CN117866084A - Fully human monoclonal antibody for resisting novel coronavirus and application thereof - Google Patents

Fully human monoclonal antibody for resisting novel coronavirus and application thereof Download PDF

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CN117866084A
CN117866084A CN202410031706.8A CN202410031706A CN117866084A CN 117866084 A CN117866084 A CN 117866084A CN 202410031706 A CN202410031706 A CN 202410031706A CN 117866084 A CN117866084 A CN 117866084A
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chain variable
variable region
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辛洪波
胡萍
刘苏俊
郭昊
尧志峰
俞之翔
兰凌宁
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Nanchang University
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Abstract

The invention relates to a fully human monoclonal antibody for resisting novel coronaviruses and application thereof, belonging to the technical field of bioengineering. The fully human monoclonal antibody has a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 19; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 20. The invention also provides a coding gene, an expression vector, application and a composition of the antibody. The binding molecule of the invention can prevent the infection of the susceptible cells by the novel coronavirus, is fully human, has low immunogenicity, good affinity, good treatment effect and low side effect compared with other animal-derived (such as murine) anti-novel coronavirus molecules, and simultaneously provides great guarantee for the standardized production of the antibody.

Description

Fully human monoclonal antibody for resisting novel coronavirus and application thereof
Technical Field
The invention relates to the technical field of bioengineering, in particular to a fully human monoclonal antibody with high affinity for resisting novel coronavirus SARS-CoV-2 and application thereof.
Background
The monoclonal antibody medicine industry is the sub-industry with the largest proportion in the global biological product industry, and the medicine has the clinical treatment advantages of strong specificity, quick curative effect, short half-life, small side effect and the like, and represents the latest development direction of the medicine treatment field. The market ratio of monoclonal antibodies in the global biological product industry has risen from 2.5% in 1997 to 34.7% in 2015, and the global market size has reached $916 billion by 2015. The 2010-2015 is also the high-speed development period of the monoclonal antibody drug industry in China, according to the prediction of the consultant in China, the market scale of the monoclonal antibody drug industry in China reaches 280 hundred million yuan by 2020, the average compound growth rate in 2016-2020 reaches 30%, and the acceleration level of 9.84% of the global monoclonal antibody drug industry in the future 5 years is predicted by far exceeding Research and Markets. Monoclonal antibody technology has been rapidly developed in recent years, from the original animal antibodies, chimeric antibodies to partially humanized antibodies, to finally fully humanized antibodies, the immunogenic response and side effects of monoclonal antibodies have been greatly reduced, and the poor therapeutic effects have been significantly improved. In view of the immunogenicity of heterologous proteins/antibodies and the therapeutic effects, therapeutic monoclonal antibodies are evolving towards humanized and fully human monoclonal antibodies, and in particular the development of fully human monoclonal antibodies will be the main direction in the field of antibody drug development. In addition, the main techniques for therapeutic fully human monoclonal antibody drug development include 1) complete antibodies produced by human monoclonal antibody transgenic mice; 2) Partially recombinant antibodies such as scFv, fab, diabody, dAb, etc. produced by the Phage riboname/mRNA and Yeast cell displays technology; 3) A complete antibody produced by a human hybridoma or a human B lymphocyte cell line. The current production of human monoclonal antibodies mainly comes from transgenic mice and expression production from phage and other systems. However, the incomplete monoclonal antibodies produced by recombinant techniques have poor application prospects due to their weak specificity and affinity for target tissues/cells.
