CN114591424A

CN114591424A - Specific antibody of new coronavirus S protein NTD region and preparation method and application thereof

Info

Publication number: CN114591424A
Application number: CN202011418754.0A
Authority: CN
Inventors: 刘剑峰; 徐涛; 张胜蓝; 徐小兰
Original assignee: Huazhong University of Science and Technology; Bioisland Laboratory
Current assignee: Huazhong University of Science and Technology; Bioisland Laboratory
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2022-06-07
Anticipated expiration: 2040-12-07
Also published as: CN114591424B

Abstract

The present invention relates to an antigen binding protein, antibody or antibody active fragment obtained by immunizing camelid with NTD region of S protein of SARS-Cov-2. The invention relies on the immune system of camelid to screen, identify and prepare the antibody which specifically recognizes and combines with the NTD region of the S protein on the surface of the new coronavirus, the obtained antibody has high affinity, and the antibody specifically recognizes and combines with the NTD region, and the region is a part with larger sequence difference between the S protein of the new coronavirus and other coronaviruses, and can be used as a mark for distinguishing the new coronavirus from other coronaviruses. The development of antibodies aiming at the NTD region of the S protein is expected to specifically distinguish new coronavirus from other coronavirus, and is beneficial to determining patients and carrying out treatment measures according to symptoms.

Description

Specific antibody of new coronavirus S protein NTD region and preparation method and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a specific antibody of a new coronavirus S protein NTD region and a preparation method and application thereof.

Background

Antibodies are proteins secreted primarily by plasma cells and used by the immune system to identify and neutralize foreign substances, such as bacteria, viruses, etc., called antigens. The binding of antibodies to antigens relies entirely on noncovalent interactions, and this specific binding mechanism allows the antibodies to capture foreign microorganisms as well as infected cells, further induce other immune mechanisms to attack them, or directly neutralize their targets. Antibodies and antibody-related products have been widely used in the research fields of life science and medicine, and many experimental techniques derived based on antigen-antibody specific binding lay important foundations for scientific research and clinical treatment, such as immunodiagnosis, immunoblotting, enzyme-linked immunosorbent, flow cytometry analysis, and the like.

In the process of researching new coronavirus, the antibody is a very important research tool, and has great value and significance for patient diagnosis, virus analysis and research and the like. Since the onset of an outbreak, development of new coronavirus-specific antibodies has begun around the world. According to recent literature reports, mouse monoclonal antibodies that specifically recognize and bind to the S protein on the surface of new coronavirus have been developed by several research organizations in the world, and their main technical schemes are: (1) preparing new coronavirus antigens, such as inactivated new coronavirus particles, separating and extracting different components of new coronavirus, and the like; (2) injecting the antigen into a rat or a mouse, and generating an antibody by means of an immune system of the animal; (3) collecting animal blood, separating, extracting serum, and further separating to obtain components containing specific antibody.

The above-mentioned technology for producing antibodies against S protein on the surface of a new coronavirus based on the immune system of an animal such as a mouse, and separating and extracting the antibodies has the following disadvantages: (1) antibodies have limited specificity: the S protein has a large structure and complex spatial folding, the traditional mouse monoclonal antibody is difficult to identify the complex spatial structure of the S surface, and researchers are difficult to obtain antibodies aiming at each structural domain; (2) the stability of the antibody is poor: the mouse monoclonal antibody can maintain the characteristics of the concentration, the specificity and the like of the antibody only by storing, transporting, testing, verifying and the like at a low temperature (4 ℃), and is not beneficial to the development of large-scale application in research; (3) the scale batch production finished product is high: the mouse monoclonal antibody is a full-length immunoglobulin, needs to be separated, purified and lifted after being subjected to recombinant expression in expensive expression systems such as mammalian cells and the like, is complex to operate, has high cost, and is not beneficial to large-scale batch production.

Disclosure of Invention

The invention overcomes the defects of the prior art, designs and implements an effective and feasible antibody screening and preparation technical scheme, obtains the antibody which can specifically recognize and combine with the NTD region of the S protein on the surface of the new coronavirus, and performs batch production preparation and application thereof.

The above object of the present invention is achieved by the following embodiments.

In a first aspect, the present invention provides an antigen binding protein, antibody or antibody active fragment obtained by immunizing a camelid with the NTD region of the S protein of SARS-Cov-2.

In some embodiments, the camelid is selected from dromedary, bactrian, llama, alpaca and llama, preferably alpaca.

In some embodiments, the antibody is a nanobody and the antibody-active fragment is a nanobody-active fragment.

In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

In some embodiments, the antigen binding protein, antibody or antibody active fragment binds to the NTD region of the S protein of SARS-Cov-2 at a kd value of 4100nM or less, preferably 500nM or less, more preferably 120nM or less, more preferably 65nM or less, more preferably 10nM or less, more preferably 6nM or less, more preferably 1nM or less.

In some embodiments, the NTD region of the S protein of SARS-Cov-2 has an amino acid sequence as set forth in SEQ ID NO 1. Further preferably, the S protein NTD region of SARS-Cov-2 is prepared by a method comprising the following steps: connecting the nucleotide sequence of the NTD region of the S protein of the SARS-Cov-2 with the nucleotide sequence of the coding transmembrane structure in series, and constructing into a vector plasmid; the vector plasmid is transfected into a eukaryotic cell line for expression, so that the NTD region of the S protein of SARS-Cov-2 is displayed outside the cell membrane in the form of a transmembrane protein.

In a second aspect, the present invention provides a method of constructing an antibody library, the method comprising the steps of:

(1) immunizing camelid with NTD region of S protein of SARS-Cov-2 as antigen, collecting peripheral venous blood of immunized animal, and separating to obtain lymphocyte;

(2) extracting total mRNA of the lymphocytes, performing reverse transcription on the total mRNA into cDNA, and amplifying the cDNA;

(3) and inserting the amplified DNA into a virus expression vector, transforming the virus expression vector into bacteria, and collecting bacterial colonies to obtain an antibody library.

In some embodiments, the immunizing of step (1) is performed by subcutaneous injection. The frequency of immunization is preferably 3-5 times. The venous peripheral blood is preferably collected before and after the last immunization, respectively.

In some embodiments, the viral expression vector of step (3) is a phage expression vector.

In some embodiments, the bacterium of step (3) is escherichia coli.

In a third aspect, the present invention provides an antibody library obtained by the above method for constructing an antibody library, or a polyclonal antibody produced by expression of the antibody library.

In a fourth aspect, the present invention provides a method of constructing a library of antigen-specific antibodies, the method comprising the steps of: screening the antibody library of the third aspect to obtain an antigen-specific antibody library.

In some embodiments, the method of constructing an antigen-specific antibody library comprises the steps of:

(i) culturing the antibody library to release viruses;

(ii) incubating the virus with an antigen, removing the virus non-specifically bound to the antigen, and retaining the virus specifically bound to the antigen;

(iii) infecting bacteria with the virus specifically bound to the antigen, collecting colonies, and obtaining an antigen-specific antibody library.

In some embodiments, the bacterium of step (iii) is escherichia coli.

In a fifth aspect, the present invention provides an antigen-specific antibody library obtained by the above-described method for constructing an antigen-specific antibody library, or a polyclonal antibody specifically binding to an antigen produced by expression of the antigen-specific antibody library.

In a sixth aspect, the present invention provides a method of preparing an antigen binding protein, antibody or antibody active fragment, said method comprising the steps of: screening the antibody library of the third aspect to obtain an antigen binding protein, an antibody or an antibody active fragment that specifically binds to an antigen.

In some embodiments, the method of making an antigen binding protein, antibody or antibody active fragment comprises the steps of:

(a) culturing the antibody library to release viruses;

(b) incubating the virus with an antigen, removing the virus non-specifically bound to the antigen, and retaining the virus specifically bound to the antigen;

(c) infecting bacteria with the virus specifically bound to the antigen, smearing the infected bacteria on a plate culture medium for culture, and selecting a single colony.

In some embodiments, the bacterium of step (c) is escherichia coli.

In some embodiments, the single colony may be expanded for culture prior to antigen-specific binding identification.

In some embodiments, the single colony may be expanded and then subjected to step (d): the DNA is extracted, transformed into host cells and expressed to obtain monoclonal antibodies.

In a seventh aspect, the present invention provides an antigen-binding protein, an antibody or an antibody active fragment obtained by the above-described method for producing an antigen-binding protein, an antibody or an antibody active fragment.

