anti-SAR-COV-2 antibody or antigen binding fragment thereof and application thereof
阅读说明：本技术 一种抗sar-cov-2抗体或其抗原结合片段及其应用 (anti-SAR-COV-2 antibody or antigen binding fragment thereof and application thereof ) 是由 万晓春 李俊鑫 何燕 于 2020-06-08 设计创作，主要内容包括：本发明涉及一种抗SAR-COV-2抗体或其抗原结合片段及其应用,具体公开了,其具有SEQ ID No：1-3所示的重链互补决定区,以及SEQ ID No：4-6所示的轻链互补决定区,或具有SEQ ID No：7-9所示的重链互补决定区,以及SEQ ID No：10-12所示的轻链互补决定区,或具有SEQ ID No：13-15所示的重链互补决定区,以及SEQ ID No：16-18所示的轻链互补决定区,或具有SEQ ID No：19-21所示的重链互补决定区,以及SEQ ID No：22-24所示的轻链互补决定区。本发明的抗体为人源化抗体,其副作用低,亲和力和特异性高。(The invention relates to an anti-SAR-COV-2 antibody or an antigen binding fragment thereof and application thereof, and particularly discloses an anti-SAR-COV-2 antibody or an antigen binding fragment thereof, which has the amino acid sequence shown in SEQ ID No: 1-3, and SEQ ID No: 4-6, or a light chain complementarity determining region having a sequence set forth in SEQ ID No: 7-9, and the heavy chain complementarity determining region of SEQ ID No: 10-12, or a light chain complementarity determining region having a sequence set forth in SEQ ID No: 13-15, and SEQ ID No: 16-18, or a light chain complementarity determining region having a sequence set forth in SEQ ID No: 19-21, and SEQ ID No: 22-24. The antibody of the present invention is a humanized antibody, and has low side effects and high affinity and specificity.)
1. An isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof that specifically binds to SAR-COV-2 surface S protein; it has three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) of any one of the following groups:
1)9g8
HCDR1：GGSITTSSDY SEQ ID No：1；
HCDR2：IYYSGRT SEQ ID No：2；
HCDR3：ARRLTYYYDSSGYANWYFDL SEQ ID No：3；
LCDR1：QRFSTF SEQ ID No：4；
LCDR:2：AAS SEQ ID No：5；
LCDR 3: QQSYSIPYS SEQ ID No: 6; or
2)6J19
HCDR1：GFTFSSYS SEQ ID No：7；
HCDR2：ISSSGTFI SEQ ID No：8；
HCDR3：ARERFVGVLDI SEQ ID No：9；
LCDR1：SSNIGRST SEQ ID No：10；
LCDR:2：SSY SEQ ID No：11；
LCDR 3: AAWDDSLNGPV SEQ ID No: 12; or
3)7N13
HCDR1：GFTFSSYS SEQ ID No：13；
HCDR2：ISSSSSTM SEQ ID No：14；
HCDR3：ARGVGATGELFDY SEQ ID No：15；
LCDR1：QGIGNE SEQ ID No：16；
LCDR:2：AAS SEQ ID No：17；
LCDR 3: LQDYNYPRT SEQ ID No: 18; or
4)8L19
HCDR1：GFTFSNYS SEQ ID No：19；
HCDR2：ISTTGTYT SEQ ID No：20；
HCDR3：ARPYYYGSGSPDY SEQ ID No：21；
LCDR1：QSISTF SEQ ID No：22；
LCDR:2：AAS SEQ ID No：23；
LCDR 3: HQTYSKPWT SEQ ID No: 24; or
5)6M9
HCDR1：GFTFRNYD SEQ ID No：25；
HCDR2：ISGSGIDT SEQ ID No：26；
HCDR3：VRGLAGAFDY SEQ ID No：27；
LCDR1：QSVTSGY SEQ ID No：28；
LCDR:2：GTS SEQ ID No：29；
LCDR3：QQHRSSPMYS SEQ ID No：30。
2. An isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof, having a heavy chain variable region and a light chain variable region as shown below;
1)9g8
heavy chain variable region:
QLQLQESGPGLVKPSETLSLTCTVSGGSITTSSDYWGWIRQPPGKGLEWIGSIYYSGRTYYNPSLKSRVTISVDTSKNDFSLKLSSVTAADTAVYYCARRLTYYYDSSGYANWYFDLWGRGTLVTVSS SEQ ID No：31
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQRFSTFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPYSFGQGTKLEIKR SEQ ID No: 32, a first step of removing the first layer; or
2)6J19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSSSEQ ID No：33
light chain variable region:
QSVLTQPPSASGTPGERVTISCSGSSSNIGRSTVSWYQQLPGTAPKLLMYSSYQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG SEQ ID No: 34; or
3)7N13
Heavy chain variable region:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTMYYGDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVGATGELFDYWGQGTLVTASSSEQ ID No：35
light chain variable region:
AIQMTQSPSSLSASVGDRVTITCRATQGIGNELGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIKR SEQ ID No: 36; or
4)8L19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAVSGFTFSNYSMNWVRQAPGKGLEWVSSISTTGTYTHYAGSVKGRFTISRDNAKNSLFLRMNSLRAEDTAVYYCARPYYYGSGSPDYWGQGTLVTVSSSEQ ID No：37
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQSISTFLNWYQQKPGKAPNLLIYAASSLQRGVPSRFTGSGSGTDFTLTISSLQPEDFATYYCHQTYSKPWTFGRGTKVEIER SEQ ID No: 38; or
5)6M9
Heavy chain variable region:
EVQLLESGGGLVKPGGSLRLSCAASGFTFRNYDINWVRQAPGKGLEWVSSISGSGIDTYYGDSVEGRFTVSRDNAESSVLLEMNSLRADDTAVFYCVRGLAGAFDYWGQGTLVTVSSSEQ ID No：39
light chain variable region:
EIVLTQSPGTLSLSPGERATLSCRASQSVTSGYLAWYQQKPGQAPRLLIHGTSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHRSSPMYSFGQGSKLEIKR SEQ ID No：40。
3. the antibody or antigen-binding fragment thereof according to any one of claim 1 or claim 2, which is a humanized antibody or antigen-binding fragment thereof.
4. A nucleotide sequence characterized in that: encoding the antibody or antigen binding fragment thereof of any one of claims 1-3.
5. A carrier, characterized by: comprising the nucleotide sequence of claim 4.
6. A host cell, characterized in that: comprising the vector of claim 5.
7. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3.
8. A detection reagent comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3.
9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 as a detection reagent for: enzyme linked immunosorbent assay, immunoblotting, flow cytometry, immunohistochemical assay or immuno-PCR.
10. A pharmaceutical composition of the isolated antibody or antigen-binding fragment thereof of any one of claims 1-3 and a pharmaceutically acceptable excipient.
11. Use of an anti-SAR-COV-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or a pharmaceutical composition according to claim 10, for the manufacture of a medicament for preventing, treating or ameliorating at least one symptom or indication of SAR-COV-2 infection;
preferably, the at least one symptom or indication is selected from the group consisting of: novel coronavirus pneumonia, pulmonary inflammation, alveolar injury, fever, cough, dyspnea, hypoxemia, acute respiratory distress syndrome, septic shock, coagulation dysfunction, metabolic acidosis, nasal obstruction, watery nasal discharge, angina, diarrhea, organ failure, septic shock and death.
Technical Field
The invention belongs to the field of immunological antibodies, and particularly relates to an anti-SAR-COV-2 (COVID-19) fully human monoclonal antibody and application thereof.
Background
In 2018, 8 of ten worldwide popular drugs are all humanized or humanized monoclonal antibody drugs. The first one is Humira, a fully human monoclonal antibody, which is a monoclonal antibody and is sold in 100 hundred million over 6 years. Since the first monoclonal antibody drug was marketed in 1986, the monoclonal antibody drugs underwent the stages of murine monoclonal antibody drugs (such as Orthoclone OKT3), chimeric monoclonal antibody drugs (Rituximab), humanized monoclonal antibody drugs (Herceptin), and fully human monoclonal antibody drugs (Humira). Because human bodies have anti-mouse antibody reaction (HAMA), murine monoclonal antibody drugs and chimeric monoclonal antibody drugs are gradually eliminated, and the monoclonal antibody drugs occupying the market at present are all humanized monoclonal antibody drugs. Compared with the internationally advanced human antibody production technology, Shenzhen and even China have great gap, mainly manifested in the weak innovation ability of the human antibody drug field, few varieties of independent research and development, no report of the market of original humanized monoclonal antibody drug exists at present, and the huge antibody drug market is occupied by foreign drug enterprises. China changes the lagging situation and strives for antibody drug markets with huge consumption potential at home and abroad, and needs to overcome the fully humanized monoclonal antibody technology urgently.