Currently, more than 20 products directed against new crown antibodies are put into use and the spike protein of the pathogen is pushed into a ten-large target list at one time. Some of which have been authorized for emergency use by the FDA, but these drugs have not been fully approved. Antibody drugs for the treatment of viral infectious diseases have been reported, as the case of antisera for the treatment of SARS and severe H5N1 hepatitis C virus infected persons has demonstrated that antibodies play an important role in the treatment of viral infections. The fully human monoclonal antibody can not only block the combination of viruses and target cells, but also kill the viruses or remove the cells infected by the viruses through the action of the equivalent cells of complement, T cells, NK cells and the like, and in addition, the monoclonal antibody has the obvious advantages of low immunogenicity, strong specificity, quick curative effect, small side effect and the like. Coronaviruses are largely divided into four subfamilies of alpha, beta, gamma and delta coronaviruses, where alpha and beta coronaviruses can infect mammals and gamma and delta coronaviruses can infect birds. There are 7 coronaviruses found to infect humans, of which SARS-CoV-2 belongs to the beta subtype of coronavirus. The Spike protein (S protein) of SARS-CoV-2 is the most important surface protein of the virus, and is closely related to the infection of the virus. The S protein contains two subunits S1 and S2, wherein the S1 subunit comprises a receptor binding region (S-RBD) which is mainly responsible for recognizing cell receptors; whereas the S2 protein contains the essential elements required for the membrane fusion process. Our bioinformatics research finds that the S1-RBD region of SARS-CoV-2 has a certain similarity with related coronaviruses such as SARS and MERS, while the S2 region is highly similar to other 6 pathogenic coronaviruses, and no monoclonal antibody drug developed against S2 protein has been developed in the market so far. Therefore, the development of the biological medicine which has good effect and can be used for treating coronavirus infection in large scale clinically has very important significance.
Disclosure of Invention
The inventor takes S2 protein with good immunogenicity on the surface of SARS-CoV-2 as antigen epitope through intensive research, expresses and purifies antigen protein after constructing plasmid through antigen protein sequence, then detects serum antibody level of recovered crowd after SARS-CoV-2 infection, and screens out a plurality of individuals with higher serum antibody level, then collects anticoagulated whole blood of these individuals, separates mononuclear cells of peripheral blood, establishes phage library, utilizes S2 protein (highly conserved, can be used for all pathogenic coronaviruses including SARS-CoV-2 and possible variant virus strains in future) as antigen protein, obtains target antibody through ELISA step by step, obtains cell culture supernatant containing target antibody through transfection HEK293T cell, finally carries out neutralization titer experiment and determines antiviral effect, thereby screening out antibody cell strain with stronger neutralization activity for novel coronavirus (SARS-CoV-2) and other pathogenic coronaviruses. The monoclonal antibody of the invention is fully human, and the heavy chain, the light chain variable region and the constant region are all derived from human genes, so the monoclonal antibody has the characteristics of low immunogenicity and high safety, and test results prove that the monoclonal antibody of the invention has the characteristic of high affinity, and the monoclonal antibody provided by the invention can obviously resist novel coronavirus (SARS-CoV-2), thus providing great guarantee for the standardized production of patent medicines of the antibody.
The early bioinformatics research shows that the RBD region sequence of the novel coronavirus (SARS-CoV-2) S1 protein has a certain similarity with related coronaviruses such as SARS and MERS, and the S2 region of the novel coronavirus has a high similarity with other discovered pathogenic coronaviruses, which suggests that the conservation degree of the S2 protein is high, and because the S2 has a high similarity in different coronaviruses, the novel coronavirus (SARS-CoV-2) provides a certain strategic reserve for preventing and treating coronavirus infection epidemic situations which can occur in the future. Therefore, when selecting antigen target, we select S2 protein as antigen epitope, finally obtain the fully human monoclonal antibody with high safety and high affinity against novel coronavirus (SARS-CoV-2).
In one aspect, the invention provides a fully human monoclonal antibody against a novel coronavirus having a heavy chain variable region and a light chain variable region:
the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs 11, 13, 15, 17 or 19; or the amino acid sequence with the same function and formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in any one of SEQ ID NO 11, 13, 15, 17 or 19;
the amino acid sequence of the light chain variable region is as shown in any one of EQ ID NOs 12, 14, 16, 18 or 20; or the amino acid sequence with the same function, which is formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in any one of 12, 14, 16, 18 or 20 of EQ ID NO.
