CN112010965A

CN112010965A - Monoclonal antibody aiming at new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof

Info

Publication number: CN112010965A
Application number: CN202010926533.8A
Authority: CN
Inventors: 董金华; 李海梅; 陈丽梅
Original assignee: Weifang Medical University
Current assignee: Weifang Medical University
Priority date: 2020-05-15
Filing date: 2020-09-04
Publication date: 2020-12-01
Anticipated expiration: 2040-09-04
Also published as: CN111995675B; CN111961133A; CN112010965B; CN111995678A; CN112010966B; CN111995676A; CN111995677A; CN111961133B; CN111995675A; CN112010966A; CN111995677B; CN111995678B; CN111995676B

Abstract

The invention discloses a monoclonal antibody aiming at new coronavirus SARS-CoV-2 and its application, the monoclonal antibody is specifically combined with new coronavirus SARS-CoV-2 spinous process protein RBD region; has wide application prospect in preparing SARS-CoV-2 detection products, preparing SARS-CoV-2 antibody inhibiting medicaments and preparing antibody medicament preparations for preventing or treating pneumonia caused by SARS-CoV-2; the monoclonal antibody aiming at the novel coronavirus SARS-CoV-2 has high titer and strong specificity, can be efficiently expressed, can be specifically combined with the spinous process protein RBD region on the surface of the novel coronavirus SARS-CoV-2, can be used for detecting the novel coronavirus, can neutralize and weaken certain toxicity of the novel coronavirus, and plays a role in preventing or/and treating the pneumonia of the novel coronavirus.

Description

Monoclonal antibody aiming at new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof

Technical Field

The invention relates to the technical field of cellular immunology and molecular biology, in particular to a monoclonal antibody aiming at a new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof.

Background

SARS-CoV-2, a genus of beta Coronavirus (Coronavir) belonging to the Coronaviridae (Coronaviridae), orthocoronaviridae (Orthocoronavirinae), the relatives of severe acute respiratory syndrome-associated Coronavirus (SARS-CoV) and the middle east respiratory syndrome-associated Coronavirus (MERS-CoV), all cause severe pneumonia symptoms. The virus is transmitted by means of droplets, contact and the like, and latent patients have transmissibility. And researches find that the patients with the new coronary pneumonia have extremely strong infectivity at the early stage of the disease process and when the symptoms are slight.

The SARS-CoV-2 virus has a diameter of 75-160 nm, its genome is continuous linear single-stranded RNA, and successively encodes nucleoprotein (nucleoprotein), envelope protein (envelope protein), membrane protein (membrane protein) and spinous process protein (spike protein, also called S-protein or S protein), in which the spinous process protein is the most important protein on its surface, and its main function is to determine host range and specificity of virus, and can be combined and fused with host cell membrane receptor to implement infection of cell. The spinous process protein includes two subunits of S1 and S2, a Receptor Binding Domain (RBD) in S1 interacts with a human SARS-CoV receptor angiotensin converting enzyme II (ACE2) molecule, and S2 contains essential elements required for a membrane fusion process to realize fusion of virus and cells. Therefore, the human monoclonal antibody of the S protein can theoretically block the combination of virus and cells, has the capability of weakening virus infection, and can be used as an antibody medicament for treating patients with new coronavirus pneumonia.

Antibodies are important glycoprotein molecules in the mammalian immune system. The antibody molecule consists of two Heavy chains (Heavy chain) and two Light chains (Light chain), wherein the Heavy chains are divided into Variable regions (VH) and three Constant regions (Constant regions of Heavy chain; CH1, CH2, CH3), and the Light chains consist of one Variable region (VL) and one Constant region (CL). The variable region has a function of specifically binding to an antigen, and is different from individual antibody, while the constant region of an antibody is determined by the species and subtype of the antibody. The heavy chain variable region of an antibody comprises three Complementarity determining regions (CDRH 1, CDRH2, CDRH3) and the light chain variable region also comprises three Complementarity determining regions (CDRL 1, CDRL2, and CDRL 3), which are also known as hypervariable regions and directly bind to an epitope.

