibody from the culture.
In order to solve the above technical problems, the seventh aspect of the present invention provides an antibody drug conjugate comprising a cytotoxic agent, and the TRPA1 antibody according to the first aspect of the present invention.
In order to solve the above technical problem, the eighth aspect of the present invention provides a pharmaceutical composition comprising the TRPA1 antibody according to the first aspect of the present invention, the chimeric antigen receptor according to the second aspect of the present invention, or the antibody drug conjugate according to the seventh aspect of the present invention, and a pharmaceutically acceptable carrier.
In order to solve the technical problem described above, the ninth aspect of the present invention provides the use of the TRPA1 antibody according to the first aspect of the present invention, the chimeric antigen receptor according to the second aspect of the present invention, the isolated nucleic acid according to the third aspect of the present invention, the recombinant expression vector according to the fourth aspect of the present invention, the transformant according to the fifth aspect of the present invention, the antibody drug conjugate according to the seventh aspect of the present invention, or the pharmaceutical composition according to the eighth aspect of the present invention for the preparation of a medicament for treating pain.
In some preferred embodiments of the invention, the pain is pain resulting from TRPA1 activation; the target point of the drug is TRPA1.
In order to solve the technical problem described above, a tenth aspect of the present invention provides a kit comprising the TRPA1 antibody according to the first aspect of the present invention, the chimeric antigen receptor according to the second aspect of the present invention, the antibody drug conjugate according to the seventh aspect of the present invention, and/or the pharmaceutical composition according to the eighth aspect of the present invention.
In order to solve the above technical problem, the eleventh aspect of the present invention provides a method for detecting TRPA1, comprising contacting a sample with the TRPA1 antibody according to the first aspect of the present invention, the chimeric antigen receptor according to the second aspect of the present invention, the antibody drug conjugate according to the seventh aspect of the present invention, the pharmaceutical composition according to the eighth aspect of the present invention, and/or the kit according to the tenth aspect of the present invention.
In some preferred embodiments of the invention, the detection is for non-diagnostic purposes.
In order to solve the above technical problem, the twelfth aspect of the present invention provides a method for treating and/or preventing pain, which comprises administering a therapeutically effective amount of the TRPA1 antibody according to the first aspect of the present invention, the chimeric antigen receptor according to the second aspect of the present invention, the isolated nucleic acid according to the third aspect of the present invention, the recombinant expression vector according to the fourth aspect of the present invention, the transformant according to the fifth aspect of the present invention, the antibody drug conjugate according to the seventh aspect of the present invention, the pharmaceutical composition according to the eighth aspect of the present invention, and/or the kit according to the tenth aspect of the present invention to a patient in need thereof.
In some preferred embodiments of the invention, the pain is pain resulting from TRPA1 activation; the target point of the drug is TRPA1.
In order to solve the technical problem described above, the thirteenth aspect of the present invention provides a TRPA1 antibody according to the first aspect of the present invention, a chimeric antigen receptor according to the second aspect of the present invention, an isolated nucleic acid according to the third aspect of the present invention, a recombinant expression vector according to the fourth aspect of the present invention, a transformant according to the fifth aspect of the present invention, an antibody drug conjugate according to the seventh aspect of the present invention, a pharmaceutical composition according to the eighth aspect of the present invention, and/or a kit according to the tenth aspect of the present invention for the treatment and/or prevention of pain.
In some preferred embodiments of the invention, the pain is pain resulting from TRPA1 activation; the target point of the drug is TRPA1.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: the anti-TRPA 1 antibody provided by the invention has relatively high affinity to TRPA1, and the screened antibody has obvious inhibition effect on AITC activated TRPA1 channel, and the IC50 is lower than 1.54 mu M and can reach 0.56 mu M at the lowest.
Drawings
FIG. 1 is a map of pEGBacMam recombinant plasmids containing TRPA1 and MBP.
FIG. 2 is a map of a pET 28a recombinant plasmid containing MSP2N 2.
FIG. 3 shows the results of TRPA1-Nanodisc protein purification.
FIG. 4 shows the results of serum titers of the 4 th and 5 th immune mice immunized with TRPA1-Nanodisc protein.