In an eighth aspect, the present invention provides an antigen binding protein, antibody or antibody active fragment that specifically recognizes and/or binds to the NTD region of the S protein of SARS-Cov-2; the antigen binding protein, antibody or antibody active fragment comprises at least one heavy chain variable region; the heavy chain variable region has: CDR1 as shown in SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16 or SEQ ID NO 17;

CDR2 as shown in SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32 or SEQ ID NO 33; and

CDR3 as shown in SEQ ID NO 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 18 and CDR3 shown in SEQ ID NO. 34.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 19 and CDR3 shown in SEQ ID NO. 35.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 20 and CDR3 shown in SEQ ID NO. 36.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 5, CDR2 shown in SEQ ID NO. 21 and CDR3 shown in SEQ ID NO. 37.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 6, CDR2 shown in SEQ ID NO. 22 and CDR3 shown in SEQ ID NO. 38.

In some embodiments, the heavy chain variable region has: CDR1 shown as SEQ ID NO. 7, CDR2 shown as SEQ ID NO. 23 and CDR3 shown as SEQ ID NO. 39.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 8, CDR2 shown in SEQ ID NO. 24 and CDR3 shown in SEQ ID NO. 40.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 25 and CDR3 shown in SEQ ID NO. 41.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 26 and CDR3 shown in SEQ ID NO. 42.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 11, CDR2 shown in SEQ ID NO. 27 and CDR3 shown in SEQ ID NO. 43.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 12, CDR2 shown in SEQ ID NO. 28 and CDR3 shown in SEQ ID NO. 44.

In some embodiments, the heavy chain variable region has: CDR1 shown as SEQ ID NO. 13, CDR2 shown as SEQ ID NO. 29 and CDR3 shown as SEQ ID NO. 45.

In some embodiments, the heavy chain variable region has: CDR1 shown as SEQ ID NO. 14, CDR2 shown as SEQ ID NO. 30 and CDR3 shown as SEQ ID NO. 46.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 15, CDR2 shown in SEQ ID NO. 31 and CDR3 shown in SEQ ID NO. 47.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 16, CDR2 shown in SEQ ID NO. 32 and CDR3 shown in SEQ ID NO. 48.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 17, CDR2 shown in SEQ ID NO. 33 and CDR3 shown in SEQ ID NO. 49.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 50 or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 50.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 51, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 51.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 52, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 52.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 53, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 53.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 54 or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 54.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 55, or the conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 55.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 56, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 56.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 57, or the conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 57.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 58 or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 58.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 59, or the conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 59.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 60 or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 60.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 61 or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 61.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 62, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 62.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 63, or the conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 63.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 64 or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 64.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 65, or the conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 65.

In some embodiments, the antigen binding protein, antibody or antibody active fragment comprises one of the heavy chain variable regions and lacks a light chain.

In a ninth aspect, the present invention provides a nucleotide sequence encoding an amino acid sequence as set forth in any one of SEQ ID NO 2 to SEQ ID NO 65 or an antigen binding protein, antibody or antibody active fragment as described above.

In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is as set forth in SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80 or SEQ ID NO 81.

In a tenth aspect, the present invention provides an expression vector comprising the nucleotide sequence described above.

In some embodiments, the expression vector is a phage expression vector, preferably a phage surface display screening vector.

In some embodiments, the expression vector further comprises a nucleotide sequence encoding the phage envelope protein pIII.

In an eleventh aspect, the present invention provides a virus exogenously transferred with the expression vector described above.

In some embodiments, the virus is a bacteriophage.

In a twelfth aspect, the present invention provides a host cell exogenously transformed with the expression vector described above, or infected with the virus described above.

In some embodiments, the host cell is e.

In a thirteenth aspect, the invention provides a method of expressing an antigen binding protein, antibody or antibody active fragment using a host cell as described above.

In a fourteenth aspect, the present invention provides an antigen binding protein, antibody or antibody active fragment obtained by expression using a host cell as described above.

In a fifteenth aspect, the present invention provides a humanized antigen binding protein, antibody or antibody active fragment obtained by humanizing said antigen binding protein, antibody or antibody active fragment described above.

In a sixteenth aspect, the present invention provides a protein conjugate comprising an antigen binding protein, antibody or antibody active fragment as described above or a humanized antigen binding protein, antibody or antibody active fragment as described above and a ligand.

In some embodiments, the ligand is selected from the group consisting of a radioisotope, a fluorophore, and a delivery vehicle.

In a seventeenth aspect, the present invention provides a pharmaceutical composition comprising an antigen binding protein, antibody or antibody active fragment as described above, a humanized antigen binding protein, antibody or antibody active fragment as described above, or a protein conjugate as described above.

In some embodiments, the pharmaceutical composition further comprises other active ingredients and/or adjuvants.

In an eighteenth aspect, the present invention provides a chimeric antigen receptor comprising an antigen binding protein, antibody or antibody active fragment as described above, a humanized antigen binding protein, antibody or antibody active fragment as described above.

In a nineteenth aspect, the present invention provides a chimeric antigen receptor T cell expressing the chimeric antigen receptor described above.

In a twentieth aspect, the present invention provides the use of an antigen binding protein, antibody or antibody active fragment as described above, an antibody library or polyclonal antibody as described above, an antigen specific antibody library or polyclonal antibody that specifically binds to an antigen as described above, a nucleotide sequence as described above, an expression vector as described above, a virus as described above, a host cell as described above, a humanized antigen binding protein as described above, an antibody or antibody active fragment as described above, a protein conjugate as described above, a pharmaceutical composition as described above, a chimeric antigen receptor as described above or a chimeric antigen receptor T cell as described above for the manufacture of a medicament for the prevention of SARS-Cov-2 infection and/or the treatment of a disease caused by SARS-Cov-2 infection.

In a twenty-first aspect, the invention provides a kit for the in vitro detection of SARS-Cov-2 or the S protein of SARS-Cov-2 comprising an antigen binding protein, antibody or antibody active fragment as described above or a humanized antigen binding protein, antibody or antibody active fragment as described above.

In some embodiments, the antigen binding protein, antibody or antibody active fragment is labeled with a label. Preferably, the label is selected from the group consisting of an enzyme, a chemiluminescent group and an isotopic group.

In a twenty-second aspect, the present invention provides the use of an antigen binding protein, antibody or antibody active fragment as described above, a humanized antigen binding protein, antibody or antibody active fragment as described above, a protein conjugate as described above or a kit as described above for the in vitro detection of SARS-Cov-2 or the S protein of SARS-Cov-2.

In a twenty-third aspect, the present invention provides a method for detecting SARS-Cov-2 or S protein of SARS-Cov-2 in a sample using an antigen-binding protein, antibody or antibody active fragment as described above, a humanized antigen-binding protein, antibody or antibody active fragment as described above, a protein conjugate as described above, or a kit as described above.

In a twenty-fourth aspect, the present invention provides a contrast agent for detecting SARS-Cov-2 infection, comprising an antigen binding protein, antibody or antibody active fragment as described above or a humanized antigen binding protein, antibody or antibody active fragment as described above.

Compared with the prior art, the technical scheme provided by the invention has the following remarkable advantages: (1) the kd value of the nano antibody obtained by the invention is about several nanomoles, the affinity is high, the nano antibody specifically recognizes and binds to an NTD region, and the region is a part with larger sequence difference between the S protein of the new coronavirus and other coronaviruses and can be used as a mark for distinguishing the new coronavirus from other coronaviruses; the antibody is developed aiming at the NTD region of the S protein, and the specific differentiation of new coronavirus and other coronavirus is expected, so that the diagnosis of patients and the implementation of symptomatic treatment measures are facilitated; (2) the antibody has the advantages of small relative molecular mass, high solubility, strong tissue permeability, good distributivity and rapid diffusion and metabolism, and is expected to make great contribution to the detection of new coronavirus or S protein on the surface of the new coronavirus and other related applications; (3) the antibody has simple structure, is easy to carry out genetic engineering transformation, has mature optimization strategy for enhancing the affinity of a single-domain antibody, prolonging the half-life period in vivo and coupling with other molecules for drug development, such as connecting radioactive isotopes, coupling transfer drugs, CART, fluorescence labeling high-resolution imaging and the like; (4) the heavy chain variable region sequence of the antibody has high homology with the human VH sequence, and the humanization of the single-domain antibody can be realized through a few amino acid mutations, so that the humanization is simple; (5) the antibody has high stability, acid and alkali pH resistance and high temperature resistance, can avoid the requirement that the conventional antibody needs low-temperature storage and transportation, and is beneficial to large-scale popularization and application; (6) the antibody provided by the invention can be well recombined and expressed in an escherichia coli expression system with low cost, the mass production cost is low, the yield can reach dozens of milligrams per liter of escherichia coli, the escherichia coli recombination and expression system is mature in technology, the quality control is simple, the production cost is reduced, and the large-scale production is realized.

