CN111995678A

CN111995678A - Monoclonal antibody aiming at new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof

Info

Publication number: CN111995678A
Application number: CN202010944547.2A
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: 2020-09-10
Publication date: 2020-11-27
Anticipated expiration: 2040-09-10
Also published as: CN112010966B; CN112010965B; CN111995677B; CN111995676B; CN112010965A; CN111995677A; CN111995678B; CN111995675B; CN112010966A; CN111995675A; CN111995676A; CN111961133B; CN111961133A

Abstract

The invention discloses a monoclonal antibody aiming at a new coronavirus SARS-CoV-2 spike protein RBD region and application thereof, belonging to the field of cellular immunology and the like. The monoclonal antibody is specifically combined with an RBD region of spinous process glycoprotein of a novel coronavirus SARS-CoV-2, and comprises complementarity determining regions CDRH1, CDRH2 and CDRH3 of a heavy chain variable region and complementarity determining regions CDRL1, CDRL2 and CDRL3 of a light chain variable region; the monoclonal antibody aiming at the spinous process protein RBD region of the SARS-CoV-2 of the new coronavirus 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 SARS-CoV-2 of the new coronavirus, can be used for detecting the SARS-CoV-2 of the new coronavirus, can neutralize and weaken certain toxicity of the new coronavirus, and plays a role in preventing or/and treating pneumonia of the new 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

The new Coronavirus SARS-CoV-2, the beta Coronavirus genus (Coronavir) belonging to the Coronaviridae (Coronaviridae), the orthocoronaviridae (Orthocoronaviridae), the relatives of the 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, Receptor Binding Domain (RBD) in S1, which interacts with human SARS-CoV Receptor angiotensin-converting enzyme II (ACE2) molecule, and S2 contains essential elements required for membrane fusion process to realize fusion of virus and cell. Therefore, the human monoclonal antibody of the S protein can block the combination of virus and cells, has the capability of weakening virus infection, and can be used as an antibody prodrug for treating patients with new coronavirus pneumonia.

Antibodies are important glycoprotein molecules in the mammalian immune system. The molecular structure is composed 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 are composed of a Variable region (VL) and a Constant region (CL). The variable region binds specifically to an antigen and is different from one antibody to another, 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 countries have proposed various treatment schemes for the treatment of new coronary pneumonia, but no therapeutic drugs or vaccines specific for new coronary viruses are currently on the market. 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 specifically 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:

the invention discloses a monoclonal antibody aiming at a new coronavirus SARS-CoV-2 spinous process protein RBD region, which is specifically combined with the new coronavirus SARS-CoV-2 spinous process glycoprotein RBD region and comprises complementarity determining regions CDRH1, CDRH2, CDRH3 of a heavy chain variable region and complementarity determining regions CDRL1, CDRL2 and CDRL3 of a light chain variable region; the amino acid sequences of the complementarity determining regions CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4; the amino acid sequences of the complementarity determining regions CDRL1, CDRL2 and CDRL3 of the light chain variable region are SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: 8.

a preferred monoclonal antibody aiming at the RBD region of the spinous process protein of the novel coronavirus SARS-CoV-2 is named A2, and 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 a monoclonal antibody as described in 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 a2 antibody can be inserted and the a2 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 a nucleic acid molecule as described above or an expression vector as described above.

The host cell expressing the a2 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 new coronavirus SARS-CoV-2;

contacting the sample obtained in the step I with the monoclonal antibody A2;

and thirdly, detecting the immunoreaction of the sample and the monoclonal antibody.

The invention also includes the application of the monoclonal antibody aiming at the new coronavirus SARS-CoV-2 spinous process protein RBD zone in the preparation of new coronavirus SARS-CoV-2 detection products.

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 the monoclonal antibody aiming at the new coronavirus SARS-CoV-2 spinous process protein RBD region in the preparation of the medicine for inhibiting the new coronavirus SARS-CoV-2 antibody.

The invention also includes the application of the monoclonal antibody aiming at the spinous process protein RBD region of the new coronavirus SARS-CoV-2 in the preparation of a 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", "SARS-CoV-2" and "new coronavirus SARS-CoV-2" used in the present invention can be used interchangeably.

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

the monoclonal antibody aiming at the spinous process protein RBD region of the SARS-CoV-2 of the new coronavirus 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 SARS-CoV-2 of the new coronavirus, can be used for detecting the SARS-CoV-2 of the new coronavirus, can neutralize and weaken certain toxicity of the new coronavirus, and plays a role in preventing or/and treating pneumonia of the new coronavirus.