FIGS. 5a-5h show ELISA detection results for 8 antibodies obtained by screening.
FIGS. 6a-6h show the results of FACS detection of 8 antibodies obtained by the screening.
FIG. 7 is a SDS-PAGE map of the purified 8 antibodies obtained by the screening.
FIGS. 8a-8i are the inhibition of TRPA1 channel current by positive small molecule A967079 and 8 antibodies screened.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
EXAMPLE 1 preparation of recombinant baculovirus
1.1 Obtaining recombinant baculovirus plasmid
Recombinant pEGBacMam plasmid containing the target gene (synthesized by Nanjing family Biotechnology Co., ltd., carrying EcoR1 and Not1 cleavage sites, ligated and recombined to pEGBacMam plasmid, pEGBacMam recombinant plasmid containing TRPA1 and MBP as shown in FIG. 1, pET 28a recombinant plasmid containing MSP2N2 as shown in FIG. 2) was introduced into E.coli DH10Bac competent cells (Shanghai virucide) and cultured in LB solid medium containing 50. Mu.g/mL kanamycin (aladdin), 7. Mu.g/mL gentamycin (aladdin), 10. Mu.g/mL tetracyclomycin (aladdin), 100. Mu.g/mL gal (Thermofish), 40. Mu.g/mL IPTG (aladdin) at 37℃for 48-72 hours. The recombinant baculovirus plasmid was extracted by selecting the homogeneous white spots to 5mL of LB liquid medium containing three antibiotics (50. Mu.g/mL kanamycin, 7. Mu.g/mL gentamicin, 10. Mu.g/mL tetracyclomycin) and culturing at 37℃for 12-16 hours at 200 rpm.
TRPA1 amino acid sequence (SEQ ID NO: 60):
MKRSLRKMWRPGEKKEPQGVVYEDVPDDTEDFKESLKVVFEGSAYGLQNFNKQKKLKRCDDMDTFFLHYAAAEGQIELMEKITRDSSLEVLHEMDDYGNTPLHCAVEKNQIESVKFLLSRGANPNLRNFNMMAPLHIAVQGMNNEVMKVLLEHRTIDVNLEGENGNTAVIIACTTNNSEALQILLKKGAKPCKSNKWGCFPIHQAAFSGSKECMEIILRFGEEHGYSRQLHINFMNNGKATPLHLAVQNGDLEMIKMCLDNGAQIDPVEKGRCTAIHFAATQGATEIVKLMISSYSGSVDIVNTTDGCHETMLHRASLFDHHELADYLISVGADINKIDSEGRSPLILATASASWNIVNLLLSKGAQVDIKDNFGRNFLHLTVQQPYGLKNLRPEFMQMQQIKELVMDEDNDGCTPLHYACRQGGPGSVNNLLGFNVSIHSKSKDKKSPLHFAASYGRINTCQRLLQDISDTRLLNEGDLHGMTPLHLAAKNGHDKVVQLLLKKGALFLSDHNGWTALHHASMGGYTQTMKVILDTNLKCTDRLDEDGNTALHFAAREGHAKAVALLLSHNADIVLNKQQASFLHLALHNKRKEVVLTIIRSKRWDECLKIFSHNSPGNKCPITEMIEYLPECMKVLLDFCMLHSTEDKSCRDYYIEYNFKYLQCPLEFTKKTPTQDVIYEPLTALNAMVQNNRIELLNHPVCKEYLLMKWLAYGFRAHMMNLGSYCLGLIPMTILVVNIKPGMAFNSTGIINETSDHSEILDTTNSYLIKTCMILVFLSSIFGYCKEAGQIFQQKRNYFMDISNVLEWIIYTTGIIFVLPLFVEIPAHLQWQCGAIAVYFYWMNFLLYLQRFENCGIFIVMLEVILKTLLRSTVVFIFLLLAFGLSFYILLNLQDPFSSPLLSIIQTFSMMLGDINYRESFLEPYLRNELAHPVLSFAQLVSFTIFVPIVLMNLLIGLAVGDIAEVQKHASLKRIAMQVELHTSLEKKLPLWFLRKVDQKSTIVYPNKPRSGGMLFHIFCFLFCTGEIRQEIPNADKSLEMEILKQKYRLKDLTFLLEKQHELIKLIIQKMEIISETEDDDSHCSFQDRFKKEQMEQRNSRWNTVLRAVKAKTHHLEP
