CN114591425A

CN114591425A - Specific antibody of new coronavirus S protein RBD region and preparation method and application thereof

Info

Publication number: CN114591425A
Application number: CN202011419890.1A
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: CN114591425B

Abstract

The present invention relates to an antigen binding protein, antibody or antibody active fragment obtained by immunizing camelid with RBD region of S protein of SARS-Cov-2. The invention screens, identifies and prepares the antibody of the RBD region of the S protein on the surface of the new coronavirus by relying on the immune system of the camelid, and the obtained antibody has strong specificity, high affinity, small relative molecular mass, high solubility, strong tissue permeability, good distribution, rapid diffusion and metabolism, simple structure, easy genetic engineering modification, simple humanization, high stability, acid and alkali pH resistance, high temperature resistance, lower mass production cost, easy large-scale recombinant preparation and potential clinical diagnosis and treatment value.

Description

Specific antibody of new coronavirus S protein RBD 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 RBD 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 fields of life science and medicine, and many experimental techniques derived based on antigen-antibody specific binding have laid important foundations for scientific research and clinical treatment, such as immunodiagnosis, immunoblotting, enzyme-linked immunosorbent assay, flow cytometry and the like.

The spread of new coronavirus-induced epidemics is accelerated worldwide. 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 antibodies specific for new coronavirus 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 space folding, the traditional mouse monoclonal antibody is difficult to recognize the complex space 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 low temperature (4 ℃), and is not beneficial to the development of large-scale application to 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 recombinant expression in expression systems with high cost such as mammalian cells, 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 RBD 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 S protein RBD region 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 S protein RBD region of SARS-Cov-2 at a kd value of 3000nM or less, preferably 1500nM or less, more preferably 600nM or less, more preferably 100nM or less, more preferably 10nM or less, more preferably 5nM or less, more preferably 1nM or less.

In some embodiments, the S protein RBD region of SARS-Cov-2 has an amino acid sequence as set forth in SEQ ID NO 1. Further preferably, the S protein RBD region of SARS-Cov-2 is prepared by a method comprising the following steps: connecting the nucleotide sequence of the S protein RBD region 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 an S protein RBD region of SARS-Cov-2 is displayed outside a 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 S protein RBD region 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 camelid is selected from dromedary, bactrian, llama, alpaca, and llama, preferably alpaca.

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 an antigen-specific antibody library, 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 specifically binding 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 e.

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 RBD 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 shown in SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 or SEQ ID NO 7;

CDR2 shown in SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO12 or SEQ ID NO 13; and

CDR3 shown in SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18 or SEQ ID NO. 19.

In some embodiments, the heavy chain variable region has: CDR2 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 8 and CDR3 shown in SEQ ID NO. 14.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 9 and CDR3 shown in SEQ ID NO. 15.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 10 and CDR3 shown in SEQ ID NO. 16.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 5, CDR2 shown in SEQ ID NO. 11 and CDR3 shown in SEQ ID NO. 17.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 6, CDR2 shown in SEQ ID NO. 12 and CDR3 shown in SEQ ID NO. 18.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 13 and CDR3 shown in SEQ ID NO. 19.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 20 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. 20.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 21 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. 21.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown in SEQ ID NO. 22 or conservative variants obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 22.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 23 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. 23.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 24 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. 24.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 25 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. 25.

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

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 25 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 set forth in SEQ ID NO 26.

In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is set forth in SEQ ID NO 27.

In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is set forth in SEQ ID NO 28.

In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is set forth in SEQ ID NO. 29.

In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is set forth in SEQ ID NO 30.

In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is set forth in SEQ ID NO 31.

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 humanised 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 antibody obtained by the invention has low kd value and high affinity, and can specifically recognize and bind to RBD regions; (2) the antibody is expected to be used as a new coronavirus detection tool and a new coronavirus S protein detection tool, and after the antibody is combined with an S protein RBD, the antibody is expected to block or destroy the membrane fusion between virus particles and host cells, so that the antibody is expected to treat diseases caused by the new coronavirus, and has potential clinical diagnosis and treatment values; (3) the antibody has the advantages of small relative molecular mass, high solubility, strong tissue permeability, good distributivity, 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; (4) the antibody has simple structure, is easy to carry out genetic engineering modification, has a mature optimization strategy for enhancing the affinity of the single-domain antibody, prolonging the half-life period in vivo and being coupled with other molecules for drug development, such as connecting radioactive isotopes, coupling transfer drugs, CART, fluorescence labeling high-resolution imaging and the like; (5) the heavy chain variable region sequence of the antibody has high homology with a human VH sequence, and the humanization of a single-domain antibody can be realized by a few amino acid mutations; (6) 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; (7) 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 result of detecting the affinity of the monoclonal antibody RBD _2A10 with an antigen;