Preferably, the fully human monoclonal antibody has any one of the following sets of heavy chain variable regions and light chain variable regions:
(i) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 11 (R2P 1-D8-H); the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 12 (R2P 1-D8-L);
(ii) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 13 (R2P 2-D10-H); the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 14 (R2P 2-D10-L);
(iii) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 15 (R2P 2-G5-H); the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16 (R2P 2-G5-L);
(iv) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17 (R2P 2-D7-H); the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 18 (R2P 2-D7-L);
(v) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 19 (R2P 2-F8-H); the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 20 (R2P 2-F8-L).
The fully human monoclonal antibodies composed of the heavy chain variable region and the light chain variable region can specifically bind to novel coronavirus (SARS-CoV-2) S2 protein.
In another aspect of the invention, there is provided a nucleic acid molecule encoding the fully human monoclonal antibody described above.
Preferably, the nucleic acid molecule has a nucleotide sequence of the heavy chain variable region as set forth in any one of SEQ ID NOs 1, 3, 5, 7 or 9; and a nucleotide sequence of a light chain variable region as set forth in any one of SEQ ID NOs 2, 4, 6, 8 or 10.
More preferably, the nucleic acid molecule has any one of the following sets of heavy chain variable regions and light chain variable regions:
(i) The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 1 (R2P 1-D8-H); the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 2 (R2P 1-D8-L);
(ii) The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 3 (R2P 2-D10-H); the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 4 (R2P 2-D10-L);
(iii) The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 5 (R2P 2-G5-H); the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 6 (R2P 2-G5-L);
(iv) The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 7 (R2P 2-D7-H); the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 8 (R2P 2-D7-L);
(v) The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 9 (R2P 2-F8-H); the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 10 (R2P 2-F8-L).
In another aspect of the invention, there is provided an expression vector comprising the nucleic acid molecule described above.
The expression vector may comprise, in addition to the nucleic acid molecules described above, suitable promoter or control sequences. The expression vector may be used to transform an appropriate host cell to enable expression of the protein.
In another aspect of the invention, a host cell is provided, said host cell comprising an expression vector as described above.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: bacterial cells such as E.coli, streptomyces; salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, NSO or Bowes melanoma cells, etc. Host cells particularly suitable for use in the present invention are eukaryotic host cells, particularly mammalian cells, such as 293T cells.
In another aspect of the present invention, there is provided a method for preparing the above fully human monoclonal antibody, comprising the steps of:
s1, expression and purification of an antigen protein sequence: expressing purified S2 protein from colibacillus system for screening SARS-CoV-2 monoclonal antibody medicine;
s2, phage display, and obtaining a specific antibody sequence: constructing libraries of blood samples of a plurality of rehabilitators, performing 4-5 rounds of phage biopanning and antibody verification by ELISA (enzyme-linked immunosorbent assay) to obtain specific antibody sequences, and performing expression and purification of recombinant antibodies to obtain antibodies with high affinity with S2 protein;
s3, pseudo virus neutralization experiment of novel coronavirus: and (3) selecting an antibody with higher affinity with the S2 protein in the step S2, performing gradient dilution, incubating the antibody with the novel coronavirus pseudovirus, adding the antibody into HEK293T-ACE2 cells, replacing a fresh complete culture medium after 6 hours, continuously culturing the cells for 48 hours, and judging the neutralization efficiency of the antibody by detecting the activity of cell luciferase.
The fully human monoclonal antibody screened by the human peripheral blood mononuclear cell specific screening method is an antibody produced by activating an autoimmune system in a human body by using a live virus protein (antigen), has obvious advantages compared with the antibody prepared by a hybridoma technology, a transgenic mouse and other technologies, has the advantages of low immunogenicity, high affinity, strong specificity, high antibody production diversity, high safety, good pharmacokinetics, low dosage, high efficacy, quick curative effect and the like, has no resource and large-scale production limit, is easier to realize standardized production and quality control, and solves the problem of potential pollution sources which are difficult to avoid by blood source products.