Various treatment schemes are provided for treating the new coronary pneumonia in various countries, but no specific treatment medicine or vaccine for the new coronary pneumonia is available on the market at present, the medicine for resisting the new coronary pneumonia mainly comprises small-molecular medicines of Reiciclovir, chloroquine and hydroxychloroquine, the combination of lopinavir and ritonavir and the combination of lopinavir, ritonavir and interferon have no obvious treatment effect, and some medicines even have serious toxic and side effects. The antibody medicine plays an important role in the treatment of infectious diseases, autoimmune diseases, tumors and the like, and has very important significance in developing monoclonal antibodies aiming at the novel coronavirus SARS-CoV-2 under the conditions that the SARS-CoV-2 vaccine is difficult to develop and has long period, and the side effect of the traditional medicine is great or even has no effect.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a monoclonal antibody aiming at the RBD region of the spike protein of the novel coronavirus SARS-CoV-2 and application thereof.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a monoclonal antibody (A7) against the SARS-CoV-2 of new coronavirus, which binds specifically to RBD region of spinous process protein of SARS-CoV-2 of new coronavirus; comprises complementarity determining regions CDRH1, CDRH2, CDRH3 of the heavy chain variable region and complementarity determining regions CDRL1, CDRL2, CDRL3 of the light chain variable region; the amino acid sequences of the complementarity determining regions CDRH1, CDRH2, CDRH3 of the heavy chain variable region are respectively shown in SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification; the amino acid sequences of the complementarity determining regions CDRL1, CDRL2, CDRL3 of the light chain variable region are respectively shown in SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: shown in fig. 8.

Preferably, the monoclonal antibody against the novel coronavirus SARS-CoV-2 (A7), wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO: 1, the amino acid sequence of the light chain variable region of the monoclonal antibody is SEQ ID NO: 5.

the invention also includes an isolated nucleic acid molecule encoding the monoclonal antibody (A7) of any one of the above.

The invention also includes an expression vector comprising a nucleic acid molecule as described above, which expression vector comprises, in addition to the nucleic acid molecule as described above, an expression control sequence operably linked to the sequence of said nucleic acid molecule.

An expression vector refers to a nucleic acid vehicle into which a polynucleotide encoding the a7 antibody can be inserted and the a7 antibody expressed. The vector may be transformed, transduced or transfected into a host cell so that the genetic material elements it carries are expressed within the host cell. Types of vectors include bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. In principle, any vector may be used as long as it is replicable and stable in the host. In addition to the origin of replication, the expression vector may contain a marker gene and other translational regulatory elements.

The invention also includes a host cell comprising the nucleic acid molecule or the expression vector described above.

The host cell expressing the a7 antibody can 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: escherichia coli, Streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, 293 cells or Bowes melanoma cells.

The invention also includes a method for detecting the level of the novel coronavirus SARS-CoV-2 for non-diagnostic purposes, comprising the steps of:

extracting a sample containing novel coronavirus SARS-CoV-2;

contacting the sample obtained in the step I with any one of the monoclonal antibodies;

and detecting the immune reaction between the sample and the antibody.

The invention also includes the application of any monoclonal antibody aiming at the novel coronavirus SARS-CoV-2 in the preparation of the novel coronavirus SARS-CoV-2 detection product.

The detection product includes, but is not limited to, a detection reagent, a kit, a chip or a test paper. Any assay product capable of detecting SARS-CoV-2 comprising a binding molecule as described above is included within the scope of the invention.

The invention also includes the application of any monoclonal antibody aiming at the novel coronavirus SARS-CoV-2 in preparing the medicine for inhibiting the novel coronavirus SARS-CoV-2 antibody.

The invention also includes the application of any monoclonal antibody aiming at the new coronavirus SARS-CoV-2 in preparing an antibody pharmaceutical preparation for preventing or treating pneumonia caused by the new coronavirus SARS-CoV-2.

The terms "new coronavirus SARS-CoV-2" and "SARS-CoV-2 virus", "new coronavirus" and "SARS-CoV-2" used in the present invention can be used interchangeably.

The sequence specific information related to the invention is as follows:

SEQ ID NO：1：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIASSGYYTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDTDTFDYWGQGTLVTVSS；

SEQ ID NO：2：

GFTFSSYA；

SEQ ID NO：3：

IASSGYYT；

SEQ ID NO：4：

AKDTDTFDY；

SEQ ID NO：5：

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSSPSTFGQGTKVEIKR；

SEQ ID NO：6：

QSISSY；

SEQ ID NO：7：

AAS；

SEQ ID NO：8：

QQANSSPST；

compared with the prior art, the invention has the following advantages:

the monoclonal antibody aiming at the novel coronavirus SARS-CoV-2 has high titer and strong specificity, can be efficiently expressed, can be specifically combined with the spinous process protein RBD region on the surface of the novel coronavirus SARS-CoV-2, can be used for detecting the novel coronavirus SARS-CoV-2, can neutralize and weaken certain toxicity of the novel coronavirus, and plays a role in preventing or/and treating the novel coronavirus pneumonia.