FIG. 2 is a schematic diagram showing the result of detecting the affinity of the monoclonal antibody RBD _2B10 with an antigen;

FIG. 3 is a diagram showing the result of detecting the affinity of the monoclonal antibody RBD _2E2 for the antigen;

FIG. 4 is a schematic diagram showing the result of detecting the affinity of the monoclonal antibody RBD _3D9 with an antigen;

FIG. 5 is a schematic diagram showing the result of detecting the affinity between the monoclonal antibody RBD _4G6 and the antigen;

FIG. 6 is a schematic diagram showing the result of detecting the affinity of the monoclonal antibody RBD _4G8 with an 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.

RBD area: the S1 subunit of the S protein of the new coronavirus mainly comprises an NTD region and an RBD region; wherein, the RBD region is called as a receptor binding region and is a region necessary for S protein to invade host cells; the literature reports show that after the new coronavirus particles enter a human body, the S protein on the surface of the virus particles is combined with angiotensin converting enzyme (ACE2) on the surface of a cytoplasmic membrane, and the RBD region of the S protein and ACE2 have membrane fusion effect, so that the virus particles invade the cells of the human body.

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 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

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

In the prior art, the conventional method for preparing the antigen is to recombine, express and purify an RBD region of an S protein as the antigen and then immunize animals such as mice, rabbits, monkeys and the like. 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 RBD region of the recombinantly expressed and purified S protein is not a membrane protein and is not present in the native state, and thus the resulting antibody is not necessarily able to correctly recognize the antigen.

Aiming at the RBD structure domain of the S protein of the novel coronavirus, the method for preparing the antigen specifically comprises the following steps:

(1) connecting in series, constructing DNA sequence of RBD region (i.e. 319-541 st amino acid, specifically: RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF) of coding S protein and an additional transmembrane domain to pCaggs vector plasmid;

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

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

The DNA sequence for constructing the RBD region of the coding S protein adopted in the embodiment is specifically shown in SEQ ID NO:1, and specifically comprises the following steps:

CGGGTGCAGCCTACAGAGTCTATTGTGCGGTTCCCAAACATCACAAACCTGTGCCCTTTCGGCGAGGTGTTCAACGCCACCCGGTTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCTCTAACTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCTCTTTCTCTACATTCAAGTGCTACGGCGTGTCCCCTACAAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCTTTCGTGATTAGAGGCGACGAGGTGAGGCAGATTGCCCCCGGCCAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACAGGCTGCGTGATCGCCTGGAACTCTAACAACCTGGACTCTAAGGTGGGCGGCAACTACAACTACCTGTACAGACTGTTCCGGAAGTCTAACCTGAAGCCATTCGAGAGGGACATTAGCACCGAGATTTACCAGGCCGGCTCTACCCCATGCAACGGCGTGGAGGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTCCAGCCTACAAACGGCGTGGGCTACCAGCCTTACCGGGTGGTGGTGCTGTCTTTCGAGCTGCTCCACGCCCCCGCCACAGTGTGCGGCCCAAAGAAGAGCACAAACCTCGTGAAGAACAAGTGCGTGAACTTC

the RBD region of the S protein provided in this example is capable of being expressed and present on the surface of a cell membrane, approaching the native conformation and state of the S protein on the surface of a 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) performing 4 times of immunization on alpaca, injecting antigen into animal body subcutaneously, recording the first immunization as the first day, and performing subsequent immunization respectively 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 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 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:

(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 6 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 an S protein RBD region.

The results of the detection of the affinity between the antibodies and the RBD region of the antigen S protein corresponding to the 6 groups of monoclonal colonies are shown in FIGS. 1 to 6, and the results of the affinity values kd are shown in Table 3 below.

Table 3: results of affinity testing

	kd(nM)
		RBD_2A10	590
RBD_2B10	1171
		RBD_2E2	2.9
RBD_3D9	0.66
		RBD_4G6	8
RBD_4G8	2678

From the above results, it was found that the antibody obtained by the selection of the monoclonal colonies of the present example bound to the RBD region of the S protein of SARS-Cov-2 at a kd value of 3000nM or less, preferably 1500nM or less, more preferably 600nM or less, more preferably 100nM or less, more preferably 10nM or less, more preferably 5nM or less, and more preferably 1nM or less.