Drawings

FIG. 1 is a schematic diagram showing the detection result of the affinity between monoclonal antibody NTD _1F7 and antigen;

FIG. 2 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _2E6 with antigen;

FIG. 3 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _3E10 with antigen;

FIG. 4 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _3B10 with antigen;

FIG. 5 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _4G3 with antigen;

FIG. 6 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _2A9 with antigen;

FIG. 7 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _1F6 with antigen;

FIG. 8 is a schematic diagram showing the detection result of the affinity between monoclonal antibody NTD _4H12 and antigen;

FIG. 9 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _2B5 with antigen;

FIG. 10 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _3E9 with antigen;

FIG. 11 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _1C8 with antigen;

FIG. 12 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _4F10 for antigen;

FIG. 13 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _1B2 for antigen;

FIG. 14 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _1G7 with antigen;

FIG. 15 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _2B3 for antigen;

FIG. 16 is a schematic diagram showing the result of detecting the affinity of monoclonal antibody NTD _3G11 for antigen;

in each of the above figures, the left figure is the flow cytometric analysis result, where black is the incubation result of the antibody with the HEK293T cell that does not overexpress the antigen, white is the incubation result of the antibody with the HEK293T cell that overexpresses the antigen, the abscissa is the fluorescence intensity, and the ordinate is the normalized cell number; the right panel measures antibody affinity values for flow cytometry.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Definition of

SARS-Cov-2: the international committee for virus classification names new coronaviruses (new coronaviruses for short).

S protein: the envelope of the new coronavirus particle is composed of Spike protein (S protein for short), E protein and M protein, wherein the S protein is one of main components on the surface of all coronaviruses; the S protein on the surface of the new coronavirus has 1273 amino acids in total length and can be divided into two parts of S1 and S2, and after the virus invades cells, the S protein is cut into a S1 subunit and a S2 subunit.

NTD region: the S1 subunit of the S protein of the novel coronavirus mainly comprises an NTD region and an NTD region; wherein, NTD region refers to N-terminal structure domain, is region with large difference of SARS-Cov-2 and SARS-Cov and other coronavirus amino acid sequence, and can be used as mark for distinguishing new coronavirus and other coronavirus. The development of antibodies aiming at the NTD region of the S protein is expected to specifically distinguish new coronavirus from other coronavirus, and is beneficial to the accurate diagnosis of patients and the implementation of treatment measures according to symptoms.

kd value: the dissociation constant (kd) is a specific type of equilibrium constant that measures the tendency of a larger object to separate (dissociate) from another smaller component, and is the reciprocal of the association constant in mol/L (M) or nmol/L (nM). A smaller kd value indicates a stronger binding ability of the two substances.

Nanobodies: an antibody naturally devoid of light chains, found in the peripheral blood of camelids, which antibody comprises only one heavy chain variable domain (VHH) and two conventional CH2 and CH3 regions, but does not readily adhere to each other, or even aggregate, as readily as artificially engineered single chain antibody fragments; the VHH structure which is cloned and expressed independently has the structural stability which is equivalent to that of the original heavy chain antibody and the binding activity with antigen, and is the minimum unit which is known to be combined with target antigen; the VHH crystal is 2.5nm, 4nm long and has a molecular weight of only 15KDa, so the VHH crystal is also called a Nanobody (Nb). Compared with the traditional animals such as mice, rabbits and the like which can only recognize the polypeptide with flat antigen surface, the immune system in the camelid animal body can recognize the complex spatial structure of the antigen surface and can generate the nano antibody with high specificity and high affinity.

According to the technical scheme of the invention, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, see the following table 1:

TABLE 1

Residue of	Conservative substitutions	Residue of	Conservative substitutions
				Ala	Ser	Leu	Ile；Val
Arg	Lys	Lys	Arg；Gln
				Asn	Gln；His	Met	Leu；Ile
Asp	Glu	Phe	Met；Leu；Tyr
				Gln	Asn	Ser	Thr；Gly
Cys	Ser	Thr	Ser；Val
				Glu	Asp	Trp	Tyr
Gly	Pro	Tyr	Trp；Phe
				His	Asn；Gln	Val	Ile；Leu
Ile	Leu；Val

Furthermore, because of the degeneracy of bases, substitutions can be made to bases of a polynucleotide sequence without altering the activity or function of the polynucleotide sequence, see table 2 below:

TABLE 2

Example 1: preparation of antigens

The conventional practice for preparing antigens in the prior art is to recombinantly express and purify the NTD region of the S protein as an antigen and then to deimmunize animals such as mice, rabbits, monkeys, etc. However, the above prior art has the following disadvantages: the immune system in the bodies of animals such as mice, rabbits, monkeys, etc. has limited capability and cannot generate a specific spatial structure for recognizing antigens; naturally, the S protein is a membrane protein located on the surface of viral particles, but the NTD domain of the recombinantly expressed and purified S protein is not a membrane protein and is not present in the native state, and therefore the resulting antibody is not necessarily able to correctly recognize the antigen.

Aiming at the NTD structural domain of the S protein of the new coronavirus, the method for preparing the antigen specifically comprises the following steps:

(1) connecting in series, constructing DNA sequence of NTD region (i.e. amino acids 1-304, specifically: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIH VSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDP FLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSK HTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFL LKYNENGTITDAVDCALDPLSETKCTLK) of S protein and an additional transmembrane domain into pCaggs carrier plasmid;

(2) the plasmid is transfected into a HEK293T cell line by using liposome to be over-expressed, so that an NTD region of the S protein is displayed outside a cell membrane in a form of transmembrane protein;

(3) collection of about 4X 10⁸Cell is treated through ultrasonic crushing, homogenizing and ultracentrifugation to obtain cell membrane extract as antigen.

The DNA sequence for constructing the NTD region of the coding S protein adopted in the embodiment is specifically shown as SEQ ID NO:1, and specifically comprises the following steps: ATGTTCGTGTTCCTCGTGCTCCTGCCTCTGGTGTCTAGCCAGTGCGTGAACCTGACCACACGGACC CAGCTCCCTCCCGCCTACACAAACTCTTTCACCCGGGGCGTGTACTACCCCGACAAGGTGTTCCGG TCTAGCGTGCTCCACTCTACACAGGACCTGTTCCTCCCTTTCTTCAGCAACGTGACATGGTTCCAC GCCATCCACGTGTCTGGCACAAACGGCACAAAGCGGTTCGACAACCCCGTGCTCCCTTTCAACGA CGGCGTGTACTTCGCCAGCACCGAGAAGTCTAACATTATCCGGGGCTGGATTTTCGGCACCACAC TCGACTCTAAGACACAGTCCCTCCTGATTGTGAACAACGCCACAAACGTGGTGATTAAGGTGTGC GAGTTCCAGTTCTGCAACGACCCTTTCCTGGGCGTGTACTACCACAAGAACAACAAGTCTTGGAT GGAGTCTGAGTTCAGAGTGTACTCTAGCGCCAACAACTGCACCTTCGAGTACGTGTCCCAGCCTTT CCTCATGGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACCTGAGAGAGTTCGTGTTCAAGAAC ATTGACGGCTACTTCAAGATTTACTCTAAGCACACCCCAATTAACCTCGTGAGGGACCTCCCTCAG GGCTTCTCCGCCTTAGAACCACTGGTGGACCTCCCTATTGGCATTAACATCACACGCTTCCAGACA CTGCTCGCCCTCCACCGGTCTTACCTGACCCCAGGCGACTCTAGCTCTGGCTGGACAGCCGGCGCC GCCGCCTACTACGTGGGCTACCTGCAGCCTAGGACCTTCCTCCTGAAGTACAACGAGAACGGCAC AATTACCGACGCCGTGGACTGCGCCCTGGACCCACTGTCCGAGACAAAGTGCACACTGAAG

The NTD region of the S protein provided in this example is capable of being expressed and present on the surface of the cell membrane, close to the native conformation and state of the S protein on the surface of the viral particle.