The human monoclonal antibody has high specificity and obvious curative effect on inflammation, cancer, influenza, especially coronavirus infection, etc. COVID-19 is an acute respiratory infectious disease caused by SAR-COV-2 coronavirus, which causes global pandemics in 2020 and seriously threatens human lives and properties. By 22 days 05/2020, 5113375 people are infected with SAR-COV-2 and 330052 people die globally, so far there is still a lack of effective drugs and vaccines. When the new coronavirus invades cells, the specific molecule Spike protein (Spike, S protein) expressed by the virus is required to be combined with a receptor on human cells so as to infect the cells and further expand the cells. The human antibody for neutralizing the virus is a certain specific antibody generated by human B lymphocyte, and can be combined with the antigen on the surface of the virus, so that the virus is prevented from adhering to a target cell receptor, the virus is prevented from invading the cell, and the SAR-COV-2 influenza can be effectively prevented and treated. However, no effective human specific antibody is available at present.
Disclosure of Invention
To solve the above problems, the present invention provides an anti-SAR-COV-2 antibody or an antigen-binding fragment thereof, which specifically neutralizes SAR-COV-2.
In one aspect, the invention provides an isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof, which specifically binds to SAR-COV-2 surface S protein; it has three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) of any one of the following groups:
1)9g8
HCDR1：GGSITTSSDY SEQ ID No：1；
HCDR2：IYYSGRT SEQ ID No：2；
HCDR3：ARRLTYYYDSSGYANWYFDL SEQ ID No：3；
LCDR1：QRFSTF SEQ ID No：4；
LCDR:2：AAS SEQ ID No：5；
LCDR 3: QQSYSIPYS SEQ ID No: 6; or
2)6J19
HCDR1：GFTFSSYS SEQ ID No：7；
HCDR2：ISSSGTFI SEQ ID No：8；
HCDR3：ARERFVGVLDI SEQ ID No：9；
LCDR1：SSNIGRST SEQ ID No：10；
LCDR:2：SSY SEQ ID No：11；
LCDR 3: AAWDDSLNGPV SEQ ID No: 12; or
3)7N13
HCDR1：GFTFSSYS SEQ ID No：13；
HCDR2：ISSSSSTM SEQ ID No：14；
HCDR3：ARGVGATGELFDY SEQ ID No：15；
LCDR1：QGIGNE SEQ ID No：16；
LCDR:2：AAS SEQ ID No：17；
LCDR 3: LQDYNYPRT SEQ ID No: 18; or
4)8L19
HCDR1：GFTFSNYS SEQ ID No：19；
HCDR2：ISTTGTYT SEQ ID No：20；
HCDR3：ARPYYYGSGSPDY SEQ ID No：21；
LCDR1：QSISTF SEQ ID No：22；
LCDR:2：AAS SEQ ID No：23；
LCDR 3: HQTYSKPWT SEQ ID No: 24; or
5)6M9
HCDR1：GFTFRNYD SEQ ID No：25；
HCDR2：ISGSGIDT SEQ ID No：26；
HCDR3：VRGLAGAFDY SEQ ID No：27；
LCDR1：QSVTSGY SEQ ID No：28；
LCDR:2：GTS SEQ ID No：29；
LCDR3：QQHRSSPMYS SEQ ID No：30。
In another aspect, the invention provides an isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof, having a heavy chain variable region and a light chain variable region as shown below;
1)9g8
heavy chain variable region:
QLQLQESGPGLVKPSETLSLTCTVSGGSITTSSDYWGWIRQPPGKGLEWIGSIYYSGRTYYNPSLKSRVTISVDTSKNDFSLKLSSVTAADTAVYYCARRLTYYYDSSGYANWYFDLWGRGTLVTVSS SEQ ID No：31
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQRFSTFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPYSFGQGTKLEIKR SEQ ID No: 32, a first step of removing the first layer; or
2)6J19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS SEQ ID No：33
light chain variable region:
QSVLTQPPSASGTPGERVTISCSGSSSNIGRSTVSWYQQLPGTAPKLLMYSSYQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG SEQ ID No: 34; or
3)7N13
Heavy chain variable region:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTMYYGDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVGATGELFDYWGQGTLVTASS SEQ ID No：35
light chain variable region:
AIQMTQSPSSLSASVGDRVTITCRATQGIGNELGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIKR SEQ ID No: 36; or
4)8L19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAVSGFTFSNYSMNWVRQAPGKGLEWVSSISTTGTYTHYAGSVKGRFTISRDNAKNSLFLRMNSLRAEDTAVYYCARPYYYGSGSPDYWGQGTLVTVSS SEQ ID No：37
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQSISTFLNWYQQKPGKAPNLLIYAASSLQRGVPSRFTGSGSGTDFTLTISSLQPEDFATYYCHQTYSKPWTFGRGTKVEIER SEQ ID No: 38; or
5)6M9
Heavy chain variable region:
EVQLLESGGGLVKPGGSLRLSCAASGFTFRNYDINWVRQAPGKGLEWVSSISGSGIDTYYGDSVEGRFTVSRDNAESSVLLEMNSLRADDTAVFYCVRGLAGAFDYWGQGTLVTVSS SEQ ID No：39
light chain variable region:
EIVLTQSPGTLSLSPGERATLSCRASQSVTSGYLAWYQQKPGQAPRLLIHGTSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHRSSPMYSFGQGSKLEIKR SEQ ID No：40。
in the technical scheme of the invention, the antibody or the antigen-binding fragment thereof is a humanized antibody, and more preferably a fully humanized antibody.
In the technical scheme of the invention, the antibody is a monoclonal antibody or a polyclonal antibody, and preferably a monoclonal antibody.
In the technical scheme of the invention, the antibody or the antigen binding fragment thereof specifically binds to SAR-COV-2 surface S protein.
In a further aspect of the invention there is provided a nucleotide sequence encoding an antibody or antigen-binding fragment thereof as hereinbefore described.
In a further aspect of the invention there is provided a vector comprising the nucleotide sequence as hereinbefore described.
In a further aspect the invention provides a host cell comprising the aforementioned vector or vectors, preferably the host cell is prokaryotic or eukaryotic, more preferably selected from the group consisting of yeast cells, mammalian cells or other cells suitable for the production of antibodies or antigen binding fragments thereof.
In a further aspect of the invention, there is provided a kit comprising an antibody or antigen-binding fragment thereof as described above.
In yet another aspect, the invention provides a detection reagent comprising an antibody or antigen-binding fragment thereof as described above.
In a further aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof as described above as a detection reagent for: enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western Blot), flow cytometry (FACS), Immunohistochemical (IHC) assay, or immuno-PCR.
In the above-mentioned immunological assay, the antibody or antigen-binding fragment thereof may be attached alone or in combination with a conjugate such as horseradish peroxidase (HRP), Alkaline Phosphatase (AP), Biotin (Biotin), Fluorescein Isothiocyanate (FITC), Cy3, Cy5, magnetic beads and agarose, by coupling via a chemical bond, electrostatic adsorption or hydrophilic-hydrophobic adsorption.
In the technical scheme of the invention, the detection reagent can be used for detection for non-diagnosis or treatment purposes.
In a further aspect of the invention, there is provided a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable excipient.
In an embodiment of the invention, wherein the antibody or antigen-binding fragment thereof blocks or reduces binding of S protein of SAR-COV-2 to a cell surface receptor of the subject, preferably a cellular angiotensin converting enzyme-related carboxypeptidase (ACE 2).