Respectively carrying out amplification culture on the 6 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 6 groups of monoclonal antibody microbial colonies are specifically shown in tables 4 to 9 below:

table 4: amino acid sequence and nucleotide sequence of monoclonal antibody RBD _2A10

Table 5: amino acid sequence and nucleotide sequence of monoclonal antibody RBD _2B10

Table 6: amino acid sequence and nucleotide sequence of monoclonal antibody RBD _2E2

Table 7: amino acid sequence and nucleotide sequence of monoclonal antibody RBD _3D9

Table 8: amino acid sequence and nucleotide sequence of monoclonal antibody RBD _4G6

Table 9: amino acid sequence and nucleotide sequence of monoclonal antibody RBD _4G8

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, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

SEQUENCE LISTING

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

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

<130> RYP2010929.3

<160> 31

<170> PatentIn version 3.5

<210> 1

<211> 669

<212> DNA

<213> SARS-Cov-2

<400> 1

cgggtgcagc ctacagagtc tattgtgcgg ttcccaaaca tcacaaacct gtgccctttc 60

ggcgaggtgt tcaacgccac ccggttcgcc tctgtgtacg cctggaaccg gaagcggatc 120

tctaactgcg tggccgacta ctccgtgctg tacaactccg cctctttctc tacattcaag 180

tgctacggcg tgtcccctac aaagctgaac gacctgtgct tcaccaacgt gtacgccgac 240

tctttcgtga ttagaggcga cgaggtgagg cagattgccc ccggccagac aggcaagatc 300

gccgactaca actacaagct gcccgacgac ttcacaggct gcgtgatcgc ctggaactct 360

aacaacctgg actctaaggt gggcggcaac tacaactacc tgtacagact gttccggaag 420

tctaacctga agccattcga gagggacatt agcaccgaga tttaccaggc cggctctacc 480

ccatgcaacg gcgtggaggg cttcaactgc tacttcccac tgcagtccta cggcttccag 540

cctacaaacg gcgtgggcta ccagccttac cgggtggtgg tgctgtcttt cgagctgctc 600

cacgcccccg ccacagtgtg cggcccaaag aagagcacaa acctcgtgaa gaacaagtgc 660

gtgaacttc 669

<210> 2

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody RBD _2A10

<400> 2

Gly Phe Thr Phe Asp Asp Tyr Asp Met

1 5

<210> 3

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody RBD _2B10

<400> 3

Gly Phe Thr Phe Asp Asp Tyr Thr Ile

1 5

<210> 4

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody RBD _2E2

<400> 4

Gly Arg Thr Phe Ser Thr Tyr Ala Val

1 5

<210> 5

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody RBD _3D9

<400> 5

Gly Gly Thr Phe Ser Val Tyr Ser Met

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody RBD _4G6

<400> 6

Gly Arg Thr Phe Ser Thr Tyr Ala Met

1 5

<210> 7

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 of monoclonal antibody RBD _4G8

<400> 7

Gly Ser Ile Phe Ser Ile Arg Ala Met

1 5

<210> 8

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody RBD _2A10

<400> 8

Thr Cys Asp Gly Gly Thr Thr Tyr

1 5

<210> 9

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody RBD _2B10

<400> 9

Ser Cys Ile Ser Ser Val Asp Gly Thr Thr His

1 5 10

<210> 10

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody RBD _2E2

<400> 10

Leu Trp Asn Ser Gly Gly Thr Tyr

1 5

<210> 11

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody RBD _3D9

<400> 11

Ser Trp Asn Gly Glu Asn Ser Tyr

1 5

<210> 12

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody RBD _4G6

<400> 12

Ile Trp Ser Tyr Gly Gly Thr Tyr

1 5

<210> 13

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 of monoclonal antibody RBD _4G8