In another aspect of the invention, the use of the fully human monoclonal antibody or binding fragment thereof in the manufacture of a reagent or kit for detecting novel coronavirus infection, and in the manufacture of a medicament for treating, preventing, novel coronavirus (SARS-CoV-2) infection is provided.
The invention aims at using S2 protein as epitope antigen, utilizing established monoclonal antibody screening platform to rapidly develop specific fully human monoclonal antibody for SARS-CoV-2 and other pathogenic coronaviruses including variant strains which may appear in future, so as to provide effective medicine for preventing and treating coronavirus infection. According to the regulations of medicine registration management method in China, the research product belongs to the 1 st class of biological products for treatment, is a research and development variety of raw medicines, and has great social significance and application value.
In another aspect of the invention, there is provided a composition comprising a therapeutically effective amount of an antibody mixture of one or more antibodies of said fully human monoclonal antibodies, and a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable" as used herein means that the molecular entity and composition do not produce adverse, allergic or other untoward reactions when properly administered to an animal or human. As used herein, a "pharmaceutically acceptable carrier" should be compatible with, i.e., capable of being blended with, the monoclonal antibodies or fragments thereof of the present invention without substantially reducing the efficacy of the composition in the usual manner.
Specific examples of some substances which may be pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc.
The composition of the present invention may be formulated into various dosage forms as required, and the dosage beneficial to the patient may be determined by the physician according to the type, age, weight and general condition of the patient, the mode of administration, etc. The administration may be, for example, injection or other therapeutic means.
The pharmaceutical composition may comprise two or more monoclonal antibodies or fragments thereof having neutralizing activity against the novel coronavirus.
In another aspect of the invention, a kit for detecting novel coronavirus (SARS-CoV-2) is provided, comprising said monoclonal antibody or fragment thereof.
Based on the monoclonal antibody or the fragment thereof, a kit for conveniently, rapidly and accurately detecting novel coronavirus (SARS-CoV-2) can be prepared. As a detection mode of the invention, an indirect ELISA method is adopted, an antigen to be detected is coated on a solid phase carrier, and the monoclonal antibody or the fragment thereof is utilized for detection.
As a preferred mode of the present invention, the monoclonal antibody or a fragment thereof is an antibody, which can be detected according to the principle of the double antibody sandwich method. The conventional method of double antibody sandwich method is to fix the primary antibody (such as monoclonal antibody of the invention) on the carrier, then make the primary antibody react with antigen, then react with the secondary antibody (the secondary antibody carries detectable signal or can combine with the substance carrying detectable signal) after washing, finally make chemiluminescence or enzyme-linked chromogenic reaction to detect signal. The double antibody sandwich method is particularly suitable for detection of antigens having two or more epitopes.
For convenience in detection, the kit may contain, in addition to the monoclonal antibody or fragment thereof of the present invention, other detection reagents or auxiliary reagents such as those conventionally used in ELISA kits, the characteristics of which and the methods of formulating them are well known to those skilled in the art, such as color-developing agents, labels, secondary antibodies, anti-antibodies, sensitizers, etc. It will be appreciated by those skilled in the art that various variants of the detection kit are encompassed by the present invention, provided that the monoclonal antibody or fragment thereof of the present invention is utilized therein as a reagent for recognizing the novel coronavirus (SARS-CoV-2).
Further, instructions for use may be included in the kit for use in describing the method of use of the reagents loaded therein.
After obtaining the monoclonal antibody or fragment thereof provided by the present invention, various immunological correlation methods can be used to detect the S2 protein of SARS-CoV-2 in the sample, thereby knowing whether the donor of the sample to be tested is infected with the novel coronavirus (SARS-CoV-2), and all such methods are included in the present invention. Preferably, the method is for non-disease diagnosis purposes.