In a further aspect, the invention provides the use of an anti-SAR-COV-2 antibody or antigen-binding fragment thereof in the manufacture of a medicament for the prevention, treatment or amelioration of at least one symptom or indication of SAR-COV-2 infection.
In the technical scheme of the invention, the medicine is an oral or injection preparation.
In a further aspect, the invention provides a method of preventing, treating or ameliorating at least one symptom or indication of SAR-COV-2 infection, the method comprising administering to a subject an antibody or antigen-binding fragment thereof of any of the foregoing or a pharmaceutical composition of the foregoing.
In the technical solutions of the present invention, wherein the at least one symptom or indication is selected from the group consisting of: pulmonary inflammation, alveolar injury, fever, cough, dyspnea, hypoxemia, acute respiratory distress syndrome, septic shock, coagulation dysfunction, metabolic acidosis, nasal congestion, runny nose, sore throat, diarrhea, organ failure, septic shock, and death.
In the technical aspect of the present invention, wherein the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered in combination with a second therapeutic agent. Wherein the second therapeutic agent is selected from the group consisting of: anti-inflammatory drugs (such as corticosteroids and non-steroidal anti-inflammatory drugs), antiviral drugs, different antibodies against the S protein of SAR-COV-2, vaccines for SAR-COV-2, antibiotics, dietary supplements such as antioxidants and any other palliative treatment of SAR-COV-2 infection, drugs to alleviate the above symptoms or indications.
In the technical aspect of the present invention, wherein the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.
Advantageous effects
(1) The anti-SAR-COV-2 antibody disclosed by the invention can be combined with the S protein of SAR-COV-2 virus in a targeted manner, has high specificity, and can effectively block the combination of the S protein on the surface of the SAR-COV-2 virus and a cell surface receptor of a subject.
(2) Compared with the mouse antibody, the gene of the fully human antibody is completely derived from the human gene, has no components of other species, does not generate toxic and side effects such as anti-mouse antibody and the like in a human body, has better biocompatibility, and is more suitable and has more potential to become a macromolecular medicament for treating influenza virus.
(3) Compared with the method for preparing the SAR-COV-2 virus resisting humanized monoclonal antibody by using the phage display technology provided by the prior art, the method for developing the SAR-COV-2 virus resisting antibody by using the single B cell has the advantages of simple and rapid operation, high affinity and specificity of the produced humanized antibody and the like.
Drawings
FIG. 1 is a graph showing the results of flow-based assay of example 1 in which NTH-3T3 expresses CD 40L.
FIG. 2 is a graph showing the results of sorting memory B cells by flow cytometry in example 1.
FIG. 3 is a graph showing the results of ELISA experiments in example 1.
FIG. 4 is a graph showing the results of agarose gel electrophoresis in example 2.
Detailed Description
For a clearer understanding of the technical features, objects and advantages of the present application, reference will now be made to the following detailed description of the embodiments of the present application, with the understanding that the examples are provided for illustration only and are not intended to limit the scope of the present application. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
As used herein, the term "antibody" refers to a molecule comprising at least one antigen binding site that immunospecifically binds to a particular antigen target of interest. Thus, the term "antibody" includes, but is not limited to, full length antibodies and/or variants thereof, fragments thereof, peptide antibodies and variants thereof, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, and antibody mimetics that mimic the structure and/or function of an antibody or designated fragments or portions thereof, including single chain antibodies and fragments thereof. Binding of an antibody to a target can result in a variety of effects, such as, but not limited to, modulation, reduction, increase, antagonism, agonism, alleviation, slowing, blocking, inhibition, elimination and/or interference with at least one target activity or binding, or receptor activity or binding, of such binding in vitro, in situ and/or in vivo. Thus, antibodies of the present disclosure encompass antibody fragments capable of binding a biomolecule (e.g., an antigen or receptor) or a portion thereof, including but not limited to Fab, Fab ', and F (ab ')2, pFc ', Fd, single domain antibodies (sdAb), variable fragments (Fv), single chain variable fragments (scFv), or disulfide-linked fvs (sdfv); a bifunctional or bivalent bifunctional antibody; a linear antibody; a single chain antibody molecule; multispecific antibodies formed from antibody fragments. The antibody may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor amounts of mutations that may naturally occur. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibodies also have the advantage that they can be synthesized uncontaminated with other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.
As used herein, the term HCDR has the same meaning as the heavy chain complementarity determining region and LCDR has the same meaning as the light chain complementarity determining region.
As used herein, monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular anti-class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of these antibodies, exhibit the desired biological activity.
As used herein, the term "SAR-COV-2," also known as "novel coronavirus," refers to a newly-occurring virus that causes novel coronavirus pneumonia (COVID-19).
As used herein, the S protein refers to the Spike protein (Spike protein) on coronavirus, and SARS-CoV-2 recognizes ACE2 on the cell surface in the human body through the Spike protein on the virus surface and infects host cells. The S protein on the surface of the coronavirus SARS-CoV-2 can be blocked to effectively inhibit the virus from adhering to a target cell receptor and prevent the virus from invading cells.
The term "humanized antibody" as used herein includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The humanized antibodies of the invention may comprise amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or in vitro site-specific mutagenesis or by in vivo somatic mutation).
The term anti-antigen-binding fragment, etc., as used herein includes any naturally occurring, enzymatically available, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The term "antigen-binding fragment" of an antibody as used herein refers to one or more fragments of the S protein of SAR-COV-2 that have the ability to bind.
In one aspect, the invention provides an isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof that specifically binds to SAR-COV-2 surface S protein; it has three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) of any one of the following groups:
1)9g8
HCDR1：GGSITTSSDY SEQ ID No：1；
HCDR2：IYYSGRT SEQ ID No：2；
HCDR3：ARRLTYYYDSSGYANWYFDL SEQ ID No：3；
LCDR1：QRFSTF SEQ ID No：4；
LCDR:2：AAS SEQ ID No：5；
LCDR 3: QQSYSIPYS SEQ ID No: 6; or
2)6J19
HCDR1：GFTFSSYS SEQ ID No：7；
HCDR2：ISSSGTFI SEQ ID No：8；
HCDR3：ARERFVGVLDI SEQ ID No：9；
LCDR1：SSNIGRST SEQ ID No：10；
LCDR:2：SSY SEQ ID No：11；
LCDR 3: AAWDDSLNGPV SEQ ID No: 12; or
3)7N13
HCDR1：GFTFSSYS SEQ ID No：13；
HCDR2：ISSSSSTM SEQ ID No：14；
HCDR3：ARGVGATGELFDY SEQ ID No：15；
LCDR1：QGIGNE SEQ ID No：16；
LCDR:2：AAS SEQ ID No：17；
LCDR 3: LQDYNYPRT SEQ ID No: 18; or
4)8L19
HCDR1：GFTFSNYS SEQ ID No：19；
HCDR2：ISTTGTYT SEQ ID No：20；
HCDR3：ARPYYYGSGSPDY SEQ ID No：21；
LCDR1：QSISTF SEQ ID No：22；
LCDR:2：AAS SEQ ID No：23；
LCDR 3: HQTYSKPWT SEQ ID No: 24; or
5)6M9
HCDR1：GFTFRNYD SEQ ID No：25；
HCDR2：ISGSGIDT SEQ ID No：26；
HCDR3：VRGLAGAFDY SEQ ID No：27；
LCDR1：QSVTSGY SEQ ID No：28；
LCDR:2：GTS SEQ ID No：29；
LCDR3：QQHRSSPMYS SEQ ID No：30。
Another aspect provides an isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof, having a heavy chain variable region and a light chain variable region as shown below;
1)9g8
heavy chain variable region:
QLQLQESGPGLVKPSETLSLTCTVSGGSITTSSDYWGWIRQPPGKGLEWIGSIYYSGRTYYNPSLKSRVTISVDTSKNDFSLKLSSVTAADTAVYYCARRLTYYYDSSGYANWYFDLWGRGTLVTVSS SEQ ID No：31
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQRFSTFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPYSFGQGTKLEIKR SEQ ID No: 32, a first step of removing the first layer; or
2)6J19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS SEQ ID No：33
light chain variable region:
QSVLTQPPSASGTPGERVTISCSGSSSNIGRSTVSWYQQLPGTAPKLLMYSSYQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG SEQ ID No: 34; or
3)7N13
Heavy chain variable region:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTMYYGDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVGATGELFDYWGQGTLVTASS SEQ ID No：35
light chain variable region:
AIQMTQSPSSLSASVGDRVTITCRATQGIGNELGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIKR SEQ ID No: 36; or
4)8L19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAVSGFTFSNYSMNWVRQAPGKGLEWVSSISTTGTYTHYAGSVKGRFTISRDNAKNSLFLRMNSLRAEDTAVYYCARPYYYGSGSPDYWGQGTLVTVSS SEQ ID No：37
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQSISTFLNWYQQKPGKAPNLLIYAASSLQRGVPSRFTGSGSGTDFTLTISSLQPEDFATYYCHQTYSKPWTFGRGTKVEIER SEQ ID No: 38; or
5)6M9
Heavy chain variable region:
EVQLLESGGGLVKPGGSLRLSCAASGFTFRNYDINWVRQAPGKGLEWVSSISGSGIDTYYGDSVEGRFTVSRDNAESSVLLEMNSLRADDTAVFYCVRGLAGAFDYWGQGTLVTVSS SEQ ID No：39
light chain variable region:
EIVLTQSPGTLSLSPGERATLSCRASQSVTSGYLAWYQQKPGQAPRLLIHGTSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHRSSPMYSFGQGSKLEIKR SEQ ID No：40。
in some embodiments, the antibody is a humanized antibody, and in particular embodiments, a fully humanized antibody is preferred.