The phage display technology inserts exogenous DNA into the gene of phage coding coat protein, so that the expression product corresponding to the exogenous DNA fragment is fused in the coat protein of the phage to form fusion protein, and the fusion protein is displayed on the surface of the phage. Has the following remarkable advantages: direct physical connection between the genotype and the phenotype is established, so that the screening is simple, convenient and efficient. The invention screens an antibody which can be combined with the S protein of the new coronavirus SARS-CoV-2 from a synthetic antibody library Tomlinson I + J phage display antibody library, and the antibody has important application value in the aspects of detecting the new coronavirus and weakening virus toxicity.

Drawings

FIG. 1 is a schematic diagram showing the results of the enzyme-linked immunosorbent assay of polyclonal antibodies obtained in each step of panning;

FIG. 2 is a schematic diagram showing the results of an enzyme-linked immunosorbent assay for monoclonal antibodies;

FIG. 3 is a polyacrylamide gel electrophoresis analysis diagram of the A7-Fab antibody fragment;

FIG. 4 is a schematic diagram of the principle of A7-Fab antigen-specific enzyme-linked immunosorbent assay;

FIG. 5 is a schematic diagram showing the result of antigen-specific detection of A7-Fab;

FIG. 6 is a schematic diagram of the principle of enzyme-linked immunosorbent assay for inhibiting ACE2 and RBD binding by A7-Fab;

FIG. 7 shows the results of enzyme-linked immunosorbent assay in which A7-Fab blocks the binding of ACE2 to RBD.

Detailed Description

The present invention aims at providing monoclonal antibody against new coronavirus SARS-CoV-2 spike protein RBD region and its application, and the present invention is further illustrated by the following examples. The examples of the invention are intended to be illustrative and not limiting, and simple modifications thereof in accordance with the principles of the invention are intended to be within the scope of the claims.

Example 1

Amplification of phage display antibody libraries

After thawing frozen 0.5mL of TG-1 E.coli frozen stock containing Tomlinson I + J phage display antibody library phagemid (MRC HGMP resource center, UK), 25mL of 2YT (1.6% Tryptone, 1% Yeast Extract, 0.5% NaCl) medium was added and cultured to OD₆₀₀At 0.4, add 10⁹cfu auxiliary phage KM13, after 1 hour of 37 ℃ infection, 3000g centrifugation for 30 minutes, abandoning the supernatant, using 2YT culture medium 50mL suspension thallus containing 100 ug/mL ampicillin, 50 ug/mL kanamycin and 0.1% glucose, shaking the thallus at 30 ℃ and 250rpm for 16 hours, 5000g and 30 minutes next day to centrifuge the culture solution, separating and recovering 40mL supernatant, adding 10mL PEG/NaCl solution into the supernatant, mixing uniformly, placing on ice for 30 minutes, 5000g and 30 minutes centrifugation, abandoning the supernatant, adding 2mL sterilized PBS solution to dissolve the precipitate, using as phage display antibody library solution, titrating the phage display antibody library by using Escherichia coli, and preparing the antibody library with the titer of 10¹²cfu/mL。

Second, panning of phage display antibody library

At 9Adding 100 μ L of PBS containing SARS-CoV-2 virus S1 protein 10 μ g/mL into 10 wells of 6-well microplate, incubating overnight at 4 deg.C, discarding antigen solution the next time, adding 200 μ L of PBS containing 2% skimmed milk powder into each well, incubating at 25 deg.C for 2 hr for sealing, washing with PBST for 3 times, adding 100 μ L of phage solution (R0; containing 10 μ L of phage solution into each well)⁹cfu phage) were incubated at room temperature for 2 hours, and after washing with PBST, phage bound to viral S1 protein were eluted by adding 100 μ L of trypsin per well.