TRPA1 base sequence (SEQ ID NO: 61):
ATGAAGCGCAGCCTGAGGAAGATGTGGCGCCCTGGAGAAAAGAAGGAGCCCCAGGGCGTTGTCTATGAGGATGTGCCGGACGACACGGAGGATTTCAAGGAATCGCTTAAGGTGGTTTTTGAAGGAAGTGCATATGGATTACAAAACTTTAATAAGCAAAAGAAATTAAAAAGATGTGACGATATGGACACCTTCTTCTTGCATTATGCTGCAGCAGAAGGCCAAATTGAGCTAATGGAGAAGATCACCAGAGATTCCTCTTTGGAAGTGCTGCATGAAATGGATGATTATGGAAATACCCCTCTGCATTGTGCTGTAGAAAAAAACCAAATTGAAAGCGTTAAGTTTCTTCTCAGCAGAGGAGCAAACCCAAATCTCCGAAACTTCAACATGATGGCTCCTCTCCACATAGCTGTGCAGGGCATGAATAATGAGGTGATGAAGGTCTTGCTTGAGCATAGAACTATTGATGTTAATTTGGAAGGAGAAAATGGAAACACAGCTGTGATCATTGCGTGCACCACAAATAATAGCGAAGCATTGCAGATTTTGCTTAAAAAAGGAGCTAAGCCATGTAAATCAAATAAATGGGGATGTTTCCCTATTCACCAAGCTGCATTTTCAGGTTCCAAAGAATGCATGGAAATAATACTAAGGTTTGGTGAAGAGCATGGGTACAGTAGACAGTTGCACATTAACTTTATGAATAATGGGAAAGCCACCCCTCTCCACCTGGCTGTGCAAAATGGTGACTTGGAAATGATCAAAATGTGCCTGGACAATGGTGCACAAATAGACCCAGTGGAGAAGGGAAGGTGCACAGCCATTCATTTTGCTGCCACCCAGGGAGCCACTGAGATTGTTAAACTGATGATATCGTCCTATTCTGGTAGCGTGGATATTGTTAACACAACCGATGGATGTCATGAGACCATGCTTCACAGAGCTTCATTGTTTGATCACCATGAGCTAGCAGACTATTTAATTTCAGTGGGAGCAGATATTAATAAGATCGATTCTGAAGGACGCTCTCCACTTATATTAGCAACTGCTTCTGCATCTTGGAATATTGTAAATTTGCTACTCTCTAAAGGTGCCCAAGTAGACATAAAAGATAATTTTGGACGTAATTTTCTGCATTTAACTGTACAGCAACCTTATGGATTAAAAAATCTGCGACCTGAATTTATGCAGATGCAACAGATCAAAGAGCTGGTAATGGATGAAGACAACGATGGGTGTACTCCTCTACATTATGCATGTAGACAGGGGGGCCCTGGTTCTGTAAATAACCTACTTGGCTTTAATGTGTCCATTCATTCCAAAAGCAAAGATAAGAAATCACCTCTGCATTTTGCAGCCAGTTATGGGCGTATCAATACCTGTCAGAGGCTCCTACAAGACATAAGTGATACGAGGCTTCTGAATGAAGGTGACCTTCATGGAATGACTCCTCTCCATCTGGCAGCAAAGAATGGACATGATAAAGTAGTTCAGCTTCTTCTGAAAAAAGGTGCATTGTTTCTCAGTGACCACAATGGCTGGACAGCTTTGCATCATGCGTCCATGGGCGGGTACACTCAGACCATGAAGGTCATTCTTGATACTAATTTGAAGTGCACAGATCGCCTGGATGAAGACGGGAACACTGCACTTCACTTTGCTGCAAGGGAAGGCCACGCCAAAGCCGTTGCGCTTCTTCTGAGCCACAATGCTGACATAGTCCTGAACAAGCAGCAGGCCTCCTTTTTGCACCTTGCACTTCACAATAAGAGGAAGGAGGTTGTTCTTACGATCATCAGGAGCAAAAGATGGGATGAATGTCTTAAGATTTTCAGTCATAATTCTCCAGGCAATAAATGTCCAATTACAGAAATGATAGAATACCTCCCTGAATGCATGAAGGTACTTTTAGATTTCTGCATGTTGCATTCCACAGAAGACAAGTCCTGCCGAGACTATTATATCGAGTATAATTTCAAATATCTTCAATGTCCATTAGAATTCACCAAAAAAACACCTACACAGGATGTTATATATGAACCGCTTACAGCCCTCAACGCAATGGTACAAAATAACCGCATAGAGCTTCTCAATCATCCTGTGTGTAAAGAATATTTACTCATGAAATGGTTGGCTTATGGATTTAGAGCTCATATGATGAATTTAGGATCTTACTGTCTTGGTCTCATACCTATGACCATTCTCGTTGTCAATATAAAACCAGGAATGGCTTTCAACTCAACTGGCATCATCAATGAAACTAGTGATCATTCAGAAATACTAGATACCACGAATTCATATCTAATAAAAACTTGTATGATTTTAGTGTTTTTATCAAGTATATTTGGGTATTGCAAAGAAGCGGGGCAAATTTTCCAACAGAAAAGGAATTATTTTATGGATATAAGCAATGTTCTTGAATGGATTATCTACACGACGGGCATCATTTTTGTGCTGCCCTTGTTTGTTGAAATACCAGCTCATCTGCAGTGGCAATGTGGAGCAATTGCTGTTTACTTCTATTGGATGAATTTCTTATTGTATCTTCAAAGATTTGAAAATTGTGGAATTTTTATTGTTATGTTGGAGGTAATTTTGAAAACTTTGTTGAGGTCTACAGTTGTATTTATCTTCCTTCTTCTGGCTTTTGGACTCAGCTTTTACATCCTCCTGAATTTACAGGATCCCTTCAGCTCTCCATTGCTTTCTATAATCCAGACCTTCAGCATGATGCTAGGAGATATCAATTATCGAGAGTCCTTCCTAGAACCATATCTGAGAAATGAATTGGCACATCCAGTTCTGTCCTTTGCACAACTTGTTTCCTTCACAATATTTGTCCCAATTGTCCTCATGAATTTACTTATTGGTTTGGCAGTTGGCGACATTGCTGAGGTCCAGAAACATGCATCATTGAAGAGGATAGCTATGCAGGTGGAACTTCATACCAGCTTAGAGAAGAAGCTGCCACTTTGGTTTCTACGCAAAGTGGATCAGAAATCCACCATCGTGTATCCCAACAAACCCAGATCTGGTGGGATGTTATTCCATATATTCTGTTTTTTATTTTGCACTGGGGAAATAAGACAAGAAATACCAAATGCTGATAAATCTTTAGAAATGGAAATATTAAAGCAGAAATACCGGCTGAAGGATCTTACTTTTCTCCTGGAAAAACAGCATGAGCTCATTAAACTGATCATTCAGAAGATGGAGATCATCTCTGAGACAGAGGATGATGATAGCCATTGTTCTTTTCAAGACAGGTTTAAGAAAGAGCAGATGGAACAAAGGAATAGCAGATGGAATACTGTGTTGAGAGCAGTCAAGGCAAAAACACACCATCTTGAGCCTTAG
MSP2N2 amino acid sequence (SEQ ID NO: 62):
STFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQE
MSP2N2 base sequence (SEQ ID NO: 63):
TCTACCTTCAGTAAACTTCGCGAACAACTGGGCCCCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGCGTCAGGAAATGTCCAAAGATTTAGAAGAGGTGAAGGCCAAGGTTCAGCCATATCTCGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCGTCAAAAGGTGGAACCGCTGCGTGCGGAACTGCAAGAGGGGGCACGCCAAAAACTCCATGAGCTCCAAGAGAAGCTCAGCCCATTAGGCGAAGAAATGCGCGATCGCGCCCGTGCACATGTTGATGCACTCCGGACTCATTTGGCGCCGTATTCGGATGAACTTCGCCAGCGTTTGGCCGCACGTCTCGAGGCGCTGAAAGAAAACGGGGGTGCCCGCTTGGCTGAGTACCACGCGAAAGCGACAGAACACCTGAGCACCTTGAGCGAAAAAGCGAAACCGGCGCTGGAAGATCTACGCCAGGGCTTATTGCCTGTTCTTGAGAGCTTTAAAGTCAGTTTTCTGTCAGCTCTGGAAGAATATACTAAAAAGCTGAATACCCAGGGTACCCCCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGCGTCAGGAAATGTCCAAAGATTTAGAAGAGGTGAAGGCCAAGGTTCAGCCATATCTCGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCGTCAAAAGGTGGAACCGCTGCGTGCGGAACTGCAAGAG
1.2 preparation of recombinant baculovirus