Example 2: alpaca immune injection

This example immunizes alpaca with the antigen prepared in example 1. The method comprises the following specific steps:

(1) the antigen prepared in example 1 was divided into 4 portions on average;

(2) 4 times of immunization is carried out on the alpaca in an accumulated way, the antigen is injected into the animal body subcutaneously, the first immunization is recorded as the first day, and the subsequent immunizations are respectively carried out on the 10 th day, the 19 th day and the 28 th day; on day 28, about 200mL of alpaca venous peripheral blood was collected before the fourth immunization injection, and on day 42, 14 days after the fourth immunization, about 300mL of alpaca venous peripheral blood was collected.

Compared with the traditional immunization technical scheme of animal antibodies such as mice and rabbits, the method provided by the embodiment collects a large amount of alpaca vein peripheral blood, and is beneficial to obtaining highly diversified nano antibodies through subsequent screening.

Example 3: construction of antibody libraries

Two batches of alpaca venous peripheral blood collected in example 2 were used as raw materials to construct a highly diverse nanobody library. The method for treating the peripheral blood of the alpaca veins of two batches is the same, and specifically comprises the following steps:

(1) separating lymphocytes from the peripheral blood of alpaca veins by using a density gradient centrifugation method;

(2) extracting total mRNA of the lymphocyte and performing reverse transcription to obtain cDNA;

(3) using proper DNA primer, using the cDNA as template, obtaining VHH fragment of alpaca immunoglobulin IgG2 and IgG3 by Polymerase Chain Reaction (PCR) amplification, namely DNA fragment of nano antibody;

(4) connecting the DNA of the VHH to a phage surface display screening vector to form a VHH-pIII fusion protein expression vector plasmid library; wherein pIII is a protein present on a bacteriophage surface flagellum;

(5) transforming the DNA connecting product to TG1 competent bacteria by an electric transformation method, and collecting all colonies after proper culture, namely the nano antibody library of the alpaca.

Compared with the traditional method for separating the antibody from the serum or the lymphocyte of the animal such as the mouse, the rabbit and the like, the method can obtain and store all nano antibody fragments (namely the library) of the alpaca for a long time, and can continuously support the follow-up continuous screening and development of the nano antibody.

Example 4: phage surface display screening specific nano antibody

In this embodiment, the nanobody library obtained in example 3 is used as a source, and the phage surface display screening is performed to obtain the antigen-specific nanobody. The method comprises the following specific steps:

(1) taking a proper amount of the cryopreserved nano antibody library obtained in the embodiment 3, inoculating the cryopreserved nano antibody library to a bacterial culture medium, adding a proper amount of helper phage after proper culture, and continuously culturing under a proper amount of conditions;

(2) extracting the amplified phage in the bacterial culture supernatant by a PEG-NaC method;

(3) incubating the phage appropriately with the antigen; if the antigen is a cell membrane protein, the phage may be incubated with the enriched antigen-overexpressing cell whole or cell membrane extract; if the antigen is intracellular protein or secretory protein, the antigen can be fixed in a test tube, a micropore plate and other media in advance, and then incubated with the phage;

(4) elutriation: discarding the phage, rinsing the antigen for a proper number of times by using PBS buffer solution, elutriating and removing the phage non-specifically combined with the antigen, and reserving the phage specifically combined with the antigen;

(5) and (3) elution: and treating the bacteriophage specifically bound with the antigen by using an acidic glycine solution to dissociate and retain the bacteriophage from the antigen.

Thus, the phage expressing the specific nano antibody is obtained.

Example 5: construction of an antigen-specific antibody library

This example uses the phage obtained in example 4 to construct an antigen-specific nanobody library. The method comprises the following specific steps:

(1) infecting and culturing the phage with the specific nano antibody to Escherichia coli in a proper state, but not adding auxiliary phage;

(2) after the bacteriophage is completely infected, the specific nano antibody exists in the escherichia coli in the form of DNA plasmid, and all the escherichia coli are collected to form the antigen-specific nano antibody library.

The library obtained in this example can be returned as a raw material to example 4 for phage surface display screening.

Example 6: obtaining of monoclonal antibody colonies

This example used the phage obtained in example 4 to obtain monoclonal nanobody colonies. The method comprises the following specific steps:

(1) infecting the phage expressing the specific nano antibody with escherichia coli cultured to a proper state, but not adding auxiliary phage;

(2) after the bacteriophage is completely infected, the escherichia coli is evenly smeared on a bacterial culture dish for culture, and then the monoclonal colony containing the DNA plasmid of the nano antibody can be obtained.

Example 7: identification of Positive monoclonal antibodies

This example identifies the monoclonal colonies obtained in example 6. The method comprises the following specific steps:

(1) selecting 16 groups of the monoclonal colonies to be cultured in a micropore plate respectively;

(2) adding IPTG to induce expression of VHH-pIII (namely the fusion protein containing the nano antibody);

(3) collecting bacterial culture supernatant containing the nano antibody, and incubating with the antigen;

(4) and (3) detecting whether the monoclonal nano antibody is combined with antigen by using a flow cytometry analysis method, namely whether the monoclonal nano antibody can be combined with HEK293T cells over-expressing the NTD region of the S protein.

The results of the detection of the affinity between the antibodies and the NTD region of the antigen S protein corresponding to the 16 groups of monoclonal colonies are shown in FIGS. 1 to 16, and the results of the affinity values kd are shown in Table 3 below.

Table 3: results of affinity assay

	kd(nM)
		NTD_1F7	494.00
NTD_2E6	0.79
		NTD_3E10	3.52
NTD_3B10	3.20
		NTD_4G3	49.59
NTD_2A9	63.65
		NTD_1F6	0.60
NTD_4H12	0.98
		NTD_2B5	0.79
NTD_3E9	5.79
		NTD_1C8	0.10
NTD_4F10	0.09
		NTD_1B2	4006.00
NTD_1G7	114.20
		NTD_2B3	2.96
NTD_3G11	37.82

From the above results, it is understood that the binding kd value of the antibody binding to the NTD region of the S protein of SARS-Cov-2 obtained by selecting the monoclonal colonies in this example is 4100nM or less, preferably 500nM or less, more preferably 120nM or less, more preferably 65nM or less, more preferably 10nM or less, more preferably 6nM or less, more preferably 1nM or less.

Respectively carrying out amplification culture on the 16 groups of monoclonal colonies, extracting DNA plasmids, carrying out DNA sequencing to obtain a nucleotide sequence of the antibody, and translating to obtain a complete amino acid sequence. Specifically, the amino acid sequences and nucleotide sequences corresponding to the 16 groups of monoclonal antibody microbial colonies are specifically shown in tables 4 to 19 below:

table 4: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _1F7

Table 5: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _2E6

Table 6: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _3E10

Table 7: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _3B10

Table 8: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _4G3

Table 9: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _2A9

Table 10: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _1F6

Table 11: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _4H12

Table 12: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _2B5

Table 13: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _3E9

Table 14: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _1C8

Table 15: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _4F10

Table 16: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _1B2

Table 17: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _1G7

Table 18: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _2B3

Table 19: amino acid sequence and nucleotide sequence of monoclonal antibody NTD _3G11

Example 8: small batch antibody production and preparation

The DNA plasmid of the nanobody obtained in example 7 was transformed into BL21(DE3) competent cells, and the monoclonal nanobody was expressed and purified in small batches with the aid of an E.coli expression system, with a batch yield of about several milligrams.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> biosampal laboratory; huazhong university of science and technology

<120> specific antibody of new coronavirus S protein NTD region, preparation method and application thereof