<400> 13

Thr Gly Gly Gly Ala Ser

1 5

<210> 14

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody RBD _2A10

<400> 14

Ala Lys Phe Ser Ala Asn Asp Pro Arg Glu

1 5 10

<210> 15

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody RBD _2B10

<400> 15

Ala Ala Thr Arg Gly Thr Tyr Tyr Asp Gly Ser Gln Tyr Leu Arg Asp

1 5 10 15

Pro Tyr Gly Met His

20

<210> 16

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody RBD _2E2

<400> 16

Ala Ala Asp Gly Ala Pro Ala Pro Tyr Thr Ile Val Glu Leu Asp Asp

1 5 10 15

Gly Glu Phe Tyr Thr

20

<210> 17

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody RBD _3D9

<400> 17

Ala Ala Ala Ser Thr Ser Ala Arg Gly Met Ser Val Leu Thr Pro Lys

1 5 10 15

Leu Gly

<210> 18

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody RBD _4G6

<400> 18

Ala Ala Asp Gly Ala Pro Ala Pro Tyr Thr Ile Val Glu Leu Asn Asp

1 5 10 15

Arg Gly Leu Tyr Lys

20

<210> 19

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody RBD _4G8

<400> 19

Ser Ala Asp Val Ile Glu Gly Asp Tyr Gly Thr Leu Leu Met Asp

1 5 10 15

<210> 20

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody RBD _2A10

<400> 20

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 Asp Asp Tyr

20 25 30

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

35 40 45

Ser Val Ile Thr Cys Asp Gly Gly Thr Thr Tyr Tyr Ala Glu Ser Val

50 55 60

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

65 70 75 80

Leu Leu Ile Asp Ser Leu Lys Ser Glu Asp Thr Asp Val Tyr Tyr Cys

85 90 95

Ala Lys Phe Ser Ala Asn Asp Pro Arg Glu His Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser

115

<210> 21

<211> 132

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody RBD _2B10

<400> 21

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 Asp Asp Tyr

20 25 30

Thr Ile Gly Trp Phe Arg Gln Val Pro Gly Asn Glu Arg Glu Gly Gly

35 40 45

Glu Gly Val Ser Cys Ile Ser Ser Val Asp Gly Thr Thr His Tyr Ala

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Tyr Cys Ala Ala Thr Arg Gly Thr Tyr Tyr Asp Gly Ser Gln Tyr

100 105 110

Leu Arg Asp Pro Tyr Gly Met His Tyr Trp Gly Lys Gly Thr Gln Val

115 120 125

Thr Val Ser Ser

130

<210> 22

<211> 129

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody RBD _2E2

<400> 22

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 Thr Tyr

20 25 30

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

35 40 45

Ala Ala Ile Leu Trp Asn Ser Gly Gly Thr Tyr Tyr Thr His Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Asp Gly Ala Pro Ala Pro Tyr Thr Ile Val Glu Leu Asp Asp

100 105 110

Gly Glu Phe Tyr Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 23

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody RBD _2E2

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Ala Ser Thr Ser Ala Arg Gly Met Ser Val Leu Thr Pro Lys

100 105 110

Leu Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 24

<211> 129

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody RBD _4G6

<400> 24

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 Arg Thr Phe Ser Thr Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Asp Gly Ala Pro Ala Pro Tyr Thr Ile Val Glu Leu Asn Asp

100 105 110

Arg Gly Leu Tyr Lys Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 25

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody RBD _4G8

<400> 25

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 Val Ser Gly Ser Ile Phe Ser Ile Arg

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Val Ile Glu Gly Asp Tyr Gly Thr Leu Leu Met Asp Leu Trp Gly

100 105 110

Arg Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 26

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody RBD _2A10

<400> 26

gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatt cacttttgat gattatgaca tgagctgggt ccgacaggct 120

ccagagaagg ggctggagtg gatctctgtt atcacctgtg atggtggtac gacttactat 180

gcagaatccg tgaaaggccg attctccatc tccagagaca acgccaagaa cacgctgtat 240

ctgctaattg acagtctgaa atctgaggac acggacgtgt attactgcgc aaaattctcg 300

gctaacgacc caagggagca ctggggccag gggacccagg tcactgtctc ctct 354

<210> 27

<211> 396

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody RBD _2B10

<400> 27

caggtgcagc tggtgcagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatt cactttcgat gattatacca taggctggtt ccgccaggtc 120

ccagggaatg agcgtgaggg gggtgagggg gtctcatgta ttagtagtgt tgatggtact 180

acacactatg cagagtccgt gaagggccga ttcaccatct ccagtgacaa cgccaagaac 240

acggtgtatc tgcaaatgaa cagcctgaaa cccgaggaca cggccgttta ttactgtgca 300

gcgaccaggg gtacatacta tgatggtagt caatacctta gggatccgta cggcatgcac 360

tactggggca aagggaccca ggtcactgtc tcctca 396

<210> 28

<211> 387

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody RBD _2E2

<400> 28

caggtgcagc tgcaggagtc ggggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcag cctctggacg caccttcagt acctatgccg tgggctggtt ccgccaggct 120