Taking a cell culture 6-well plate (Nest), carrying out adherent culture on 1X 10 6/2mL GibcoTM Sf9 Insect cells in each well for 30min in a constant temperature and humidity incubator at 27 ℃, taking 100 mu L of an instruction Medium (Sf-900 TM III SFM), adding 10 mu L of a transfection reagent (Cellfectin TM II), adding 5 mu g of recombinant baculovirus plasmid into the other 100 mu L of the instruction Medium, mixing, incubating for 20 min at room temperature, uniformly dripping the transfection complex into the 6-well plate, continuously culturing for 72 h at 27 ℃, centrifuging at 6000rpm for 15 min at 4 ℃, taking supernatant, adding 2% FBS, and preserving at 4 ℃ in a dark place to obtain the P1-generation recombinant baculovirus.
And (3) infecting 2mL of insect cells with the cell density of 5X 10 5/mL sf9 by using the P1 generation recombinant baculovirus according to the proportion of 1:100, adhering to the wall for culturing for 72 hours at the temperature of 27 ℃, centrifuging at 6000rpm for 15 minutes at the temperature of 4 ℃, adding 2% FBS into the supernatant, and preserving at the temperature of 4 ℃ in the dark to obtain the P2 generation recombinant baculovirus.
Separating 40mL sf9 insect cells with the density of 1X 10 6/mL, culturing for 24 hours, taking P2 generation recombinant baculovirus to infect according to the proportion of 1:100, culturing for 96 hours at the temperature of 27 ℃ and the speed of 120rpm, centrifuging for 15 minutes at the speed of 6000rpm at the temperature of 4 ℃, filtering the supernatant by a 0.22 mu m filter device, adding 2 percent FBS, and preserving in dark at the temperature of 4 ℃ to obtain the P3 generation recombinant baculovirus.
Separating 200mL sf9 insect cells with the density of 1X 10 6/mL, culturing for 24 hours, taking the P3 generation recombinant baculovirus to infect according to the proportion of 1:100, culturing for 96 hours at the temperature of 27 ℃ and the speed of 120rpm, centrifuging for 15 minutes at the speed of 6000rpm at the temperature of 4 ℃, filtering the supernatant by a 0.22 mu m filter device, adding 2 percent FBS, and preserving at the temperature of 4 ℃ in a dark place to obtain the P4 generation recombinant baculovirus.
EXAMPLE 2 protein purification
HEK-293S cells are passaged, the concentration is 1.5X10 6/mL,37℃,8％CO2, the culture is carried out for 24 hours under the conditions of 60 percent humidity and 120rpm, the HEK-293S cells are infected by the P4 generation recombinant baculovirus according to the proportion of 1:10, sodium butyrate is added in a shaking table for 12-18 hours, and the final concentration is 10mM.