<130> RYP2010938.3

<160> 81

<170> PatentIn version 3.5

<210> 1

<211> 912

<212> DNA

<213> SARS-Cov-2

<400> 1

atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgaccaca 60

cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120

aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180

aacgtgacat ggttccacgc catccacgtg tctggcacaa acggcacaaa gcggttcgac 240

aaccccgtgc tccctttcaa cgacggcgtg tacttcgcca gcaccgagaa gtctaacatt 300

atccggggct ggattttcgg caccacactc gactctaaga cacagtccct cctgattgtg 360

aacaacgcca caaacgtggt gattaaggtg tgcgagttcc agttctgcaa cgaccctttc 420

ctgggcgtgt actaccacaa gaacaacaag tcttggatgg agtctgagtt cagagtgtac 480

tctagcgcca acaactgcac cttcgagtac gtgtcccagc ctttcctcat ggacctggag 540

ggcaagcagg gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat tgacggctac 600

ttcaagattt actctaagca caccccaatt aacctcgtga gggacctccc tcagggcttc 660

tccgccttag aaccactggt ggacctccct attggcatta acatcacacg cttccagaca 720

ctgctcgccc tccaccggtc ttacctgacc ccaggcgact ctagctctgg ctggacagcc 780

ggcgccgccg cctactacgt gggctacctg cagcctagga ccttcctcct gaagtacaac 840

gagaacggca caattaccga cgccgtggac tgcgccctgg acccactgtc cgagacaaag 900

tgcacactga ag 912

<210> 2

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _1F7

<400> 2

Gly Arg Thr Phe Ser Ile Tyr Ala Met

1 5

<210> 3

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _2E6

<400> 3

Glu Thr Ile Phe Ile Ile Gln Leu Met

1 5

<210> 4

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _3E10

<400> 4

Arg Ile Ile Phe Thr Pro Asn Val Met

1 5

<210> 5

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _3B10

<400> 5

Gly Ser Ile Ser Ser Ile Asn Ala Met

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _4G3

<400> 6

Gly Ser Gly Phe Ser Phe Ala Ala Met

1 5

<210> 7

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _2A9

<400> 7

Gly Ser Ile Tyr Arg Pro Asn Phe Val

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _1F6

<400> 8

Gly Ser Phe Phe Ser Ile His Ala Met

1 5

<210> 9

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _4H12

<400> 9

Gly Ser Ile Phe Thr Ile Asn Ala Met

1 5

<210> 10

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _2B5

<400> 10

Gly Ser Ile Phe Arg Leu Asn Ala Met

1 5

<210> 11

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _3E9

<400> 11

Gly Ser Ile Phe Arg Leu Asn Ala Met

1 5

<210> 12

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _1C8

<400> 12

Gly Ser Ile Phe Arg Leu Asn Ala Met

1 5

<210> 13

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _4F10

<400> 13

Gly Ser Ile Phe Arg Leu Asn Ser Met

1 5

<210> 14

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _1B2

<400> 14

Gly Ser Ile Phe Arg Leu Asn Ser Met

1 5

<210> 15

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _1G7

<400> 15

Gly Phe Thr Phe Asp Asp Tyr Ala Ile

1 5

<210> 16

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _2B3

<400> 16

Gly Arg Ser Phe Ser Thr Tyr Gly Val

1 5

<210> 17

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody NTD _3G11

<400> 17

Gly Phe Ser Phe Asp Asp Tyr Ala Ile

1 5

<210> 18

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _1F7

<400> 18

Asn Gly Ser Gly Gly Ser Ser Tyr

1 5

<210> 19

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _2E6

<400> 19

Arg Ser Ile Gly Thr Thr Asn

1 5

<210> 20

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _2E6

<400> 20

Ser Asp Ser Gly Ser Thr Asn

1 5

<210> 21

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _3B10

<400> 21

Thr Ile Gly Gly Val Thr Asn

1 5

<210> 22

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _4G3

<400> 22

Thr Ser Gly Gly Arg Ser Asn

1 5

<210> 23

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _2A9

<400> 23

Thr Ser Ser Gly Ser Thr Asn

1 5

<210> 24

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _1F6

<400> 24

Thr Arg Asp Tyr Ser Thr Asn

1 5

<210> 25

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _4H12

<400> 25

Thr Arg Arg Gly Ser Thr Asn

1 5

<210> 26

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _2B5

<400> 26

Thr Pro Gly Gly Ser Thr Asn

1 5

<210> 27

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _3E9

<400> 27

Thr Ser Gly Gly Ser Thr Asn

1 5

<210> 28

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _1C8

<400> 28

Thr Ser Gly Gly Ser Thr Asn

1 5

<210> 29

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _4F10

<400> 29

Thr Gly Gly Gly Ser Thr Asn

1 5

<210> 30

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _1B2

<400> 30

Asp Ser Thr Ala Arg Asn Val Tyr

1 5

<210> 31

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _1G7

<400> 31

Ser Asn Ser Asp Gly

1 5

<210> 32

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _2B3

<400> 32

Arg Tyr Ser Ala Asp Asn Thr Thr

1 5

<210> 33

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody NTD _3G11

<400> 33

Cys Ser Ser Asn Asp Cys Thr His

1 5

<210> 34

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _1F7

<400> 34

Asn Ala Asp Val Gly Glu Pro Pro Val Gly Tyr Glu

1 5 10

<210> 35

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _2E6

<400> 35

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

1 5 10

<210> 36

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _3E10

<400> 36

Asn Ala Arg Gly Pro Val Pro Gly Arg Pro Ala Gly Asp

1 5 10

<210> 37

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _3B10

<400> 37

Glu Gly Val Thr Ala Met Tyr Asp

1 5

<210> 38

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _4G3

<400> 38

Ile Leu Gln Arg Gly Glu Pro Pro Leu Trp Tyr Asp

1 5 10

<210> 39

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _2A9

<400> 39

Leu Val Gly Leu Gly Val Phe Pro Leu Ile Phe Glu

1 5 10

<210> 40

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _1F6

<400> 40

Asn Val Glu Val Gly Thr Pro Pro Val Gly Tyr Asp

1 5 10

<210> 41

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _4H12

<400> 41

Asn Ala Glu Val Gly Thr Pro Pro Leu Gly Trp Asp

1 5 10

<210> 42

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _2B5

<400> 42

Asn Ala Asp Tyr Gly Thr Pro Pro Ile Gly Met Asn

1 5 10

<210> 43

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _3E9

<400> 43

Asn Ala Glu Tyr Gly Thr Pro Pro Leu Gly Met Thr

1 5 10

<210> 44

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _1C8

<400> 44

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

1 5 10

<210> 45

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _4F10

<400> 45

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

1 5 10

<210> 46

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _1B2

<400> 46

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

1 5 10

<210> 47

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _1G7

<400> 47

Ala Ala Ala Pro Tyr Tyr Tyr Cys Ser Asp Phe Gly Trp Ile Ser Gly

1 5 10 15

Gly Met Asp

<210> 48

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _1G7

<400> 48

His Glu Tyr Cys Ser Gly Phe Asp Cys Leu Arg Phe Tyr Gly Met Asp

1 5 10 15

<210> 49

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody NTD _3G11

<400> 49

Ala Thr Val Gly Ser Gly Tyr Glu Cys Tyr Trp Pro Gly Gly Asp Asp

1 5 10 15

<210> 50

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _1F7

<400> 50

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Asn Gly Ser Gly Gly Ser Ser Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Thr Lys Lys Ser Leu Tyr

65 70 75 80

Leu His Met Ile Ser Leu Glu Ala Glu Asp Ala Asp Asp Tyr Tyr Cys

85 90 95

Asn Ala Asp Val Gly Glu Pro Pro Val Gly Tyr Glu Tyr Trp Gly Gln

100 105 110

Arg Thr Asn Val Thr Val Ser Ser

115 120

<210> 51

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _2E6

<400> 51

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Thr Ile Phe Ile Ile Gln

20 25 30

Leu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Arg Ser Ile Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Ser Ile Ser Arg Asp Asn Thr Lys Lys Ser Leu Tyr Leu

65 70 75 80

His Met Ile Ser Leu Glu Ala Glu Asp Ala Asp Asp Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Gly Glu Pro Pro Val Gly Tyr Glu Tyr Trp Gly Gln Arg

100 105 110

Thr Asn Val Thr Val Ser Ser

115

<210> 52

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _3E10

<400> 52

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Met Ser Cys Ala Ala Ser Arg Ile Ile Phe Thr Pro Asn

20 25 30

Val Met Gly Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

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

50 55 60

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

65 70 75 80

Gln Met Ser Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Arg Gly Pro Val Pro Gly Arg Pro Ala Gly Asp Tyr Trp Gly Lys

100 105 110

Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 53

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _3B10

<400> 53

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 Ser Ile Ser Ser Ile Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Val Val

35 40 45

Ala Asp Ile Thr Ile Gly Gly Val Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

His Thr Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Glu Gly Val Thr Ala Met Tyr Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 54

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _4G3

<400> 54

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 Ser Gly Phe Ser Phe Ala

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Val Val Thr Ser Gly Gly Arg Ser Asn Glu Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Arg Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ile