Culturing TG-1 Escherichia coli to OD600 of 0.4, collecting 4mL bacterial solution, adding 500 μ L dissolved phage solution into bacterial solution, infecting at 37 deg.C for 30min, centrifuging at 5000g for 20min, discarding supernatant, suspending thallus with 2YT culture medium containing 100 μ g/mL ampicillin, 50 μ g/mL kanamycin and 0.1% glucose, shaking at 30 deg.C and 250rpm for 16 hr; centrifuging the culture solution for 5000g and 30min the next day, separating and recovering the supernatant, adding 1/5 volumes of PEG/NaCl solution into the supernatant solution, uniformly mixing, placing on ice for 30min, centrifuging for 5000g and 30min, discarding the supernatant, and adding 200 μ L of sterilized PBS solution as phage solution (R1) after the first enrichment; repeating the steps to respectively obtain phage solutions R2 and R3; and performing enzyme-linked immunosorbent assay, and verifying the binding specificity and binding performance of the phage display antibody library obtained in the panning process and the new coronavirus S1 protein.

The enzyme-linked immunosorbent assay was performed as follows: 100. mu.L of a neocoronavirus S1 protein solution (2. mu.g/mL) or a PBS solution of bovine serum albumin BSA (2. mu.g/mL) was added to a 96-well plate, overnight at 4 ℃, the antigen solution was discarded the next day, 200. mu.L of a solution containing 2% skim milk powder was added thereto, and the plate was incubated at 25 ℃ for 2 hours and then blocked. The microplate was washed 3 times with PBS containing 0.1% Tween 20, and diluted solutions of R0 and R2 phage (10) were added⁹cfu/well), incubation at 25 ℃ for 1 hour, washing the microplate with PBST solution, addition of HRP-labeled mouse anti-M13 antibody, incubation for 1 hour, washing the plate with PBST, addition of HRP substrate TMBZ (prepared with sodium acetate solution pH6.0, containing 1/10000 diluted 30% H₂O₂) Measuring absorbance at 450nm with microplate reader after color development, drawing bar chart, and comparing each step to obtainBinding of phage antibodies to S1 protein and BSA.

The results of the enzyme-linked immunosorbent assay are shown in fig. 1, when the binding capacities of the phage libraries R0, R2 and R3 obtained in the phage panning process to BSA and S protein are compared, it is found that the binding capacity of the phage solution R2 obtained in the second panning process to S protein is significantly increased compared with R0, while the binding capacity to BSA is very weak and is not significantly changed compared with R0, which indicates that the constructed phage display antibody library is enriched in antibodies against the new coronavirus S protein.

Screening of monoclonal antibodies

Culturing TG-1 E.coli to OD₆₀₀Taking 100 mu L of elutriation sieve R2 phage antibody library dissolved out phage solution for infecting 200 mu L of escherichia coli bacterial solution, incubating for 30 minutes at 37 ℃, coating the bacterial solution on a 2YT culture medium plate containing 100 mu g/mL ampicillin, 50 mu g/mL kanamycin and 1% glucose, and culturing overnight at 37 ℃; the next day, 96 colonies were picked and inoculated onto 96-well plates, and cultured at 37 ℃ to OD₆₀₀To 0.4, M13 phage was added to each well, centrifuged at 5000g for 20 minutes after infection, the supernatant was removed, 200. mu.L of 2YT medium containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin and 0.1% glucose was added to each well, the cells were suspended, and cultured at 30 ℃/250rpm for 16 hours; centrifuging the culture solution at a speed of 5000g/30 min the next day, separating and recovering the supernatant, performing enzyme-linked immunosorbent assay, and detecting the binding specificity and binding performance of each monoclonal antibody and S protein.

The enzyme-linked immunosorbent assay was performed as follows: 100 μ L of PBS solution containing virus S protein (1 μ g/mL) was added to a 96-well plate, overnight at 4 ℃, the antigen solution was discarded the next day, 200 μ L of a solution containing 2% skim milk powder was added, incubation was performed at 25 ℃ for 2 hours, and the plate was blocked. Washing the ELISA plate with PBS solution containing 0.1% Tween 20 for 3 times, adding phage solution, incubating at 25 deg.C for 1 hr, washing the ELISA plate with PBST solution, adding HRP-labeled mouse anti-M13 antibody, incubating for 1 hr, washing the plate with PBST, adding HRP substrate TMBZ (prepared with sodium acetate solution of pH6.0, containing 1/10000 diluted 30% H)₂O₂) After developing, the absorbance at 450nm and 630nm is measured by a microplate readerAnd drawing a histogram to compare the binding performance of the phage antibody prepared by each clone with the S protein and the bovine serum albumin.