In some embodiments, the antibody is a monoclonal or polyclonal antibody, preferably a monoclonal antibody.
In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to SAR-COV-2 surface S protein.
In some embodiments, the amino acid sequence of the heavy chain variable region or the light chain variable region of the antibody may be an amino acid sequence with equivalent functions formed by replacing, deleting or adding one or more amino acids.
In some embodiments, the anti-SAR-COV-2 antibodies or antigen-binding fragments of the invention can target the S protein of SAR-COV-2 virus as demonstrated by ELISA experiments. Compared with a mouse antibody, the gene of the fully human antibody is completely derived from the human gene, has no other species of components, does not generate toxic and side effects such as anti-mouse anti-antibody and the like in a human body, has better biocompatibility, and is more suitable and has more potential to become a macromolecular drug for treating influenza virus.
In another aspect, the present application provides genes encoding anti-SAR-COV-2 fully human monoclonal antibodies described herein. In some embodiments, the gene comprises a nucleotide sequence encoding an amino acid having the amino acids set forth above.
In some embodiments, the nucleotide sequence is as follows (the following sequences are exemplary only, and one skilled in the art can design other nucleotide sequences that can be translated into the desired amino acid sequence depending on the particular amino acid sequence):
1)9g8
the nucleotide sequence encoding the heavy chain variable region is:
gtctctggtggctctatcaccactagtagtgactactggggctggatccgccagcccccagggaaggggctggagtggattgggagtatctattatagtgggagaacctactacaacccgtccctcaagagtcgagtcaccatatccgtagacacgtccaagaacgacttctctctgaagctgagctctgtgaccgccgcagacacggctgtgtattactgtgcgagacgccttacgtattactatgatagtagtggttatgcgaactggtacttcgatctctggggccgtggcaccctggtcactgtctcctca；SEQ ID No：41
the nucleotide sequence encoding the light chain variable region is:
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagaggttcagcacctttttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcacgatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtatcccgtactcttttggccaggggaccaagctggagatcaaacga SEQ ID No：42
2)6J19
the sequence encoding the heavy chain variable region is:
gaggtgcagctggtggagtctgggggaggccTggtcaAgcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagttatagtatgaagtgggtccgccaggctccagggaaggggctggagtgggtctcaaccatcagtagtagtggtactttcataaagtatgcagactcactgcagggccgattcaccatcaccagagacaacgccaagaccgcagtgtatctgcaaatgaacagcctgagagtcgaggacacggctgtttattactgtgcgagagaacgattcgttggtgttttggatatctggggccaagggacaatggtcaccgtctcttca；SEQ ID No：43
the nucleotide sequence encoding the light chain variable region is:
Cagtctgtgctgactcagccaccctcagcgtctgggacccccggggagagggtcaccatctcttgttctggaagcagctccaacatcggaaggagtactgtaagctggtaccagcagctcccaggaacggcccccaaactcctcatgtatagtagttatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgtgcagcatgggatgacagcctgaatggtccggtgttcggcggagggaccaagctgaccgtcctaggt SEQ ID No：44
3)7N13
the nucleotide sequence encoding the heavy chain variable region is:
GaggtgcagctggtggagtctgggggaggcttgGtacagcCTGgggggTCcctgagactctcctgtgcagcctctggattcaccttcagtagttatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtttcatatattagtagtagtagtagtaccatgtactacggagactctgtgaagggccgattcaccatctccagagacaatgccaagaactcactgtatctgcaaatgaacagcctgagagacgaggacacggctgtgtattactgtgcgagaggagtgggagccacgggggaactctttgactactggggccagggaaccctggtcaccgcctcctca：SEQ ID No：45
the nucleotide sequence encoding the light chain variable region is:
gccaTccagatgacccaGTCtccatCctccctgtctgcatCtgtaggagacagagtcaccatcacttgccgggcaactcagggaattggaaatgaattagggtggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttacaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatTttgcaacttattactgtctacaagattacaattaccctcgtacgttcggccaagggaccaaggtggaaatcaaacga SEQ ID No：46
4)8L19
the nucleotide sequence encoding the heavy chain variable region is:
gaggtgcagctggtggagtctgggggaggccTggtcaAgcctggggggtccctgagactctcctgtgcagtctctggattcaccttcagtaactatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatccattagtactactggtacttacacacactacgccggctcagtgaagggccgattcaccatctccagagacaatgccaagaactcgctgtttttgcgaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaggccctattactatggttcggggagtcctgactactggggccagggaaccctggtcaccgtctcctca SEQ IDNo：47
the nucleotide sequence encoding the light chain variable region is:
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcacctttttgaattggtatcagcagaagcccgggaaagcccctaatctcctgatctatgctgcatccagtttgcaacgtggggtcccatcaaggttcactggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaccagacttacagtaagccctggacgttcggccgagggaccaaggtggaaatcgaacga SEQ ID No：48
5)6M9
the nucleotide sequence encoding the heavy chain variable region is:
Gaggtgcagctgttggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcaggaactatgacatcaactgggtccgccaggctccagggaaggggctggagtgggtctcatcgattagtggtagtggtattgacacatactacggagactcagtggagggccgattcaccgtctccagagacaacgccgagagctcagtattactggagatgaacagcctgagagccgacgatacggctgtattttactgtgtgaggggtctggctggcgcctttgactactggggccagggaaccctggtcaccgtctcctca SEQ ID No：49
the nucleotide sequence encoding the light chain variable region is:
GaaattGtGttgacGcagTCtccagGcAccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttaccagcggctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatccatggtacatccaggagggccactggcatcccagacaggttcagtggcagtgggtctgggacagatttcactctcaccatcagcagactggagcctgaagattttgcagtctattactgtcagcagcatcgtagctcacccatgtacagttttggccaggggagcaagctggagatcaaacga SEQ ID No：50
in another aspect, the invention provides a vector comprising a nucleotide sequence as described above.
In a further aspect, the present application provides a cell comprising a nucleotide sequence as described above or a vector as described above.
In still another aspect, the present application provides a method for producing the SAR-COV-2-resistant fully human monoclonal antibody or a bioactive fragment derived from the monoclonal antibody and capable of specifically binding to SAR-COV-2, comprising culturing genetically engineered cells containing the above genes or the above vectors encoding the heavy and light chains of the SAR-COV-2-resistant fully human monoclonal antibody or directly culturing the above cells, collecting and purifying to obtain the SAR-COV-2-resistant fully human monoclonal antibody.