85 90 95

Leu Gln Arg Gly Glu Pro Pro Leu Trp Tyr Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 55

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _2A9

<400> 55

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 Pro Gly Ser Ile Tyr Arg Pro Asn

20 25 30

Phe Val Gly Trp Tyr Arg Gln Ala Pro Gly Thr Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ser Ser Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Leu

85 90 95

Val Gly Leu Gly Val Phe Pro Leu Ile Phe Glu His Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 56

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _1F6

<400> 56

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Phe Phe Ser Ile His

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Arg Asp Tyr Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Val Glu Val Gly Thr Pro Pro Val Gly Tyr Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 57

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _4H12

<400> 57

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 Ser Ile Phe Thr Ile Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Arg Arg Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Glu Val Gly Thr Pro Pro Leu Gly Trp Asp Ser Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 58

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _2B5

<400> 58

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Phe Arg Leu Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val

35 40 45

Ala Thr Ile Thr Pro Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Ile Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Tyr Gly Thr Pro Pro Ile Gly Met Asn Tyr Trp Gly Lys Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 59

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _3E9

<400> 59

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Glu Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Arg Leu Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Glu Tyr Gly Thr Pro Pro Leu Gly Met Thr Tyr Trp Gly Lys Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 60

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _1C8

<400> 60

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Arg Leu Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Asp Ile Thr Ser Gly Gly Ser Arg Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Ala Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Tyr Gly Thr Pro Pro Leu Gly Met Thr Tyr Trp Gly Lys Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 61

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _4F10

<400> 61

Gln Val Gln Leu Gln 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 Ser Ile Phe Arg Leu Asn

20 25 30

Ser Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Gly Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Tyr Gly Thr Pro Pro Leu Gly Met Thr Tyr Trp Gly Ala Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 62

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _1B2

<400> 62

Gln Val Gln Leu Val Gln 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 Gly Ser

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asp Ser Thr Ala Arg Asn Val Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Gly Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Asp Val Gly Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

100 105 110

Gln Val Thr Val Ser Ser

115

<210> 63

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _1G7

<400> 63

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Asn Ser Asp Gly Ile Thr Ser Val Lys Gly Arg Phe

50 55 60

Pro Ile Ser Ser Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn

65 70 75 80

Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ala Pro

85 90 95

Tyr Tyr Tyr Cys Ser Asp Phe Gly Trp Ile Ser Gly Gly Met Asp Tyr

100 105 110

Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 64

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _2B3

<400> 64

Glu Val Gln Leu Val Glu Ser Gly Gly Arg Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Ser Thr Tyr

20 25 30

Gly Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Arg Tyr Ser Ala Asp Asn Thr Thr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Trp

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

His Glu Tyr Cys Ser Gly Phe Asp Cys Leu Arg Phe Tyr Gly Met Asp

100 105 110

Asp Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 65

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody NTD _3G11

<400> 65

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 Ser Phe Asp Asp Tyr

20 25 30

Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Cys Ile Cys Ser Ser Asn Asp Cys Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Asn Thr Val Tyr

65 70 75 80

Met Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Val Gly Ser Gly Tyr Glu Cys Tyr Trp Pro Gly Gly Asp Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 66

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _1F7

<400> 66

caggtgcagc tgcaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcag cctctggacg caccttcagt atctatgcca tgggctggtt ccgccaggct 120

ccggggaagg agcgtgagtt tgtagcagct attaacggga gtggtggtag ctcatactat 180

gccgactccg tgaaaggccg attctccatc tccagagaca acaccaagaa gtcgctgtat 240

ctgcacatga tcagcctgga agctgaggac gcggacgact attactgcaa tgcggatgtt 300

ggggaaccgc ccgtggggta tgagtactgg ggccagcgga ccaacgtcac tgtcgcctca 360

tcg 363

<210> 67

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _2E6

<400> 67

caggtgcagc tggtgcagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctgaaac catcttcatt atccagctca tgggctggta ccgccaggct 120

ccagggaagc agcgcgagtt ggtcgctgct attcgtagta ttggtaccac aaactatgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240

caaatgaaca acctgaaacc tgaagacacg gccgtctatt actgtaatgc gcgaggggac 300

tacggcttgg ggcccgagta tgactactgg ggccagggga cccaggtcac tgtctcctca 360

<210> 68

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _3E10

<400> 68

caggtgcagc tggtgcagtc tgggggaggc ttggtgcagc ctggggggtc tctgagaatg 60

tcctgtgcag cctcgcgaat cattttcact cccaatgtca tgggctggta ccgccaggtt 120

ccagggaagc agcgcgagtt ggtcgcactt atttctgata gtggtagtac aaactatgca 180

gactccgtga agggccgatt caccatctcc agagacgccg ccagggacgc ggtatatctg 240

caaatgagcg gcctgaaacc tgaggacacg gccgtctatt attgtaatgc ccgcgggcca 300

gtacctggca gaccggcggg ggactattgg ggcaaaggga cccaggtcac tgtctcctca 360

<210> 69

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _3B10

<400> 69

gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctgggggatc tctgagactc 60

tcctgtgcag cctctggaag catctccagt atcaatgcca tgggctggta ccgccaggct 120

ccagggaagc agcgcgaggt ggtcgcagat atcactatag gtggtgtcac aaactatgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240

cacaccgtgt atctgcaaat gaacaaccta aaacctgagg acacggccgt ctattactgt 300

gagggggtta ctgcgatgta tgactactgg ggccagggga cccaggtcac cgtctcctca 360

<210> 70

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _4G3

<400> 70

gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag cggcttcagt ttcgctgcca tgggctggta ccgccaggct 120

ccagggaaac agcgcgaatt ggtcgcagtt gttactagtg gtggtcggtc aaacgaagca 180

gactccgtga agggccgatt caccatctcc aaagacaatg ccaagaacac ggtgtatcta 240

caaatgaaca gactaacccc tgaggacacg gccgtctatt attgtattct ccaacggggg 300

gagccacccc tgtggtatga ctactggggc caggggaccc aggtcactgt ctcctca 357

<210> 71

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _2A9

<400> 71

gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cccctggaag catctatagg cccaatttcg tgggctggta ccgtcaggct 120

ccagggacgc agcgcgagtt ggtcgcaact attactagta gtggtagcac aaactatgcg 180

gactccgtga agggccgatt caccatctcc agagaaaacg ccaaaaacac ggtgtatctg 240

caaatgaaca gcctgaaacc tgaggacacg gccgtctatt attgtcttgt aggcctcggt 300

gttttccctt tgatatttga acactggggc caggggaccc aggtcactgt ctcctca 357

<210> 72

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _1F6

<400> 72

gaggtgcagc tggtggagtc tgggggaggc ttggcacagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag tttcttcagt atccatgcca tgggctggta ccgccaggct 120

ccagggaagc agcgcgagtt ggtcgcaact attactcgtg attatagcac aaactatgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac gatatatctg 240

caaatgaaca gcctagaacc tgaggacacg gccgtctatt actgtaatgt agaagttggg 300

acaccgcccg tgggatatga ctactggggc caggggaccc aggtcactgt ctcctca 357

<210> 73

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _4H12

<400> 73

gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag catcttcacg atcaatgcca tgggctggta ccgccaggct 120