Experiments show that 7 micropores are dark in color, the absorbances of the micropores are 1.40, 1.30, 1.35, 1.05, 1.10, 0.80 and 1.68 respectively, plasmids of antibodies in the micropores with the absorbances of 1.68 are extracted after thalli of the corresponding micropores are subjected to amplification culture, the antibodies are named as monoclonal antibodies A7 according to the positions (A row and 7 column) of the micropores in the experiments, and the antibodies are compared with amino acid sequences of the antibodies registered in an antibody gene library, so that the same sequences as the antibody genes are not found, and the antibodies are novel antibodies. The details of the amino acid sequence of the antibody are as follows.

The heavy chain variable region sequence of the A7 antibody is SEQ ID NO: 1, CDRH1 sequence of SEQ ID NO: 2; the CDRH2 sequence is SEQ ID NO: 3; the CDRH3 sequence is SEQ ID NO: 4;

the variable region sequence of the light chain of the A7 antibody is SEQ ID NO: 5, CDRL1 sequence is SEQ ID NO: 6; the CDRL2 sequence is SEQ ID NO: 7; the CDRL3 sequence is SEQ ID NO: 8;

A7-VH (antibody heavy chain variable region):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIASSGYYTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDTDTFDYWGQGTLVTVSS(SEQ ID NO：1)；

CDRH1：GFTFSSYA(SEQ ID NO：2)；

CDRH2：IASSGYYT(SEQ ID NO：3)；

CDRH3：AKDTDTFDY(SEQ ID NO：4)；

A7-VL (antibody light chain variable region):

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSSPSTFGQGTKVEIKR(SEQ ID NO：5)；

CDRL1：QSISSY(SEQ ID NO：6)；

CDRL2：AAS(SEQ ID NO：7)；

CDRL3：QQANSSPST(SEQ ID NO：8)；

fourth, antigen specificity of monoclonal antibody

Adding 100 mu L of enzyme label plate into 96-hole enzyme label plateThe new coronavirus S protein, S-RBD protein and BSA protein solution with the concentration of 1 mu g/mL respectively are used for staying overnight at 4 ℃, the protein solution is discarded the next day, 200 mu L of 2% skimmed milk powder solution is added, incubation is carried out for 2 hours at 25 ℃, and the ELISA plate is sealed; after washing the microplate 3 times with PBS containing 0.1

% Tween

20, 100. mu.L of diluted phage display antibody solution was added to each well, incubated at 25 ℃ for 1 hour, the microplate was washed with PBST solution, HRP-labeled mouse anti-M13 antibody was added, and after incubation for 1 hour, the microplate was washed with PBST, and HRP substrate TMBZ (prepared with sodium acetate solution pH6.0, containing 1/10000 diluted 30% H) was added₂O₂) And after color development, measuring the absorbance at 450nm by using an enzyme-labeling instrument, drawing a histogram, and comparing the binding performance of the phage antibody and the envelope protein.

The results of the ELISA are shown in FIG. 2, and the A7 antibody binds to the S protein, also binds to the S-RBD protein, and does not bind to the coated BSA, indicating that the A7 antibody is an antibody recognizing the RBD region of the S protein, and has specificity for binding to the RBD of the S protein of the novel coronavirus SARS-CoV-2.

Fifthly, expression and purification of A7-Fab fragment

Performing Polymerase Chain Reaction (PCR) by using the A7 antibody plasmid obtained by panning as a template, wherein the Reaction system is 50 μ L, and the Reaction conditions are as follows: after denaturation at 94 ℃ for 2 minutes, at 94 ℃ for 30 seconds, at 55 ℃ for 30 seconds, at 68 ℃ for 1 minute, after reaction for 30 cycles, the PCR product was examined with 1% agarose gel and the desired gene fragment was recovered, after purification of the PCR product, PCR was treated with restriction enzyme EcoRv/HindIII, after purification it was linked with pUQ2 treated with the same enzyme with Ligation High v.2 (Toyobo Co., Ltd.), E.coli XL0-Gold was transformed, plated and cultured overnight, on the next day a single clone was picked, PCR was cloned, a clone containing the gene fragment was picked, the colony was cultured and the plasmid was extracted, and sequencing was carried out to confirm the success of cloning.