After 72 hours of incubation, cells were collected by centrifugation at 6000rpm for 15 minutes at 4℃and cell pellet was resuspended with Lysis buffer(50mM HEPES(Sigma-Aldrich,V900477),pH7.4,150mM NaCl(Adamas,82999B),1％ Protease Inhibitor Cocktail,EDTA-Free,0.5％LMNG/0.05％CHS(Anatrace,NG310-CH210),1mM TCEP( Michelin, T819166)), and the membrane was turned over at 4℃for 3 hours. After completion of the membrane dissolution, the supernatant was combined with MBP affinity chromatography packing (NEB, E8021L) at 4℃for 2.5 hours by tumbling at 40000rpm for 45 minutes, the combined supernatant was passed through a gravity column, the hetero proteins were eluted with Wash buffer (25mM HEPES pH7.4, 150mM NaCl,1mMTCEP,0.01% LMNG/0.001% CHS), and the hetero buffer (25mM HEPES,pH7.4, 150mM NaCl,0.01% LMNG/0.001% CHS, 1mM TCEP,40mM Maltose (Adamas, 73054C)) and concentrating the protein of interest with a ultrafiltration tube (Millopore, UFC 910096) and packaging in the quantitative ratio trpa1:msp2n2:soybean lipid=1:3:200 substances. The required Soybean Polar Lipid Extract Polar (Avanti) was calculated, the protein loading needle was used to draw the mother liquor (25 mg/mL of soybean mother liquor), the soybean mother liquor was hit to the bottom of a clean dry glass tube, and then the chloroform in the soybean mother liquor in the glass tube was blow-dried with a nitrogen blower, which resulted in a pale yellow residue at the bottom of the glass tube. Adding SEC buffer, and ultrasound for 10min, wherein the liquid in the glass tube is firstly white turbid and then gradually clarified to become light yellow solution, adding TRPA1, MSP2N2 and soybean into the EP tube proportionally, incubating on ice for 1h, then adding 100mg/mL Bio-Beads SM-2 (Bio-Beads TM SM-2resin, bio-Rad) for removing the detergent in the solution, the mixture was turned over overnight at 4 ℃. The Bio-beads SM-2 was removed by gravity column filtration and the filtered protein solution was concentrated to a volume of 500. Mu.L; 13000rpm,4 ℃ for 10min, performing gel filtration chromatography, wherein the type of gel column is Superrose 6 Increate 10/300GL (cytova), the buffer is SEC buffer (25 mM HEPES, pH7.4, 150mM NaCl), collecting protein sample, testing A280 concentration, and performing SDS-PAGE gel electrophoresis to detect the size and purity of target protein, wherein the SDS-PAGE gel electrophoresis chart is shown in figure 3.
EXAMPLE 3 animal immunization
Immunization of animals (usually mice) with antigen is also a crucial step in the first step of the monoclonal antibody preparation process. Whether the animal has good immune response to the antigen can generate the antibody with high titer and good specificity directly determines the difficulty of later screening monoclonal hybridoma cell strains and the effectiveness of the obtained antibody.
Antigens for immunization we used proteins produced by the protein preparation method of TRPA1-nanodisc in examples 1 and 2. The immunized animals are selected from balb/c and 6-8 weeks female mice, the initial immunization TRPA1-nanodics antigen dosage is 100 mug protein/animal, the animal is mixed with Freund's complete adjuvant according to the volume ratio of 1:1, the insufficient antigen volume is replaced by buffer for purifying the corresponding protein, and the animal is immunized by conventional back multipoint subcutaneous injection after emulsification by an oscillator. And then, carrying out immunization once every 14 days, mixing the antigen with 50 mug/dose of half of the antigen of the primary immunization with Freund's incomplete adjuvant according to the volume ratio of 1:1, replacing the part of the antigen with buffer for purifying the corresponding protein, emulsifying, and carrying out conventional back multipoint subcutaneous injection to immunize animals. In general, after the third immunization is completed, the blood of the mice is taken at intervals of one week, after the serum is separated, the serum diluted by ELISA (TRPA 1-nanodics antigen is coated, gradient (1:100 is started, 3 times diluted 10 times) is added after 2% BAS is blocked) is incubated for 1h, goat anti-mouse HRP (SA 00001-1, protein) is added for 1h, a chromogenic solution is added for developing for 10min, a stop solution is added for stopping the reaction, the absorbance value of OD450nm is tested, the titer of the serum is tested, after each time the serum of the mice is tested at intervals of 7 days, the serum is generally diluted for 1:10000, the OD450nm value is positive, the immune effect is better (figure 4), and the subsequent experiments can be carried out.