ccagggaagc agcgcgagct ggtcgcaact attactcgtc gtggtagcac aaactatgca 180

gactccgtga agggtcgatt caccatctcc agagacaacg ccaagaacat aatctatctg 240

caaatgaaca gcctgaaacc tgaggacacg gccgtctatt attgtaatgc cgaggtggga 300

actcctcccc tcggatggga ttcctggggc caggggaccc aggtcaccgt ctcctca 357

<210> 74

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _2B5

<400> 74

gaggtgcagc tggtggagtc tgggggaggc tcggtgcagc ctggggggtc tctgagactc 60

tcctgtgtcg cctctggaag catcttcaga ctcaatgcca tgggctggta ccgccaggct 120

ccagggaagc agcgcgactt ggtcgctact attacgcctg gtggtagcac aaactatgca 180

gactccgtga agggccgatt caccatctcc atagacaacg ccaagaacac ggtgtatctc 240

caaatgaaca gcctgaaacc tgaggacacg gccgtctatt actgtaatgc cgactacgga 300

actcccccca tcggcatgaa ctactggggc aaagggaccc aggtcaccgt ctcctca 357

<210> 75

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _3E9

<400> 75

caggtgcagc tggtgcagtc tgggggaggc ttggtggagc ctggggggtc tttgagactc 60

tcctgtgcag cctctggaag catcttcaga ctcaacgcca tgggctggta ccgccaggct 120

ccagggaagc agcgcgagtt ggtcgctacg attactagtg gtggtagcac aaactatgca 180

gactccgtga agggccgatt caccatctcc agagacagcg ccaaggacac cttgtatctg 240

caaatgaaca gcctgaaacc cgaggacacg gccgtctatt actgtaatgc cgagtacggt 300

actccccccc tcggcatgac ctactggggc aaagggaccc aggtcaccgt ctcctca 357

<210> 76

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _1C8

<400> 76

caggtgcagc tgcaggagtc ggggggaggc ttggtgcagc ctgggggctc tctgacactc 60

tcctgtgcag cctctggaag catcttcaga ctcaacgcca tgggctggta ccgccaggct 120

ccagggaagc agcgcgagtt ggtcgctgac attactagtg gtggtagcag aaactatgca 180

gactccgtga agggccgatt caccatctcc agagacgccg ccaagaacac ggtgtatctg 240

caaatgaacg gcctgaaacc cgaggacacg gccgtctact actgtaatgc cgactacggt 300

actccccccc tcggcatgac ctactggggc aaagggaccc aggtcactgt ctcctca 357

<210> 77

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _4F10

<400> 77

caggtgcagc tgcaggagtc ggggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag catcttcaga ctcaactcca tgggctggta ccgccagact 120

ccagggaagc agcgcgagtt ggtcgctgct attactggtg gtggtagcac aaactatgca 180

gactccgtga agggccgatt caccatctcc agagacaatg ccaggaacac ggtgtatctg 240

caaatgaaca gcctgaaacc cgaggacacg gccgtctatt actgtaatgc cgactacggt 300

actcccccgc tcggcatgac ctactggggc gctgggaccc aggtcactgt ctcctca 357

<210> 78

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _1B2

<400> 78

caggtgcagc tggtgcagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatt caccttcagc ggctcctcaa tgcattgggt ccgtcaggct 120

ccagggaagg ggctcgagtg ggtgtcaggt atagattcca ctgctcgtaa cgtatactat 180

gcagactccg tgaagggtcg attcaccgtc tccagagaca acggcaagaa cacactgtat 240

ctgcaaatga acagtctgaa atctgaggac acggccgtgt attactgtgt aaaagatgta 300

ggatactacg gcatggacta ctggggcaaa gggacccagg tcactgtctc ctca 354

<210> 79

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _1G7

<400> 79

caggtgcagc tggtgcagtc tgggggaggc ttggtgcagc cgggggggtc tctgagactc 60

tcctgtgtag cctctggatt cactttcgat gattatgcca taggctggtt ccgccaggcc 120

ccagggaagg agcgtgaggg ggtctcatgt attagtaata gtgatggtat tacctccgtg 180

aagggccgat tccccatctc cagtgacaac gccaagaaca cggtgtatct gcaaatgaac 240

agcctgaaac ctgaggacac ggccgtttat tactgtgcag ccgccccgta ttactattgc 300

tcagacttcg ggtggatctc gggcggcatg gactactggg gcaaagggac ccaggtcacc 360

gtctcctca 369

<210> 80

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _2B3

<400> 80

caggtgcagc tggtgcagtc tgggggaggc ttggtgcagc cgggggggtc tctgagactc 60

tcctgtgtag cctctggatt cactttcgat gattatgcca taggctggtt ccgccaggcc 120

ccagggaagg agcgtgaggg ggtctcatgt attagtaata gtgatggtat tacctccgtg 180

aagggccgat tccccatctc cagtgacaac gccaagaaca cggtgtatct gcaaatgaac 240

agcctgaaac ctgaggacac ggccgtttat tactgtgcag ccgccccgta ttactattgc 300

tcagacttcg ggtggatctc gggcggcatg gactactggg gcaaagggac ccaggtcacc 360

gtctcctca 369

<210> 81

<211> 372

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody NTD _3G11

<400> 81

gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgaggctc 60

tcctgtgcag cctctggctt ctctttcgac gattatgcca taggctggtt ccgccaggcc 120

ccagggaagg agcgtgaggg ggtctcatgt atctgtagta gtaatgattg cacacactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca ctgccaagaa cacggtgtac 240

atgcaaatga acagcctgaa acctgaggac acggccgttt attattgtgc gacagtcggg 300

tcaggctatg agtgttactg gccgggcggg gatgactact ggggccaggg gacccaggtc 360

accgtctcct ca 372

Claims

1. An antigen binding protein, antibody or antibody active fragment obtained by immunizing camelidae with the NTD region of the S protein of SARS-Cov-2.

2. The antigen binding protein, antibody or antibody active fragment according to claim 1, wherein the camelid is selected from the group consisting of dromedary, bactrian camels, llamas, alpacas and llamas.

3. The antigen binding protein, antibody or antibody active fragment of claim 1, wherein the antibody is a nanobody and the antibody active fragment is a nanobody active fragment;

and/or, the antibody is a monoclonal antibody or a polyclonal antibody.

4. The antigen-binding protein, antibody or antibody-active fragment according to any one of claims 1 to 3, wherein said antigen-binding protein, antibody or antibody-active fragment binds to the NTD region of the S protein of SARS-Cov-2 at a kd value of 4100nM or less, preferably 500nM or less, more preferably 120nM or less, more preferably 65nM or less, more preferably 10nM or less, more preferably 6nM or less, more preferably 1nM or less.

5. The antigen-binding protein, antibody or antibody active fragment according to claim 1 or 4, wherein the nucleotide sequence of the NTD region encoding the S protein of SARS-Cov-2 is shown in SEQ ID NO 1;

preferably, the S protein NTD region of SARS-Cov-2 is prepared by a method comprising the following steps: connecting the nucleotide sequence of the NTD region of the S protein of the SARS-Cov-2 with the nucleotide sequence of the coding transmembrane structure in series, and constructing a vector plasmid; the vector plasmid is transfected into a eukaryotic cell line for expression, so that the NTD region of the S protein of SARS-Cov-2 is displayed outside the cell membrane in the form of a transmembrane protein.

6. A method of constructing an antibody library comprising the steps of:

(3) inserting the amplified DNA into a virus expression vector, preferably a phage expression vector, transforming into bacteria, preferably escherichia coli, and collecting colonies to obtain an antibody library;

preferably, in the step (1), the camelid is alpaca, and/or the immunization adopts a subcutaneous injection mode, and/or the immunization times are 3-5 times, and/or the venous peripheral blood of the immunized animal is collected before and after the last immunization respectively.

7. An antibody library obtained by the method of claim 6, or polyclonal antibodies produced by expression of said antibody library.

8. A method of constructing an antigen-specific antibody library comprising the steps of: screening the antibody library of claim 7 to obtain an antigen-specific antibody library;

preferably, the method comprises the following steps:

(i) culturing the antibody library to release viruses;

(iii) infecting bacteria, preferably Escherichia coli, with the virus specifically binding to the antigen, collecting colonies, and obtaining an antigen-specific antibody library.

9. An antigen-specific antibody library obtained by the method of claim 8, or polyclonal antibodies specifically binding to an antigen produced by expression of the antigen-specific antibody library.

10. A method of producing an antigen binding protein, antibody or active fragment of an antibody comprising the steps of: screening the antibody library of claim 7 to obtain an antigen binding protein, antibody or antibody active fragment that specifically binds to an antigen;

preferably, the method comprises the following steps:

(a) culturing the antibody library to release viruses;

(c) infecting bacteria, preferably escherichia coli, with the virus specifically bound to the antigen, smearing the infected bacteria to a plate culture medium for culture, selecting a single colony, and optionally performing antigen-specific binding identification on the single colony;

more preferably, the method further comprises the following steps: (d) DNA of the single colony is extracted, transformed into a host cell and expressed.

11. An antigen binding protein, antibody or antibody active fragment obtained by the method of claim 10.

12. An antigen binding protein, antibody or antibody active fragment that specifically recognizes and/or binds to the NTD region of the S protein of SARS-Cov-2, wherein the antigen binding protein, antibody or antibody active fragment comprises at least one heavy chain variable region; the heavy chain variable region has:

CDR1 as shown in SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16 or SEQ ID NO 17;

CDR3 as shown in SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48 or SEQ ID NO 49;

preferably, the antigen binding protein, antibody or antibody active fragment comprises one of the heavy chain variable regions and lacks a light chain.