The heavy chain variable region gene of the A7 antibody was PCR-amplified, treated with AgeI/XhoI and cloned into pUQ2 following the same procedure as for cloning the light chain, to construct a Fab expression vector.

Transforming and expressing a host strain SHuffle T7 express by using a successfully constructed recombinant plasmid, culturing for 14h at 37 ℃ on an LB plate culture medium, and selecting a single bacteriumInoculating to 4mL LB liquid medium containing final concentration of 100. mu.g/mL ampicillin, culturing overnight at 37 deg.C at 200rpm, transferring the overnight cultured bacterial liquid to 100mL LB medium containing final concentration of 100. mu.g/mL ampicillin the next day, culturing at 30 deg.C and 200rpm for 3h, measuring absorbance value when OD is OD₆₀₀Stopping culturing when the concentration is between 0.4 and 0.5, adding isopropyl thiogalactoside (IPTG) into the bacterial liquid to make the final concentration be 0.4mmol/L, and continuously culturing at 16 ℃ and 200rpm for 18 hours;

centrifuging the cultured bacterial solution at 6000g for 20min, collecting thallus, adding Na 8mmol/L₂HPO₄·12H₂O、47.9mmol/L NaH₂PO₄·2H₂Performing ultrasonic treatment with power of 120W, working time of 3s and interval time of 2s for 20min in O, 300mmol/L NaCl and TALON buffer solution with pH of 7.0 under ice water, standing for 30min, centrifuging at 4 ℃ for 20min at 6000 Xg, and collecting supernatant;

after 100. mu.L of TALON Metal Affinity Resin (TAKARA Bio) was combined with 9mL of the supernatant at 4 ℃ for 30 minutes using a shaker at 40rpm, the supernatant mixture was applied to a gravity purification column using 8mmol/LNa₂HPO₄·12H₂O、47.9mmol/L NaH₂PO4·2H₂Washing the purification column with O, 300mmol/L NaCl, 5mmol/L imidazole and TALON Washing Buffer (pH7.0), and then Washing with 8mmol/L Na₂HPO₄·12H₂O、47.9mmol/L NaH₂PO₄·2H₂O, 300mmol/L NaCl, 500mmol/L imidazole and Elution Buffer with pH7.0, samples of the specifically bound A7-Fab protein were collected in order of Elution, and polyacrylamide gel electrophoresis (SDS-PAGE) was performed on the purified protein to detect the content of the purified protein.

The experimental results are as follows: the electrophoresis results are shown in FIG. 3, wherein lane M is protein Marker (Beijing Solebao Biotechnology Co., Ltd.), and lane A7-Fab is the collected Fab fragment of the antibody. Two protein bands appear near 28kDa as indicated by the arrows, the antibody variable and first constant regions in the Fab fragment, the light chain of the antibody. The results show that the A7-Fab antibody fragment was well purified.

Sixthly, detecting the antigen specificity of the A7-Fab antibody fragment by enzyme-linked immunosorbent assay

The schematic diagram of the ELISA test method is shown in FIG. 4 (in the figure, BSA is bovine serum albumin; S protein is new coronavirus S protein), purified S1 protein (2 mug/mL) and BSA (2 mug/mL) are coated on the ELISA plate, the ELISA plate is incubated overnight at 4 ℃, the coating solution is poured off the next day, 2% skimmed milk powder dissolved in PBS is added, and the ELISA plate is sealed. After 2 hours of blocking, the plate was washed, a purified A7-Fab antibody solution was added to each well, and after 1 hour of incubation at room temperature, the plate was washed again, an anti-histidine-tag antibody (Nippon Fuji reagent Co., Ltd.) labeled with HRP was added thereto, and after 1 hour of incubation at room temperature, the plate was washed, and then a substrate was added thereto for color development, and absorbance at 450nm was measured.

The results are shown in fig. 5, the a7-Fab antibody reacts with the experimental group (S1 protein) and develops color, the absorbance of the antibody is 1.3, the absorbance of the antibody is 0.05 at 450nm compared with the control group (bovine serum albumin BSA), and a significant difference exists, which proves that the a7-Fab antibody fragment can be specifically bound with the new coronavirus S1 protein.