In another aspect of the invention, there is provided a method of non-therapeutically inhibiting a novel coronavirus (SARS-CoV-2), said method comprising administering to a patient an effective amount of said monoclonal antibody or fragment thereof.
In another aspect of the present invention, there is provided a method for non-therapeutic detection of novel coronavirus (SARS-CoV-2), wherein the monoclonal antibody or fragment thereof is contacted with a sample to be tested, and the presence and amount of the novel coronavirus (SARS-CoV-2) are obtained by detecting the binding of the monoclonal antibody or fragment thereof to the sample to be tested.
As used herein, the term "sample to be tested" encompasses a variety of sample types, including blood and other body fluid samples of biological origin, solid tissue samples such as biopsy tissue samples or tissue cultures, or cells derived therefrom or their progeny. The term also includes samples that have been treated by any means after they have been obtained, for example by treatment with reagents, solubilization, or enrichment for certain components such as proteins or polynucleotides. The term encompasses various clinical samples from any species, as well as cultured cells, cell supernatants, and cell lysates.
Compared with the prior art, the invention has the beneficial effects that:
the invention successfully prepares the fully human monoclonal antibody for resisting the novel coronavirus SARS-CoV-2, the monoclonal antibody has the characteristic of high affinity, and the monoclonal antibody can specifically bind the novel coronavirus SARS-CoV-2 and can obviously resist the novel coronavirus SARS-CoV-2.
Compared with the murine antibody, the gene of the fully human antibody is fully derived from human genes, has no other species components, does not generate toxic or side effects such as anti-mouse antibody and the like in human body, has better biocompatibility, is more suitable and has better potential to become a macromolecular medicament for treating novel coronavirus SARS-CoV-2, and provides great guarantee for the standardized production of the antibody.
Drawings
FIG. 1 is a technical roadmap of the invention;
FIG. 2 shows the expression and purification patterns of five antibody proteins of the present invention, wherein MW is the molecular weight marker, IN is the negative control, FT is the flow-through, W is the wash solution, and E is the eluent;
FIG. 3 is a graph showing purification patterns of five antibody proteins of the present invention, wherein MW is a molecular weight marker.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The experimental procedure, without specific conditions noted in the examples, is generally followed by conventional conditions, such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
FIG. 1 is a technical scheme of the invention, comprising construction of antigen protein sequence vectors, expression of a prokaryotic system, purification and verification, acquisition of S2 antigen proteins, phage display, multiple rounds of panning, phage screening, detection of polyclonal antibodies by Elisa after each round, screening of monoclonal antibodies, gradual detection, specific antibody sequence analysis, expression and purification of recombinant antibodies, new coronavirus-antibody neutralization experiments, acquisition of anti-SARS-CoV-2 fully humanized monoclonal antibodies.
Examples: preparation of fully human monoclonal antibodies to novel coronaviruses (SARS-CoV-2)
Construction of S2 phage library
1.1 Acquisition of peripheral blood mononuclear cells
Fresh blood 3.5mL was drawn from volunteers infected with the novel coronavirus and placed in an anticoagulation tube. Diluting fresh anticoagulated whole blood with PBS in equal volume, slowly adding into lymphocyte separation liquid (domestic Solarbio, cat. No. P8610), centrifuging, taking out carefully, sucking the second cloud-like mononuclear cell layer, washing in a centrifuge tube containing PBS, centrifuging, discarding supernatant, washing with PBS for 1 time, and collecting cells as peripheral blood mononuclear cells (periphery blood mononuclear cell, PBMC).
1.2 Extracting monocyte total RNA and constructing phage library
Total RNA was extracted from the cells, synthesized by reverse transcription, and the human antibody heavy (VH) and light (V and V) variable region genes were amplified by direct PCR and semi-nested PCR. The VH genes and VL genes were ligated into human single chain antibody (scFv) genes using a modified overlap extension PCR method, and scFv library genes were cloned into phage vectors.