EXAMPLE 4 cell fusion
Cell fusion is the most important link of a hybridoma method, whether specific antibodies can be screened is directly influenced by the fusion efficiency, mice with titers reaching fusion requirements are taken, 100 mu g of celiac antigens are reinforced 3 days before fusion, spleen cells are taken, washed twice with serum-free and resistance-free DMEM (20 mL) culture medium, centrifuged, resuspended in 2mL of fusion buffer solution, and counted for later use. Myeloma cell 1 the day before fusion: after 2 passages and collection of SP2/0 mouse myeloma cells (China academy of sciences cell bank, TCM 18) on the day of fusion, the culture medium was removed by centrifugation, washed twice with serum-free, resistance-free DMEM (20 mL), resuspended in 2mL of fusion buffer after centrifugation, and counted for later use. Spleen cells and SP2/0 were taken 8X 10 7 cells each into a new 50mL out centrifuge tube, 20mL of fusion buffer was added, centrifuged together (5 minutes at 500 Xg) 2 times at a ratio of 1:1, then resuspended in 8mL of fusion buffer and added to an electrofusion cuvette for electrofusion, the electrofusion instrument was a BTX ECM2001 fusion instrument with fusion parameters of 48V,40s of alternating current, pulse voltage: 2070V,30 μs, PF,7S. Standing for 10min, adding the whole fusion solution into 600mL of prepared culture medium, wherein the main components in the culture medium are (75% DMEM+20% FBS+1% PS+2% HAT (50×) +1% Gln+1% OPI), and standing and culturing at 37 ℃ for 2h. After mixing, 42 plates were spread with 150 μl per well. The medium with HT added was changed completely on day four of the fusion, and the supernatant was taken on day 7 for testing.
EXAMPLE 5 monoclonal cell selection
Screening monoclonal cells mainly comprises the steps of detecting the supernatant of hybridoma cells, wherein the detection method mainly comprises ELISA and FACS, wherein the ELISA mainly comprises the steps of testing the affinity of the hybridoma supernatant to immune TRPA1-nanodisc antigen, coating 2 mug/mL of TRPA1-nanodisc antigen, adding the hybridoma supernatant for incubation for 1h after 2% BAS is blocked, adding goat anti-mouse HRP for incubation for 1h, adding a chromogenic solution for developing for 10min, adding a stop solution for stopping reaction, testing the absorbance value of OD450 nm), testing the titer of serum, screening positive hybridoma supernatant, carrying out flow screening, wherein a flow cytometer is beckman coulter CytoFLEX, the screened cell line is HEK293S-TRPA1, the cells to be tested are cultured in suspension according to a proper proportion, taking 10 mu l of cells to mix with 10 mu l of trypan blue, adding 5X 10 5 cells into a 96-hole V-shaped dilution plate for counting. Centrifugation at 1500rpm for 5 min, plate removal of medium, incubation with ELISA positive hybridoma supernatant at 4℃for 1h, washing 3 times with PBS, and addition of 1:200 dilution of secondary antibody (jackson, allophycocyanin-AffiniPure F (ab') 2Fragment Donkey Anti-Mouse lgG (H+L)), PBS wash 3 times after incubation for 30 min at 4℃and finally adding 150 μl PBS to resuspend cells per well, transfer to 1.5mL EP tube, or directly loading with microwell plates, beckman CytoFLEX detection, outputting MFI values using Cytoexpert, analyzing data by difference from positive and negative controls to obtain positive results, performing two rounds of subcloning by limiting dilution method, and final screening to obtain positive monoclonal cells as shown in FIGS. 5a-5H and 6 a-6H.
EXAMPLE 6 monoclonal antibody preparation
The obtained positive monoclonal cells were expanded, cultured in suspension in 50mL of serum-free medium, which was inoculated at a density of 3X 10 5～5×105/mL, the cell amount of the hybridomas was counted every day, typically for 4-5 days, the supernatant was collected by centrifugation, 200. Mu.L of the filler of ProteinA was added, the binding was reversed for 2-3 hours, affinity purification (PBS pH 7.3 equilibrated solution, glycine pH 3.0 eluent), the purified antibodies were dialyzed against PBS, the buffer was replaced, the concentration was determined and the purity of the antibodies was detected by SDS-PAGE, and the SDS-PAGE results are shown in FIG. 7.