Seventhly, enzyme-linked immunosorbent assay verifies that A7-Fab antibody fragment blocks the combination of ACE2 and S1 protein

A schematic diagram of an enzyme-linked immunosorbent assay principle of A7-Fab for preventing the combination of ACE2 and RBD is shown in FIG. 6 (in the diagram, ACE2-hFc is a fusion protein of Angiotensin Converting Enzyme (ACE)2 and a human antibody Fc segment, and RBD is a RBD region of a new coronavirus S protein), ACE2-hFc (2 mu g/mL) is coated on an enzyme label plate, the enzyme label plate is incubated overnight at 4 ℃, the coating solution is poured off the next day, 2% of skim milk powder dissolved in PBS is added, and the enzyme label plate is sealed. And (3) washing the plate after 2 hours of sealing, adding RBD (0.5mg/mL) and a purified A7-Fab antibody fragment solution (5mg/mL) into each hole of the experimental group, adding only an RBD protein solution into the control group, washing the ELISA plate again after 1 hour of incubation at room temperature, adding a rabbit anti-RBD antibody (1mg/mL), pouring the antibody solution after 1 hour of incubation at room temperature, adding an HRP-labeled goat anti-rabbit antibody (Shanghai Biolabe) after plate washing, carrying out floating incubation at room temperature for 1 hour, adding a substrate after plate washing, developing, and measuring the absorbance at 450 nm.

The experimental result is shown in fig. 7, in the control group without the a7-Fab antibody fragment, the absorbance of the microplate microporous sample at 450nm is 1.05, while the absorbance of the experimental group with the a7-Fab antibody fragment added thereto is 0.54, which is a significant difference between the absorbance and the absorbance, indicating that after the a7-Fab is added, the binding capacity of the RBD and ACE2 is reduced by 49%, which proves that the a7 antibody can effectively block the binding of the RBD and ACE2, and the virus starts to infect cells after the new coronavirus is bound with ACE2 on the cell surface through the RBD region of the S1 protein, i.e. the a7 antibody has the ability of neutralizing the toxicity of the new coronavirus.

Sequence listing

<110> Weifang medical college

<120> monoclonal antibody aiming at new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof

<130> 20200904A-7

<141> 2020-09-04

<150> 2020104148681

<151> 2020-05-15

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<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Gln Gln Ala Asn Ser Ser Pro Ser Thr

1 5

Claims

1. A monoclonal antibody against the novel coronavirus SARS-CoV-2, characterized in that: the monoclonal antibody is specifically combined with a new coronavirus SARS-CoV-2 spinous process protein RBD region; comprises complementarity determining regions CDRH1, CDRH2, CDRH3 of the heavy chain variable region and complementarity determining regions CDRL1, CDRL2, CDRL3 of the light chain variable region; the amino acid sequences of the complementarity determining regions CDRH1, CDRH2, CDRH3 of the heavy chain variable region are respectively shown in SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification; the amino acid sequences of the complementarity determining regions CDRL1, CDRL2, CDRL3 of the light chain variable region are respectively shown in SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: shown in fig. 8.

2. The monoclonal antibody against the novel coronavirus SARS-CoV-2 according to claim 1, wherein: the amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO: 1; the amino acid sequence of the variable region of the light chain of the monoclonal antibody is SEQ ID NO: 5.

3. an isolated nucleic acid molecule, wherein: the nucleic acid molecule encodes the monoclonal antibody of any one of claims 1-2.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. A method for detecting the level of a novel coronavirus SARS-CoV-2 for non-diagnostic purposes, characterized in that: the method comprises the following steps:

extracting a sample containing novel coronavirus SARS-CoV-2;

contacting the sample obtained in the step I with the monoclonal antibody of any one of claims 1-2;

and detecting the immune reaction between the sample and the antibody.

7. The use of the monoclonal antibody against the novel coronavirus SARS-CoV-2 as claimed in any one of claims 1 to 2 in the preparation of a novel coronavirus SARS-CoV-2 detection product.

8. The use of the monoclonal antibody against the novel coronavirus SARS-CoV-2 as claimed in any one of claims 1-2 in the preparation of a medicament for inhibiting the novel coronavirus SARS-CoV-2 antibody.

9. Use of the monoclonal antibody against the novel coronavirus SARS-CoV-2 according to any one of claims 1 to 2 for the preparation of a pharmaceutical preparation for preventing or treating pneumonia caused by the novel coronavirus SARS-CoV-2.