In the prior art, a method for preparing an anti-SAR-COV-2 virus humanized monoclonal antibody by adopting a phage display technology exists, although the method has the advantages of low production cost and no complicated work such as immunization, cell fusion and the like, the method has obvious defects, and the antibody obtained from a non-immune antibody library is often insufficient in affinity, limited by the conversion rate of foreign genes, insufficient in library capacity of the antibody library to cover the antibody diversity of animals and the like. Therefore, the present invention provides a method for screening an antibody, which comprises isolating a B cell secreting a functional antibody from blood of a patient, then extracting RNA and synthesizing cDNA, cloning a gene secreting a target antibody therefrom, and finally recombining and expressing a fully human monoclonal antibody. The technology is simple and quick to operate, the produced humanized antibody has high affinity and specificity, and in addition, the technology of the monoclonal antibody with the virus neutralizing or tumor killing function separated from the memory B cells can be further improved, so that the complicated operation and cost are greatly reduced.
In another aspect, the present application provides a pharmaceutical composition comprising an anti-SAR-COV-2 fully human monoclonal antibody described herein or a biologically active fragment derived therefrom that is capable of specifically binding SAR-COV-2.
In another aspect, the application provides an application of the anti-SAR-COV-2 fully human monoclonal antibody or a bioactive fragment which is derived from the monoclonal antibody and can specifically bind to SAR-COV-2, or the pharmaceutical composition in the preparation of a medicine for treating diseases caused by SAR-COV-2 virus.
In another aspect, the present application provides a kit for detecting the level of SAR-COV-2 virus, which comprises the anti-SAR-COV-2 fully human monoclonal antibody described in the present application or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding SAR-COV-2; in some embodiments, the kit further comprises a second antibody and an enzyme for detection or a fluorescent or radioactive label, and a buffer; the second antibody is, for example, an anti-antibody against a monoclonal antibody described herein.
Example 1
(1) Construction of NTH-3T3 cell line stably expressing CD40L (3T3-CD40L)
Lentivirus was used to establish 3T3-CD40L feeder cells. Constructing a lentivirus expression vector pLVX-CD40L, transfecting 293T cells, and collecting virus supernatant on the fourth day of transfection. NIH-3T3 cells were activated, cultured for 3 passages, infected with lentivirus, cultured further and passaged 3 times. Sorting cells with FITC fluorescence intensity near MFI by using a flow cytometer, adding the cells into a culture bottle again at 37 ℃ and 5% CO2The cells were cultured in an incubator and tested as shown in FIG. 1, in which 3T3 cells expressing CD40L and 3T3 cells transfected with an empty vector pLVX (with ZxGreen) were stained with anti-CD 40L with APC, respectively, and then analyzed by flow cytometry. As a result, all 3T3-CD40L feeder cells were found to express CD 40L. When the cells grow to 80% -90%, the cells are collected by digestion at a concentration of 80% per ml1×107A cell. Placing in an irradiator for 5000rads irradiation, and resuspending the cells in the frozen stock solution at a concentration of 3.5 × 10/ml7The cells are packed in 1ml of freezing tubules and frozen in liquid nitrogen (can be stored for 2 years).
(2) Sorting and activation of memory B cells
Isolation and cryopreservation of Peripheral Blood Mononuclear Cells (PBMC) from convalescent patients infected with SAR-COV-2 virus using lymph separation medium, 10-50X 10 per tube6Cells, frozen in liquid nitrogen tank. PBMC flow staining solutions were prepared, and the components thereof are shown in Table 1 below
TABLE 1 PBMC flow staining solution
Antibodies
Volume (μ L)
CD19-PE-Cy7
0.5
IgM-PE
1.0
IgA-APC
2.5
IgD-FITC
2.5
PBS-1％(wt/vol)BSA
43.5
Thawing PBMC, adding the above PBMC flow staining solution and sorting on flow cytometer, and sorting out CD19 as shown in FIG. 2+IgM–IgA–IgD–The purity of the memory B cells is required to be more than 90%, and if the purity of the memory B cells is less than 90%, the sorting process is repeated. A mixed medium for activating B cells was prepared as shown in table 2 below:
TABLE 2
Components
Volume of
Complete IMDM Medium
336mL
IL-2(10,000U mL-1)
3.5mL
IL-21(100μg mL-1)
175μL
3T3-CD40L obtained in step (1)
10mL
Adding memory B cells into mixed culture medium, mixing, diluting in 384-well plate with 1 cell per well and 50 μ l volume, standing at 37 deg.C and 5% CO2And (5) standing and culturing in an incubator. After 13 days, the supernatant was collected to obtain human monoclonal antibodies 9g8, 6J19, 7N13, 8L19 and 6M 9.
(3) Experiment of binding of human monoclonal antibody to surface antigen S protein of SAR-COV-2 virus
The surface antigen S protein of the SAR-COV-2 virus is purchased from Cassia Proteus, has immunogenicity, and the anti-S protein antibody can be used for detecting and treating the SAR-COV-2 coronavirus. ELISA experiments were performed on the 5 human monoclonal antibodies 6J19, 7N13, 8L19 and 6M9 obtained above, specifically:
(1) coating 100ng/100 mu l of HA protein of SAR-COV-2 virus in a 96-well enzyme label plate, wherein each well is 100 mu l;
(2) standing in a refrigerator at 4 deg.C overnight;
(3) washing with PBST solution for three times, adding 5% skimmed milk powder solution 200 μ l per well, and incubating at 37 deg.C for 1 hr;
(4) three washes with PBST solution, 100 μ l of normal human serum without virus infection (negative control) or supernatant, triplicate each;
(5) incubation for 1 hour at 37 ℃ followed by three washes with PBST solution;
(6) adding 100 mul of HRP-carrying anti-human IgG antibody (abcam) diluted at a ratio of 1:5000 into an enzyme-labeled plate;
(7) incubation for 1 hour at 37 ℃ followed by three washes with PBST solution;
(8) add 100. mu.l TMB substrate solution (Thermo Scientific) to each well for 5min at 37 ℃;
(9) the stop solution 2M sulfuric acid 100. mu.l was added to each well, and the absorbance was immediately measured at a wavelength of 450nm in a microplate reader. As shown in FIG. 3, ELISA experiments show that the human monoclonal antibodies 6J19, 7N13, 8L19 and 6M9 obtained by the invention can be targeted to bind to the S protein of SAR-COV-2 virus.
EXAMPLE 2 cloning of humanized monoclonal antibody Gene
The B cell capable of secreting antibody binding to SAR-COV-2 virus obtained in example 1 was lysed, and the lysate was taken for reverse transcription of RNA to obtain PCR template cDNA of human antibody gene. Primers for cloning antibody genes were designed and synthesized, genes for heavy and light chains of the antibody were cloned using cDNA as a template, and sequenced by Jinzhi corporation. Specifically, the method comprises the following steps:
(1) the lysed B cell fluid was transferred to a 96-well plate (Eppendorf, 030133366).
(2) Reverse transcription system: 150ng random primer (Invitrogen,48190-se Inhibitor(Eppendorf)and 50UIII reverse transcriptase (Invitrogen,18080-044), DEPC water was supplied to 14. mu.l/well.
(3) Reverse transcription reaction procedure: 42 ℃ for 10 min; at 25 ℃ for 10 min; 50 ℃ for 60 min; 94 ℃ for 5 min.
(4) The cDNA was stored at-20 ℃.