2. Screening monoclonal antibodies using S2 phage library display technology
S2 protein is used as a stationary phase, phage display library is used as a mobile phase, and in vitro generation is utilizedScreening was performed by panning. The specific operation process is as follows: 1mL of 50 mu g S antigen protein group (Ag group), 500 mu L of 10 mu g antigen control group (NC group), incubation at 4℃for 12h, then washing three times with 0.05% PBST, adding 5% milk to the tube, incubation at 37℃for 2h, washing three times with 0.05% PBST after blocking, diluting phage library with 1% mill-PBST, inputting library phage into the tube, incubation at 32℃for 2h, washing 3 times with 0.05% PBST, and washing 2 times with PBS. The binder was eluted with 1mL glycine-HC and then neutralized with Tris-HCl until pH 7.0. The phage were transferred to the tube, incubated at 32℃for 1 hour, and then transferred to the EP tube by applying 5mL of 5% milk/PBST on the tube, incubating at 37℃for 2 hours, followed by 3 washes with 0.05% PBST. Subsequently, the titer of eluted phage was detected, and E.coli TG1 to OD were cultured 600 The eluate was diluted by 10 μl of the diluted solution was mixed with 180 μl of E.coli TG1, incubated at 37deg.C for 30min, poured onto a 2 XYT-A (Amp 100 μg/mL) plate, and the plate was placed upside down overnight at 37deg.C.
Amplifying the eluted phage: (1) Adding 10 μl of Escherichia coli TG1 into 800 μl of 2YT culture solution, and culturing at 37deg.C to OD 600 =0.4-0.6. TG1 cultured to logarithmic phase was transferred to 10mL of 2YT-G (final concentration of 2% glucose) medium, and shake cultured at 37℃to OD 600 The eluate was added, incubated at 37℃for 30min, shaken at 37℃for 30min, 30mL of 2YT-AG broth (final concentration 0.1% Amp,2% glucose) was added, shake flask incubated at 37℃for 1h, M13KO7 (M13 KO7: tg1=20:1) was added, incubated at 37℃for 30min, shake-up at 37℃for 30min, bacterial solution centrifuged at 5000rpm for 10 min, resuspended in 40mL of 2YT-AK, incubated on a shaker at 30℃overnight, centrifuged at 8000rpm for 10 min, supernatant discarded, resuspended in 1mL of PBS, centrifuged at 12000rpm for 5 min, and the supernatant was transferred to a new 1.5mL centrifuge tube. Detection of amplified phage titer: culturing E.coli TG1 to OD 600 =0.4-0.6. The eluate was diluted and 10. Mu.L of the diluted solution was mixed with 180. Mu.L of E.coli TG 1. Incubate at 37℃for 30min, pour onto 2 XYT-A (Amp 100. Mu.g/mL) plates. The dishes were placed upside down at 37℃overnight and the specific biological screening results are shown in Table 1.
TABLE 1 biological screening results
According to the screening results of Table 1, four rounds of biopanning were performed to obtain 1.2X10 s 7 (pfu) phage library of interest.
3. Step-by-step screening of libraries using ELISA techniques
(1) Polyclonal phage ELISA detection: plates were coated with antigen (test group: 4. Mu.g/mL protein S2; control group: 0. Mu.g antigen) overnight at 4℃followed by 2 washes with 0.05% PBST, 1 wash with PBS, 2 hours blocked with 300. Mu.L of 5% milk, 3 washes with 0.05% PBS, 100. Mu.L of diluted amplified phage were added to the wells, incubated for 2 hours at 32℃3 times with 0.05% PBST, anti-M13-hrp antibody (1:400) diluted in blocking buffer, 100. Mu.L of each well, 1 hour at 32℃incubated for 3 times with 0.05% PBST, 100. Mu.L TMB was added to each well, incubated at room temperature, and 100. Mu.L 2M HCl was added to each well. The results of the screening are shown in Table 2 using a microplate reader reading set at 450-630 nm.