13. An antigen binding protein, antibody or antibody active fragment that specifically recognizes and/or binds to the NTD region of the S protein of SARS-Cov-2, wherein the antigen binding protein, antibody or antibody active fragment comprises at least one heavy chain variable region; the heavy chain variable region has:

CDR1 shown as SEQ ID NO. 2, CDR2 shown as SEQ ID NO. 18 and CDR3 shown as SEQ ID NO. 34;

or, CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 19 and CDR3 shown in SEQ ID NO. 35;

or, CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 20 and CDR3 shown in SEQ ID NO. 36;

or, CDR1 shown in SEQ ID NO. 5, CDR2 shown in SEQ ID NO. 21 and CDR3 shown in SEQ ID NO. 37;

or, CDR1 shown in SEQ ID NO. 6, CDR2 shown in SEQ ID NO. 22, and CDR3 shown in SEQ ID NO. 38;

or, CDR1 shown as SEQ ID NO. 7, CDR2 shown as SEQ ID NO. 23, and CDR3 shown as SEQ ID NO. 39;

or, CDR1 shown in SEQ ID NO. 8, CDR2 shown in SEQ ID NO. 24 and CDR3 shown in SEQ ID NO. 40;

or, CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 25 and CDR3 shown in SEQ ID NO. 41;

or, CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 26 and CDR3 shown in SEQ ID NO. 42;

or, CDR1 shown in SEQ ID NO. 11, CDR2 shown in SEQ ID NO. 27, and CDR3 shown in SEQ ID NO. 43;

or, CDR1 shown in SEQ ID NO. 12, CDR2 shown in SEQ ID NO. 28, and CDR3 shown in SEQ ID NO. 44;

or, CDR1 shown as SEQ ID NO. 13, CDR2 shown as SEQ ID NO. 29 and CDR3 shown as SEQ ID NO. 45;

or, CDR1 shown as SEQ ID NO. 14, CDR2 shown as SEQ ID NO. 30 and CDR3 shown as SEQ ID NO. 46;

or, CDR1 shown in SEQ ID NO. 15, CDR2 shown in SEQ ID NO. 31, and CDR3 shown in SEQ ID NO. 47;

or, CDR1 shown in SEQ ID NO. 16, CDR2 shown in SEQ ID NO. 32 and CDR3 shown in SEQ ID NO. 48;

or, CDR1 shown in SEQ ID NO. 17, CDR2 shown in SEQ ID NO. 33, and CDR3 shown in SEQ ID NO. 49;

14. An antigen binding protein, antibody or antibody active fragment that specifically recognizes and/or binds the NTD region of the S protein of SARS-Cov-2, characterized in that said antigen binding protein, antibody or antibody active fragment comprises at least one heavy chain variable region; the heavy chain variable region has:

an amino acid sequence shown as SEQ ID NO. 50, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 50;

or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids of the amino acid sequence shown in SEQ ID NO. 51 or the amino acid sequence shown in SEQ ID NO. 51;

or the amino acid sequence shown as SEQ ID NO. 52, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 52;

or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids of the amino acid sequence shown in SEQ ID NO. 53 or the amino acid sequence shown in SEQ ID NO. 53;

or the amino acid sequence shown as SEQ ID NO. 54, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 54;

or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids of the amino acid sequence shown in SEQ ID NO. 55 or the amino acid sequence shown in SEQ ID NO. 55;

or the amino acid sequence shown as SEQ ID NO. 56, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 56;

or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 57 or the amino acid sequence shown in SEQ ID NO. 57;

or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 58 or the amino acid sequence shown in SEQ ID NO. 58;

or the amino acid sequence shown as SEQ ID NO. 59, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 59;

or the amino acid sequence shown as SEQ ID NO. 60, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 60;

or the amino acid sequence shown as SEQ ID NO. 61, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 61;

or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids of the amino acid sequence shown in SEQ ID NO. 62 or the amino acid sequence shown in SEQ ID NO. 62;

or the amino acid sequence shown as SEQ ID NO. 63, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 63;

or the amino acid sequence shown as SEQ ID NO. 64, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 64;

or the amino acid sequence shown as SEQ ID NO. 65, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 65;

15. A nucleotide sequence encoding an amino acid sequence as set forth in any one of SEQ ID NO 2 to SEQ ID NO 65 or an antigen binding protein, antibody or antibody active fragment as set forth in any one of claims 1 to 5 or 11 to 14;

preferably, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is selected from the group consisting of: 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or 81.

16. An expression vector comprising the nucleotide sequence of claim 15;

preferably, the expression vector is a phage expression vector, preferably a phage surface display screening vector;

more preferably, the expression vector also contains a nucleotide sequence for coding the phage envelope protein pIII.

17. A virus exogenously introduced with the expression vector of claim 16; the virus is preferably a bacteriophage.

18. A host cell, preferably E.coli, which is exogenously transfected with an expression vector according to claim 8 or infected with a virus according to claim 9.

19. A method of expressing an antigen binding protein, antibody or antibody active fragment using the host cell of claim 18.

20. Expressing the obtained antigen binding protein, antibody or antibody active fragment using the host cell of claim 18.

21. A humanized antigen-binding protein, antibody or antibody active fragment obtained by humanizing the antigen-binding protein, antibody or antibody active fragment according to claim 1 to 5, 11 to 14 or 20.

22. A protein conjugate comprising the antigen binding protein, antibody or antibody active fragment of claim 1 to 5, 11 to 14 or 20 or the humanized antigen binding protein, antibody or antibody active fragment of claim 21 and a ligand;

preferably, the ligand is selected from the group consisting of radioisotopes, fluorophores, and delivery vehicles.

23. A pharmaceutical composition comprising the antigen binding protein, antibody or antibody active fragment of claims 1 to 5, 11 to 14 or 20, the humanized antigen binding protein, antibody or antibody active fragment of claim 21 or the protein conjugate of claim 22;

preferably, the pharmaceutical composition further comprises other active ingredients and/or auxiliary materials.

24. A chimeric antigen receptor comprising the antigen binding protein, antibody or antibody active fragment of claim 1 to 5, 11 to 14 or 20 or the humanized antigen binding protein, antibody or antibody active fragment of claim 21.

25. A chimeric antigen receptor T cell expressing the chimeric antigen receptor of claim 24.

26. The antigen binding protein, antibody or antibody active fragment of claim 1 to 5, 11 to 14 or 20, the antibody library or polyclonal antibody of claim 7, the antigen specific antibody library or polyclonal antibody specifically binding to an antigen of claim 9, the nucleotide sequence of claim 15, the expression vector of claim 16, use of a virus according to claim 17, a host cell according to claim 18, a humanized antigen binding protein, an antibody or an antibody active fragment according to claim 21, a protein conjugate according to claim 22, a pharmaceutical composition according to claim 23, a chimeric antigen receptor according to claim 24 or a chimeric antigen receptor T cell according to claim 25 for the manufacture of a medicament for the prevention of SARS-Cov-2 infection and/or for the treatment of a disease caused by SARS-Cov-2 infection.

27. A kit for in vitro detection of SARS-Cov-2 or the S protein of SARS-Cov-2, comprising the antigen binding protein, antibody or antibody active fragment of claim 1 to 5, 11 to 14 or 20 or the humanized antigen binding protein, antibody or antibody active fragment of claim 21;

preferably, the antigen binding protein, antibody or antibody active fragment is labeled with a label;

more preferably, the label is selected from the group consisting of an enzyme, a chemiluminescent group and an isotopic group.

28. Use of the antigen binding protein, antibody or antibody active fragment of claims 1-5, 11-14 or 20, the humanized antigen binding protein, antibody or antibody active fragment of claim 21, the protein conjugate of claim 22 or the kit of claim 27 for the in vitro detection of SARS-Cov-2 or the S protein of SARS-Cov-2.

29. A method for detecting SARS-Cov-2 or S protein of SARS-Cov-2 in a sample using the antigen binding protein, antibody or antibody active fragment of claims 1 to 5, 11 to 14 or 20, the humanized antigen binding protein, antibody or antibody active fragment of claim 21, the protein conjugate of claim 22 or the kit of claim 27.

30. A contrast agent for detecting SARS-Cov-2 infection, comprising the antigen-binding protein, antibody or antibody active fragment according to claim 1 to 5, 11 to 14 or 20 or the humanized antigen-binding protein, antibody or antibody active fragment according to claim 21.