(5) Design and synthesis of primers:
forward Primer 5 '-3' sequence (Forward Primer 5 '-3' sequence)
Heavy chain variable region PCR primers:
5′VH1 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGGTGCAG(SEQ ID NO:51)
5′VH1/5 CTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGCAG(SEQ ID NO:52)
5′VH3 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGGTGGAG(SEQ ID NO:53)
5′VH3-23 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGTTGGAG(SEQ ID NO:54)
5′VH4 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGCAGGAG(SEQ ID NO:55)
5′VH 4-34CTGCAACCGGTGTACATTCCCAGGTGCAGCTACAGCAGTG(SEQ ID NO:56)
5′VH 1-18 CTGCAACCGGTGTACATTCCCAGGTTCAGCTGGTGCAG(SEQ ID NO:57)
5′VH 1-24 CTGCAACCGGTGTACATTCCCAGGTCCAGCTGGTACAG(SEQ ID NO:58)
5′VH3-33 CTGCAACCGGTGTACATTCTCAGGTGCAGCTGGTGGAG(SEQ ID NO:59)
5′VH 3-9 CTGCAACCGGTGTACATTCTGAAGTGCAGCTGGTGGAG(SEQ ID NO:60)
5′VH4-39 CTGCAACCGGTGTACATTCCCAGCTGCAGCTGCAGGAG(SEQ ID NO:61)
5′VH 6-1 CTGCAACCGGTGTACATTCCCAGGTACAGCTGCAGCAG(SEQ ID NO:62)
3′JH 1/2/4/5 TGCGAAGTCGACGCTGAGGAGACGGTGACCAG(SEQ ID NO:63)
3′JH 3 TGCGAAGTCGACGCTGAAGAGACGGTGACCATTG(SEQ ID NO:64)
3′JH 6 TGCGAAGTCGACGCTGAGGAGACGGTGACCGTG(SEQ ID NO:65)
kappa light chain variable region PCR primer
5′Vκ 1-5 CTGCAACCGGTGTACATTCTGACATCCAGATGACCCAGTC(SEQ ID NO:66)
5′Vκ 1-9 TTGTGCTGCAACCGGTGTACATTCAGACATCCAGTTGACCC AGTCT(SEQ ID NO:67)
5′Vκ 1D-43 CTGCAACCGGTGTACATTGTGCCATCCGGATGACCCAGTC(SEQ ID NO:68)
5′Vκ 2-24 CTGCAACCGGTGTACATGGGGATATTGTGATGACCCAGAC(SEQ ID NO:69)
5′Vκ 2-28 CTGCAACCGGTGTACATGGGGATATTGTGATGACTCAGTC(SEQ ID NO:70)
5′Vκ 2-30 CTGCAACCGGTGTACATGGGGATGTTGTGATGACTCAGTC(SEQ ID NO:71)
5′Vκ 3-11 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACAC AGTC(SEQ ID NO:72)
5′Vκ 3-15 CTGCAACCGGTGTACATTCAGAAATAGTGATGACGCAGTC(SEQ ID NO:73)
5′Vκ 3-20 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACG CAGTCT(SEQ ID NO:74)
5′Vκ 4-1 CTGCAACCGGTGTACATTCGGACATCGTGATGACCCAGTC(SEQ ID NO:75)
3′Jκ 1/4 GCCACCGTACGTTTGATYTCCACCTTGGTC(SEQ ID NO:76)
3′Jκ 2 GCCACCGTACGTTTGATCTCCAGCTTGGTC(SEQ ID NO:77)
3′Jκ 3 GCCACCGTACGTTTGATATCCACTTTGGTC(SEQ ID NO:78)
3′Jκ 5 GCCACCGTACGTTTAATCTCCAGTCGTGTC(SEQ ID NO:79)
(6) Heavy and light chains of the antibody gene were PCR amplified using the KOD-Plus-Neo (TOYOBO, KOD401) kit, respectively, in a 40. mu.L system: 3.5. mu.L of cDNA, 20nM mixed primer, 4. mu.L of buffer (buffer), 4. mu.L of 2mM dNTPs, 2.4. mu.L of MgSO 24，1μL KOD。
(7) Reaction procedure: 94 ℃ for 2 min; 45 cycles: 10s at 98 ℃; at 58 ℃ for 30 s; 68 ℃ for 28 s.
(8) As a result of agarose gel analysis, it was found in FIG. 4 that the light chains of the five antibodies were about 300bp, and the heavy chains were about 400bp, respectively.
(9) Sending the PCR amplification product to Jinzhi Biotechnology Ltd for sequencing
(10) The sequences of the 5 antibody variable regions are as follows:
1)9g8
heavy chain variable region:
QLQLQESGPGLVKPSETLSLTCTVSGGSITTSSDYWGWIRQPPGKGLEWIGSIYYSGRTYYNPSLKSRVTISVDTSKNDFSLKLSSVTAADTAVYYCARRLTYYYDSSGYANWYFDLWGRGTLVTVSS SEQ ID No：31
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQRFSTFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPYSFGQGTKLEIKR SEQ ID No: 32, a first step of removing the first layer; or
2)6J19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS SEQ ID No：33
light chain variable region:
QSVLTQPPSASGTPGERVTISCSGSSSNIGRSTVSWYQQLPGTAPKLLMYSSYQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG SEQ ID No: 34; or
3)7N13
Heavy chain variable region:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTMYYGDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVGATGELFDYWGQGTLVTASS SEQ ID No：35
light chain variable region:
AIQMTQSPSSLSASVGDRVTITCRATQGIGNELGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPRTFGQGTKVEIKR SEQ ID No: 36; or
4)8L19
Heavy chain variable region:
EVQLVESGGGLVKPGGSLRLSCAVSGFTFSNYSMNWVRQAPGKGLEWVSSISTTGTYTHYAGSVKGRFTISRDNAKNSLFLRMNSLRAEDTAVYYCARPYYYGSGSPDYWGQGTLVTVSS SEQ ID No：37
light chain variable region:
DIQMTQSPSSLSASVGDRVTITCRASQSISTFLNWYQQKPGKAPNLLIYAASSLQRGVPSRFTGSGSGTDFTLTISSLQPEDFATYYCHQTYSKPWTFGRGTKVEIER SEQ ID No: 38; or
5)6M9
Heavy chain variable region:
EVQLLESGGGLVKPGGSLRLSCAASGFTFRNYDINWVRQAPGKGLEWVSSISGSGIDTYYGDSVEGRFTVSRDNAESSVLLEMNSLRADDTAVFYCVRGLAGAFDYWGQGTLVTVSS SEQ ID No：39
light chain variable region:
EIVLTQSPGTLSLSPGERATLSCRASQSVTSGYLAWYQQKPGQAPRLLIHGTSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHRSSPMYSFGQGSKLEIKR SEQ ID No：40。
correspondingly, the CDR region sequences are shown as follows:
1)9g8
HCDR1：GGSITTSSDY SEQ ID No：1；
HCDR2：IYYSGRT SEQ ID No：2；
HCDR3：ARRLTYYYDSSGYANWYFDL SEQ ID No：3；
LCDR1：QRFSTF SEQ ID No：4；
LCDR:2：AAS SEQ ID No：5；
LCDR 3: QQSYSIPYS SEQ ID No: 6; or
2)6J19
HCDR1：GFTFSSYS SEQ ID No：7；
HCDR2：ISSSGTFI SEQ ID No：8；
HCDR3：ARERFVGVLDI SEQ ID No：9；
LCDR1：SSNIGRST SEQ ID No：10；
LCDR:2：SSY SEQ ID No：11；
LCDR 3: AAWDDSLNGPV SEQ ID No: 12; or
3)7N13
HCDR1：GFTFSSYS SEQ ID No：13；
HCDR2：ISSSSSTM SEQ ID No：14；
HCDR3：ARGVGATGELFDY SEQ ID No：15；
LCDR1：QGIGNE SEQ ID No：16；
LCDR:2：AAS SEQ ID No：17；
LCDR 3: LQDYNYPRT SEQ ID No: 18; or
4)8L19
HCDR1：GFTFSNYS SEQ ID No：19；
HCDR2：ISTTGTYT SEQ ID No：20；
HCDR3：ARPYYYGSGSPDY SEQ ID No：21；
LCDR1：QSISTF SEQ ID No：22；
LCDR:2：AAS SEQ ID No：23；
LCDR 3: HQTYSKPWT SEQ ID No: 24; or
5)6M9
HCDR1：GFTFRNYD SEQ ID No：25；
HCDR2：ISGSGIDT SEQ ID No：26；
HCDR3：VRGLAGAFDY SEQ ID No：27；
LCDR1：QSVTSGY SEQ ID No：28；
LCDR:2：GTS SEQ ID No：29；
LCDR3：QQHRSSPMYS SEQ ID No：30。
Finally, the description is as follows: the above embodiments are only used for illustrating the implementation processes and features of the present application, and not for limiting the technical solutions of the present application, and although the present application is described in detail with reference to the above embodiments, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the present application, and it is intended to cover any modifications or partial substitutions within the scope of the present application.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute of Chinese academy of sciences
<120> anti-SAR-COV-2 antibody or antigen binding fragment thereof and application thereof
<130> CP120010319C
<160> 79
<170> PatentIn version 3.3
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<213> Artificial sequence
<400> 5
Ala Ala Ser
1
<210> 6
<211> 9
<212> PRT
<213> Artificial sequence
<400> 6
Gln Gln Ser Tyr Ser Ile Pro Tyr Ser
1 5
<210> 7
<211> 8
<212> PRT
<213> Artificial sequence
<400> 7
Gly Phe Thr Phe Ser Ser Tyr Ser
1 5
<210> 8
<211> 8
<212> PRT
<213> Artificial sequence
<400> 8
Ile Ser Ser Ser Gly Thr Phe Ile
1 5
<210> 9