TABLE 2 polyclonal phage ELISA detection results
Based on the test results of Table 2, four rounds of polyclonal phage selection were performed, confirming that a second round of phage was used for monoclonal selection.
(2) Monoclonal phage ELISA detection:
1) According to the experimental results, R2 phage were selected for monoclonal screening, and 192 clones were selected from the plates for detecting elution titers. These phages were cultured with shaking at 250 rpm at 37℃until OD 600 Incubation of M13KO7 infected cultures (moi=20:1) for 30min at 37 ℃, shaking for 30min at 37 ℃ =0.4-0.6. 2 XYT-AK (Amp 100. Mu.g/mL, kan 100. Mu.g/mL) was precipitated in equal volume, incubated overnight at 30℃and centrifuged, and the cultures and supernatants were used for ELISA, and plates (test group: 4. Mu.g/mL protein S2; control group: 0. Mu.g antigen) were coated with antigen overnight and wells were washed 3 times at 4 ℃.
The tubes were washed three times with 300. Mu.L of 5% mill for 2 hours at 37℃and 100. Mu.L of phage supernatant was added to the wells and incubated for 2 hours at 32℃and the tubes were washed three times. Anti-m 13-hrp antibody (1:400) was diluted in blocking buffer, 100. Mu.L per well incubated for 1 hour at 32℃and the tubes were washed three times, TMB was added to 100. Mu.L per well incubated at room temperature, and 100. Mu.L of 2MHCl per well. Plates were read using a microplate reader set at 450-620nm and then highly specific clones were sequenced.
2) Positive clone validation ELISA: mu.L of positive clone was added to 2mL of 2YT-AG medium and cultured to OD 600 =0.4-0.6. M13KO7 infected cultures (MOI=20:1) were incubated for 30min at 37℃and homogenized for 30min at 37 ℃.2 XYT-AK (Amp 100. Mu.g/mL, kan 100. Mu.g/mL) was precipitated in an isovolumetric suspension. Incubated overnight at 30℃and centrifuged, the cultures and supernatants were used for ELISA and plates were coated with antigen (test group: 4. Mu.g/mL protein S2; control group: 0. Mu.g antigen) overnight, wells were washed 3 times at 4 ℃.
The wells were blocked with 300. Mu.L of 5% mill for 2 hours at 37℃and the tubes were washed three times, 100. Mu.L of phage supernatant was added to the wells, incubated for 2 hours at 32℃and the tubes were washed three times, anti-M13-hrp antibody (1:400) was diluted in blocking buffer, 100. Mu.L of each well was added, incubated for 1 hour at 32℃and the tubes were washed three times, 100. Mu.L of room temperature was added to each well of TMB and 100. Mu.L of 2M HCl was added to each well. Plates were read using a microplate reader set at 450-620nm, then high specificity clones were sequenced, positive clone ELISA (IgG) expression was verified, antigen (test panel: 4. Mu.g/mL protein S2) was plated overnight, wells were washed 3 times at 4 ℃. Tubes were washed three times with 300. Mu.L of 5% milk at 37℃for 2 hours, 100. Mu.L of diluted IgG was added, incubated at 32℃for 2 hours, tubes were washed three times, goat anti-human IgG HRP (1:700) was diluted in blocking buffer, 100. Mu.L of each well was added, and incubated at 32℃for 1 hour, and tubes were washed three times. TMB was added at 100. Mu.L per well and incubated at room temperature, 100. Mu.L of 2M HCl was added per well, plates were read using a microplate reader set at 450-620nm, and the results of the assays are shown in Table 3.