EXAMPLE 7 monoclonal antibody functional test
The antibody function test is mainly a method for detecting calcium flow by an enzyme-labeled instrument, and the FlexStation multifunctional plate reader produced by the U.S. Molecular Devices can be used for effectively reading fluorescence intensity change caused by calcium ions flowing into cells through ion channels. HEK293T cell lines with stable TRPA1 expression were plated onto 96-well plates in advance for adherent culture, grown to appropriate density, the culture was discarded, and simultaneously a membrane permeable Calcium ion concentration indicator (FLIPR Calcium 5Assay kit) and antibody solutions of different concentrations (concentration gradient adjusted as required) prepared with extracellular buffer were added, cells were incubated in an incubator for at least 60 minutes, positive small molecule a967079, an inhibitor specific for TRPA1 channels, blank control extracellular buffer as solvent, and extracellular buffer (10% DMSO/HBSS or PBS) containing the corresponding concentration of cosolvent DMSO. Placing the 96-well plate in a full-process light-shielding place, placing the 96-well plate in a FlexStation3 multifunctional plate reader (Molecular Devices) after incubation is completed, loading an agonist AITC (propyl isothiocyanate) sample plate of TRPA1 into a sample applicator of an enzyme label instrument in advance, setting the concentration to 400 mu M (the final concentration in the well is 100 mu M), setting a test program, immediately starting to monitor the change of the fluorescence intensity in the column when one row of agonist is added, monitoring for at least two minutes, calculating the fluorescence increment of all the wells before and after the addition of the agonist according to a quantified fluorescence intensity change curve outputted by a machine after all the well plate tests are completed, carrying out homogenization treatment with the fluorescence increment of corresponding blank control wells to obtain the inhibition rate under the action of antibodies with different concentrations, setting 2 compound wells for each antibody concentration, and obtaining a inhibition curve of the inhibition rate by concentration logarithm in graphpad (Y=100/(1+10 ((LogIC-X)) and calculating the half-inhibition concentration IC 50 of an antibody inhibition channel, so as to judge the inhibition effect of the antibody on the A1, and the result is shown in fig. 8a-8 i. 33E8, 20D5, 38B3, 20E6, 4A11, 39C6, 33E6, and 36F6 have ICs 50 of 0.65. Mu.M, 1.54. Mu.M, 0.70. Mu.M, 0.86. Mu.M, 0.56. Mu.M, 0.66. Mu.M, 1.18. Mu.M, and 0.56. Mu.M, respectively.
Example 8 acquisition of antibody sequences
Screening the obtained monoclonal antibodies, resuscitating hybridoma cells, collecting cells after the density reaches 5X 10 6/mL, completing the extraction of RNA of the monoclonal cells according to FastPure Cell/Tissue Total RNA Isolation Kit of the North praise, using the extracted RNA as a template, completing the amplification of variable regions of VH and VL chains of the monoclonal antibodies by using HiScript-TS 5'/3' RACE Kit of the North praise, determining the concentration after the gel electrophoresis, completing the addition of base A at two ends of VH and VL by using 2X TAQ MASTER Mix, completing the construction of vectors by using pMDTM-T Vector Cloning Kit of TaKaRa, completing the coating, then picking up monoclonal colony culture, carrying out sequencing, and obtaining the sequences of VH and VL after the sequence alignment (shown in tables 1-8).
TABLE 1 33E8 antibody V sequence information
TABLE 2 20D5 antibody V sequence information
TABLE 3 38B3 antibody V sequence information
TABLE 4 20E6 antibody V sequence information
TABLE 5 4A11 antibody V sequence information
TABLE 6 39C6 antibody V sequence information
TABLE 7 33E6 antibody V sequence information
TABLE 8V sequence information for 36F6 antibody
Wherein the CDR sequences of the heavy chain variable region and the light chain variable region of the antibodies 33E8, 20D5, 38B3, 20E6 and 33E6 are very similar, the CDR sequences of the antibodies are summarized as follows:
SEQ ID NO. 56: GYX 1 FTDYW, wherein X 1 is T or K. SEQ ID NO. 57: IDSSDSYX 2, wherein X 2 is S or T. SEQ ID NO. 58: x 3 RGDNSGYAI, wherein X 3 is A or V. SEQ ID NO 59: QTIVHX 4 TGNTY, wherein X 4 is S or T.
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