ccaggaaagg agcgtgagtt tgttgcagct attctctgga atagtggtgg cacatattat 180

acacactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240

ctgcaaatga acagcctgaa acctgaggac acggccgttt attattgtgc agcagatggg 300

gccccggcgc cctataccat agtggaattg gacgacggtg aattctatac ctactggggc 360

caggggaccc aggtcaccgt ctcctca 387

<210> 29

<211> 378

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody RBD _3D9

<400> 29

caggtgcagc tggtgcagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtacag cctctggtgg cacctttagt gtgtattcca tgggctggtt ccgccaggct 120

ccagggaagg accgtgagtt tgtagcagct atcagctgga atggtgaaaa ctcatactat 180

acagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtcc 240

ctgcaaatga acagcctgaa acctgaggac acggccgttt attcttgtgc agcagcctcg 300

actagtgctc ggggcatgag tgtcctgacg ccgaaactgg gttcctgggg ccaggggacc 360

caggtcactg tctcctca 378

<210> 30

<211> 387

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody RBD _4G6

<400> 30

caggtgcagc tggtgcagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggacg caccttcagt acctatgcca tgagctggtt ccgccaggct 120

ccaggaaagg agcgtgagtt tgttgcagct attatctgga gttatggtgg cacatattat 180

acagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240

ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agcagatggg 300

gccccggcgc cctatacgat agtggaactt aacgaccgtg gactctataa gtactggggc 360

caggggaccc aggtcactgt ctcctca 387

<210> 31

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody RBD _4G8

<400> 31

gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag tctctggaag catcttcagt atccgtgcca tgggctggta ccgccaggct 120

ccagggaagc agcgcgagtt ggtcgcagct attactggtg gtggtgcaag ctatgcggac 180

tccgtgaagg gccgactcac catctccaca gacaacgcca acaacacggt gtctctgctg 240

atgaacagcc tgaagcctga ggacgcggcc gtctattact gtagtgcaga cgtcattgag 300

ggcgactatg ggacgttgtt aatggattta tggggccggg ggacccaggt caccgtctcc 360

tca 363

Claims

1. An antigen binding protein, antibody or antibody active fragment obtained by immunizing camelidae with the RBD 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 S protein RBD region of SARS-Cov-2 at a kd value of 3000nM or less, preferably 1500nM or less, more preferably 600nM or less, more preferably 100nM or less, more preferably 10nM or less, more preferably 5nM 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 RBD region of the S protein encoding SARS-Cov-2 is shown in SEQ ID NO 1;

preferably, the S protein RBD region of SARS-Cov-2 is prepared by a method comprising the following steps: connecting the nucleotide sequence of the S protein RBD region 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 RBD region of the S protein of SARS-Cov-2 is displayed outside the cell membrane in the form of 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 the S protein RBD region of SARS-Cov-2, wherein said antigen binding protein, antibody or antibody active fragment comprises at least one heavy chain variable region; the heavy chain variable region has:

CDR1 shown in SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 or SEQ ID NO 7;

CDR3 shown in SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18 or SEQ ID NO. 19;

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 the RBD 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:

CDR2 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 8 and CDR3 shown in SEQ ID NO. 14;

or, CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 9, and CDR3 shown in SEQ ID NO. 15;

or, CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 10 and CDR3 shown in SEQ ID NO. 16;

or, CDR1 shown in SEQ ID NO. 5, CDR2 shown in SEQ ID NO. 11, and CDR3 shown in SEQ ID NO. 17;

or, CDR1 shown in SEQ ID NO. 6, CDR2 shown in SEQ ID NO. 12, and CDR3 shown in SEQ ID NO. 18;

or, CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 13 and CDR3 shown in SEQ ID NO. 19;

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

the amino acid sequence shown as SEQ ID NO. 20 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. 20;

or the amino acid sequence shown as SEQ ID NO. 21, 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. 21;

or the amino acid sequence shown as SEQ ID NO. 22, 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. 22;

or the amino acid sequence shown as SEQ ID NO. 23, 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. 23;

or the amino acid sequence shown as SEQ ID NO. 24, 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. 24;

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. 25 or the amino acid sequence shown in SEQ ID NO. 25;

15. A nucleotide sequence encoding an amino acid sequence as set forth in any one of SEQ ID NO 2 to SEQ ID NO 25 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: 26, 27, 28, 29, 30 and 31.

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 1-5, 11-14 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.