TABLE 3 ELISA detection results of monoclonal phages
According to the test results of Table 3, 8 phages having a strong binding ability to S2 were obtained, and clones R2P1-E1, R2P1-H1, R2P1-F2, R2P1-D8, R2P2-G5, R2P2-D7, R2P2-D10 and R2P2-F8 in the tables were sent for sequencing. By analysis of the sequencing results we finally obtained five unique and correct sequences (R2P 1-D8, R2P2-G5, R2P2-D7, R2P2-D10 and R2P 2-F8). Expression and purification of five antibodies were subsequently transfected with XtenCHO cells by an Xten transfection protocol and medium and cells were collected until the viability was reduced below 50% (14 days post transfection), wherein the five antibody protein expression and purification profiles are shown in fig. 2-3.
4. New coronavirus antibody neutralization assay
The day before the experiment, HEK293T-ACE2 cells (Cat. GM-C09233) to be infected were inoculated into 96-well cell culture plates at an inoculum size of about 1X 10 4 The cell density is preferably about 30% when virus infection is performed on the next day per cell/well. Five G5 and F8 antibodies with better binding capacity are selected for pseudo-virus neutralization experiments, and are serially diluted according to the concentration of antibody mother liquor and 3 times gradient. The initial concentration of both antibodies was 3mg/mL, the diluted concentration of the first well S1 was 60. Mu.g/mL, and the final concentration of the neutralization incubation was 30. Mu.g/mL. 0.37. Mu.L/well pseudovirus: 597.78. Mu.L of complete medium was added to 2.2. Mu.L of virus stock, mixed well and dispensed into 10 EP tubes (5. Mu.L reserved) at 55. Mu.L. Antibodies G5 and F8 were mixed in equal volumes with the above pseudovirus solutions in serial dilutions, and incubated at room temperature for 1 hour. The 96-well plate with HEK293T-ACE2 cells laid in advance is taken out from the incubator, and whether the cells are polluted, density and state are observed under a lens, and the upper layer culture medium is sucked by a row gun (note that the gun head does not touch the cells at the bottom of the 96-well plate, and the 96-well plate can be sucked after being inclined). 100. Mu.L of the diluted pseudovirus antibody mixture was pipetted into a 96-well plate with HEK293T-ACE2 cells spread (96-well plate antibody layout is shown in Table 4), taking care to avoid washing up cells along the well wall. The fresh complete medium was changed for 6 hours of treatment and cultivation continued for 48 hours. 48 hours after liquid change of the infection pseudovirus by fluorescenceThe luciferase assay kit (Promega/E1910) assayed the luciferase activity on a microplate reader to determine the antibody neutralization efficiency, and the results are shown in Table 5.
Table 4 96 well plate antibody layout
TABLE 5 antibody neutralization efficiency
As can be seen from the detection results in Table 5, the OD value of the sample has a certain upward trend from high concentration to low concentration, which indicates that the antibody has a certain neutralization protection effect. The antibody can be used as a fully human monoclonal antibody of high affinity anti-novel coronavirus SARS-CoV-2, can be used for preparing medicaments for detecting, treating or preventing novel coronavirus SARS-CoV-2 infection, and can provide great guarantee for the standardized production of the antibody.
Finally, it should be emphasized that the foregoing description is merely illustrative of the preferred embodiments of the invention, and that various changes and modifications can be made by those skilled in the art without departing from the spirit and principles of the invention, and any such modifications, equivalents, improvements, etc. are intended to be included within the scope of the invention.

Claims (6)

1. A fully human monoclonal antibody against a novel coronavirus, wherein the fully human monoclonal antibody has a heavy chain variable region and a light chain variable region;
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 19; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 20.
2. A nucleic acid molecule encoding the fully human monoclonal antibody of claim 1, having the following heavy chain variable region and light chain variable region:
the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 9; the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 10.
3. An expression vector comprising the nucleic acid molecule of claim 2.
4. A host cell comprising the expression vector of claim 3.
5. Use of the fully human monoclonal antibody of claim 1 in the preparation of a reagent or kit for detecting a novel coronavirus infection, and in the preparation of a medicament for treating or preventing a novel coronavirus infection.
6. A composition comprising a therapeutically effective amount of the fully human monoclonal antibody of claim 1, and a pharmaceutically acceptable carrier.
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