<211> 11
<212> PRT
<213> Artificial sequence
<400> 9
Ala Arg Glu Arg Phe Val Gly Val Leu Asp Ile
1 5 10
<210> 10
<211> 8
<212> PRT
<213> Artificial sequence
<400> 10
Ser Ser Asn Ile Gly Arg Ser Thr
1 5
<210> 11
<211> 3
<212> PRT
<213> Artificial sequence
<400> 11
Ser Ser Tyr
1
<210> 12
<211> 11
<212> PRT
<213> Artificial sequence
<400> 12
Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val
1 5 10
<210> 13
<211> 8
<212> PRT
<213> Artificial sequence
<400> 13
Gly Phe Thr Phe Ser Ser Tyr Ser
1 5
<210> 14
<211> 8
<212> PRT
<213> Artificial sequence
<400> 14
Ile Ser Ser Ser Ser Ser Thr Met
1 5
<210> 15
<211> 13
<212> PRT
<213> Artificial sequence
<400> 15
Ala Arg Gly Val Gly Ala Thr Gly Glu Leu Phe Asp Tyr
1 5 10
<210> 16
<211> 6
<212> PRT
<213> Artificial sequence
<400> 16
Gln Gly Ile Gly Asn Glu
1 5
<210> 17
<211> 3
<212> PRT
<213> Artificial sequence
<400> 17
Ala Ala Ser
1
<210> 18
<211> 9
<212> PRT
<213> Artificial sequence
<400> 18
Leu Gln Asp Tyr Asn Tyr Pro Arg Thr
1 5
<210> 19
<211> 8
<212> PRT
<213> Artificial sequence
<400> 19
Gly Phe Thr Phe Ser Asn Tyr Ser
1 5
<210> 20
<211> 8
<212> PRT
<213> Artificial sequence
<400> 20
Ile Ser Thr Thr Gly Thr Tyr Thr
1 5
<210> 21
<211> 13
<212> PRT
<213> Artificial sequence
<400> 21
Ala Arg Pro Tyr Tyr Tyr Gly Ser Gly Ser Pro Asp Tyr
1 5 10
<210> 22
<211> 6
<212> PRT
<213> Artificial sequence
<400> 22
Gln Ser Ile Ser Thr Phe
1 5
<210> 23
<211> 3
<212> PRT
<213> Artificial sequence
<400> 23
Ala Ala Ser
1
<210> 24
<211> 9
<212> PRT
<213> Artificial sequence
<400> 24
His Gln Thr Tyr Ser Lys Pro Trp Thr
1 5
<210> 25
<211> 8
<212> PRT
<213> Artificial sequence
<400> 25
Gly Phe Thr Phe Arg Asn Tyr Asp
1 5
<210> 26
<211> 8
<212> PRT
<213> Artificial sequence
<400> 26
Ile Ser Gly Ser Gly Ile Asp Thr
1 5
<210> 27
<211> 10
<212> PRT
<213> Artificial sequence
<400> 27
Val Arg Gly Leu Ala Gly Ala Phe Asp Tyr
1 5 10
<210> 28
<211> 7
<212> PRT
<213> Artificial sequence
<400> 28
Gln Ser Val Thr Ser Gly Tyr
1 5
<210> 29
<211> 3
<212> PRT
<213> Artificial sequence
<400> 29
Gly Thr Ser
1
<210> 30
<211> 10
<212> PRT
<213> Artificial sequence
<400> 30
Gln Gln His Arg Ser Ser Pro Met Tyr Ser
1 5 10
<210> 31
<211> 128
<212> PRT
<213> Artificial sequence
<400> 31
Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Thr Ser
20 25 30
Ser Asp Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
35 40 45
Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Arg Thr Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Asp Phe
65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg Arg Leu Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Ala Asn
100 105 110
Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 32
<211> 108
<212> PRT
<213> Artificial sequence
<400> 32
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Arg Phe Ser Thr Phe
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Tyr
85 90 95
Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 33
<211> 118
<212> PRT
<213> Artificial sequence
<400> 33
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ser Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Ser Ser Ser Gly Thr Phe Ile Lys Tyr Ala Asp Ser Leu
50 55 60
Gln Gly Arg Phe Thr Ile Thr Arg Asp Asn Ala Lys Thr Ala Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Arg Phe Val Gly Val Leu Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 34
<211> 111
<212> PRT
<213> Artificial sequence
<400> 34
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Glu
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Arg Ser
20 25 30
Thr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Met Tyr Ser Ser Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95
Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
<210> 35
<211> 120
<212> PRT
<213> Artificial sequence
<400> 35
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser Ser Ser Ser Ser Thr Met Tyr Tyr Gly Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Val Gly Ala Thr Gly Glu Leu Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Ala Ser Ser
115 120
<210> 36
<211> 108
<212> PRT
<213> Artificial sequence
<400> 36
Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Thr Gln Gly Ile Gly Asn Glu
20 25 30
Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Asn Tyr Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 37
<211> 120
<212> PRT
<213> Artificial sequence
<400> 37
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Thr Thr Gly Thr Tyr Thr His Tyr Ala Gly Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe
65 70 75 80
Leu Arg Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Pro Tyr Tyr Tyr Gly Ser Gly Ser Pro Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 38
<211> 108
<212> PRT
<213> Artificial sequence
<400> 38
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Phe
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Thr Tyr Ser Lys Pro Trp
85 90 95
Thr Phe Gly Arg Gly Thr Lys Val Glu Ile Glu Arg
100 105
<210> 39
<211> 117
<212> PRT
<213> Artificial sequence
<400> 39
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr
20 25 30
Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Gly Ser Gly Ile Asp Thr Tyr Tyr Gly Asp Ser Val
50 55 60
Glu Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Glu Ser Ser Val Leu
65 70 75 80
Leu Glu Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Phe Tyr Cys
85 90 95
Val Arg Gly Leu Ala Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 40
<211> 110
<212> PRT
<213> Artificial sequence
<400> 40
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Gly
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile His Gly Thr Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Arg Ser Ser Pro
85 90 95
Met Tyr Ser Phe Gly Gln Gly Ser Lys Leu Glu Ile Lys Arg
100 105 110
<210> 41
<211> 315
<212> DNA
<213> Artificial sequence
<400> 41
gtctctggtg gctctatcac cactagtagt gactactggg gctggatccg ccagccccca 60
gggaaggggc tggagtggat tgggagtatc tattatagtg ggagaaccta ctacaacccg 120
tccctcaaga gtcgagtcac catatccgta gacacgtcca agaacgactt ctctctgaag 180
ctgagctctg tgaccgccgc agacacggct gtgtattact gtgcgagacg ccttacgtat 240
tactatgata gtagtggtta tgcgaactgg tacttcgatc tctggggccg tggcaccctg 300
gtcactgtct cctca 315
<210> 42
<211> 324
<212> DNA
<213> Artificial sequence
<400> 42
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gaggttcagc acctttttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca cgatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaacag agttacagta tcccgtactc ttttggccag 300
gggaccaagc tggagatcaa acga 324
<210> 43
<211> 354
<212> DNA
<213> Artificial sequence
<400> 43
gaggtgcagc tggtggagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt agttatagta tgaagtgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcaacc atcagtagta gtggtacttt cataaagtat 180
gcagactcac tgcagggccg attcaccatc accagagaca acgccaagac cgcagtgtat 240
ctgcaaatga acagcctgag agtcgaggac acggctgttt attactgtgc gagagaacga 300
ttcgttggtg ttttggatat ctggggccaa gggacaatgg tcaccgtctc ttca 354
<210> 44
<211> 333
<212> DNA
<213> Artificial sequence
<400> 44
cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccggggagag ggtcaccatc 60
tcttgttctg gaagcagctc caacatcgga aggagtactg taagctggta ccagcagctc 120
ccaggaacgg cccccaaact cctcatgtat agtagttatc agcggccctc aggggtccct 180
gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240
tctgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggtccggtg 300
ttcggcggag ggaccaagct gaccgtccta ggt 333
<210> 45
<211> 359
<212> DNA
<213> Artificial sequence
<400> 45
gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt agttatagca tgaactgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtttcatat attagtagta gtagtagtac catgtactac 180
ggagactctg tgaagggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240
ctgcaaatga acagcctgag agacgaggac acggctgtgt attactgtgc gagaggagtg 300
ggagccacgg gggaactctt tgactactgg ggccagggaa ccctggtcac cgcctcctc 359
<210> 46
<211> 324
<212> DNA
<213> Artificial sequence
<400> 46
gccatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaactca gggaattgga aatgaattag ggtggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccagtt tacaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtctacaa gattacaatt accctcgtac gttcggccaa 300
gggaccaagg tggaaatcaa acga 324
<210> 47
<211> 360
<212> DNA
<213> Artificial sequence
<400> 47
gaggtgcagc tggtggagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60
tcctgtgcag tctctggatt caccttcagt aactatagca tgaactgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcatcc attagtacta ctggtactta cacacactac 180
gccggctcag tgaagggccg attcaccatc tccagagaca atgccaagaa ctcgctgttt 240
ttgcgaatga acagcctgag agccgaggac acggctgtgt attactgtgc gaggccctat 300
tactatggtt cggggagtcc tgactactgg ggccagggaa ccctggtcac cgtctcctca 360
<210> 48
<211> 324
<212> DNA
<213> Artificial sequence
<400> 48
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc acctttttga attggtatca gcagaagccc 120
gggaaagccc ctaatctcct gatctatgct gcatccagtt tgcaacgtgg ggtcccatca 180
aggttcactg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaccag acttacagta agccctggac gttcggccga 300
gggaccaagg tggaaatcga acga 324
<210> 49
<211> 351
<212> DNA
<213> Artificial sequence
<400> 49
gaggtgcagc tgttggagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagg aactatgaca tcaactgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcatcg attagtggta gtggtattga cacatactac 180
ggagactcag tggagggccg attcaccgtc tccagagaca acgccgagag ctcagtatta 240
ctggagatga acagcctgag agccgacgat acggctgtat tttactgtgt gaggggtctg 300
gctggcgcct ttgactactg gggccaggga accctggtca ccgtctcctc a 351
<210> 50
<211> 330
<212> DNA
<213> Artificial sequence
<400> 50
gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttacc agcggctact tagcctggta ccagcagaaa 120
cctggccagg ctcccaggct cctcatccat ggtacatcca ggagggccac tggcatccca 180
gacaggttca gtggcagtgg gtctgggaca gatttcactc tcaccatcag cagactggag 240
cctgaagatt ttgcagtcta ttactgtcag cagcatcgta gctcacccat gtacagtttt 300
ggccagggga gcaagctgga gatcaaacga 330
<210> 51
<211> 38
<212> DNA
<213> Artificial sequence
<400> 51
ctgcaaccgg tgtacattcc caggtgcagc tggtgcag 38
<210> 52
<211> 38
<212> DNA
<213> 52
<400> 52
ctgcaaccgg tgtacattcc gaggtgcagc tggtgcag 38
<210> 53
<211> 38
<212> DNA
<213> Artificial sequence
<400> 53
ctgcaaccgg tgtacattct gaggtgcagc tggtggag 38
<210> 54
<211> 38
<212> DNA
<213> Artificial sequence
<400> 54
ctgcaaccgg tgtacattct gaggtgcagc tgttggag 38
<210> 55
<211> 38
<212> DNA
<213> Artificial sequence
<400> 55
ctgcaaccgg tgtacattcc caggtgcagc tgcaggag 38
<210> 56
<211> 40
<212> DNA
<213> Artificial sequence
<400> 56
ctgcaaccgg tgtacattcc caggtgcagc tacagcagtg 40
<210> 57
<211> 38
<212> DNA
<213> Artificial sequence
<400> 57
ctgcaaccgg tgtacattcc caggttcagc tggtgcag 38
<210> 58
<211> 38
<212> DNA
<213> Artificial sequence
<400> 58
ctgcaaccgg tgtacattcc caggtccagc tggtacag 38
<210> 59
<211> 38
<212> DNA
<213> Artificial sequence
<400> 59
ctgcaaccgg tgtacattct caggtgcagc tggtggag 38
<210> 60
<211> 38
<212> DNA
<213> Artificial sequence
<400> 60
ctgcaaccgg tgtacattct gaagtgcagc tggtggag 38
<210> 61
<211> 38
<212> DNA
<213> Artificial sequence
<400> 61
ctgcaaccgg tgtacattcc cagctgcagc tgcaggag 38
<210> 62
<211> 38
<212> DNA
<213> Artificial sequence
<400> 62
ctgcaaccgg tgtacattcc caggtacagc tgcagcag 38
<210> 63
<211> 32
<212> DNA
<213> Artificial sequence
<400> 63
tgcgaagtcg acgctgagga gacggtgacc ag 32
<210> 64
<211> 34
<212> DNA
<213> Artificial sequence
<400> 64
tgcgaagtcg acgctgaaga gacggtgacc attg 34
<210> 65
<211> 33
<212> DNA
<213> Artificial sequence
<400> 65
tgcgaagtcg acgctgagga gacggtgacc gtg 33
<210> 66
<211> 40
<212> DNA
<213> Artificial sequence
<400> 66
ctgcaaccgg tgtacattct gacatccaga tgacccagtc 40
<210> 67
<211> 46
<212> DNA
<213> Artificial sequence
<400> 67
ttgtgctgca accggtgtac attcagacat ccagttgacc cagtct 46
<210> 68
<211> 40
<212> DNA
<213> Artificial sequence
<400> 68
ctgcaaccgg tgtacattgt gccatccgga tgacccagtc 40
<210> 69
<211> 40
<212> DNA
<213> Artificial sequence
<400> 69
ctgcaaccgg tgtacatggg gatattgtga tgacccagac 40
<210> 70
<211> 40
<212> DNA
<213> Artificial sequence
<400> 70
ctgcaaccgg tgtacatggg gatattgtga tgactcagtc 40
<210> 71
<211> 40
<212> DNA
<213> Artificial sequence
<400> 71
ctgcaaccgg tgtacatggg gatgttgtga tgactcagtc 40
<210> 72
<211> 45
<212> DNA
<213> Artificial sequence
<400> 72
ttgtgctgca accggtgtac attcagaaat tgtgttgaca cagtc 45
<210> 73
<211> 40
<212> DNA
<213> Artificial sequence
<400> 73
ctgcaaccgg tgtacattca gaaatagtga tgacgcagtc 40
<210> 74
<211> 46
<212> DNA
<213> Artificial sequence
<400> 74
ttgtgctgca accggtgtac attcagaaat tgtgttgacg cagtct 46
<210> 75
<211> 40
<212> DNA
<213> Artificial sequence
<400> 75
ctgcaaccgg tgtacattcg gacatcgtga tgacccagtc 40
<210> 76
<211> 30
<212> PRT
<213> Artificial sequence
<400> 76
Gly Cys Cys Ala Cys Cys Gly Thr Ala Cys Gly Thr Thr Thr Gly Ala
1 5 10 15
Thr Tyr Thr Cys Cys Ala Cys Cys Thr Thr Gly Gly Thr Cys
20 25 30
<210> 77
<211> 30
<212> DNA
<213> Artificial sequence
<400> 77
gccaccgtac gtttgatctc cagcttggtc 30
<210> 78
<211> 30
<212> DNA
<213> Artificial sequence
<400> 78
gccaccgtac gtttgatatc cactttggtc 30
<210> 79
<211> 30
<212> DNA
<213> Artificial sequence
<400> 79
gccaccgtac gtttaatctc cagtcgtgtc 30