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 the antibody which can be combined with the S protein of the new coronavirus SARS-CoV-2 from the synthetic antibody library Tomlinson I + J phage display antibody library, and the antibodies have important application value in the aspects of detecting the new coronavirus and weakening the virus toxicity.

Drawings

FIG. 1 shows the results of enzyme-linked immunoassay for the antigen binding performance of phages obtained by panning an antibody library;

FIG. 2 shows the results of ELISA assay for the antigen specificity of monoclonal antibody A2;

FIG. 3 shows the results of ELISA assay for the antigen specificity of monoclonal antibody A3;

FIG. 4 is a schematic diagram of the principle of detecting new coronavirus using elutriated antibody;

FIG. 5 shows the results of ELISA using Fab fragment of A2 antibody in combination with A3 to detect virus S protein;

FIG. 6 is a standard graph of an ELISA assay for the detection of viral S protein using Fab fragments of the A2 antibody in combination with A3.

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.

The invention is further described with reference to specific examples.

Example 1

Amplification of a phage display antibody library:

mu.L of E.coli TG-1 (MRC HGMP resources center, UK) containing Tomlinson I + J phage display antibody library was inoculated into 25mL of 2YT medium (1.6% Tryptone, 1% Yeast Extract, 0.5% NaCl) containing 100. mu.g/mL ampicillin and 1% glucose, and cultured at 37 ℃ to OD₆₀₀At 0.4, add 10⁹cfu (Colony Formation Unit) KM13 helper phage (MRC HGMP resources center, UK), 1 hour after infection at 37 ℃, 3000g was centrifuged for 30 minutes, the supernatant was discarded, 50mL (1.6% Tryptone, 1% Yeast Extract, 0.5% NaCl) of 2YT medium containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin and 0.1% glucose was used to suspend the cells, the cells were shaken at 30 ℃ and 250rpm for 16 hours, the culture broth was centrifuged the next day at 5000g and 30 minutes, 40mL of the supernatant was separated and recovered, 10mL of PEG/NaCl solution was added to the supernatant, after mixing well, the mixture was left on ice for 30 minutes, 5000g and 30 minutes were centrifuged, the supernatant was discarded, and 2mL of sterile solution was added to dissolve the precipitate to obtain a phage display antibody library solution. The phage display antibody library was titrated with E.coli to prepare an antibody library with a concentration of 10¹²cfu/mL。

Second, panning of phage display antibody library

Adding 100 μ L of PBS solution containing SARS-CoV-2 virus S protein (Nanjing Kingsley Biotech Co., Ltd.) in 10 wells of 96-well microplate, incubating overnight at 4 deg.C, discarding antigen solution the next time, adding 200 μ L of PBS solution containing 2% skimmed milk powder in 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; each well contains 10 μ L of S protein)⁹cfu phage) were incubated at room temperature for 2 hours, and after washing with PBST, phage bound to viral S protein were eluted by adding 100 μ L trypsin per well.

Culturing TG-1 E.coli to OD₆₀₀To 0.4, 4mL of bacterial solution was taken, 500. mu.L of the dissolved phage solution was added to the bacterial solution, infected at 37 ℃ for 30 minutes, centrifuged at 5000g for 20 minutes, the supernatant was discarded, the cells were suspended in 2YT medium containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin and 0.1% glucose, and shaken at 30 ℃ and 250rpm for 16 hours; centrifuging the culture solution at 5000g for 30 min the next day, separating and recovering the supernatant,adding 1/5 volume PEG/NaCl solution into the supernatant, mixing, standing on ice for 30 min, centrifuging for 30 min at 5000g, discarding supernatant, and adding 200 μ L sterilized PBS solution as the first enriched phage solution (R1); 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 and the S protein obtained in the panning process.

The enzyme-linked immunosorbent assay was performed as follows: 100. mu.L of PBS containing a neocoronavirus S protein solution (2. mu.g/mL) or 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, 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 R0, R2 and R3 phage solutions (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) 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, the S protein and the BSA obtained in each step.

The results of the enzyme-linked immunosorbent assay are shown in fig. 1, and when the binding capacities of the phage libraries R0, R2 and R3 obtained in the phage panning process and the S protein are compared, it is found that the binding capacity of the phage solution R2 obtained in the second panning process and the S protein is significantly increased, and the binding performance to BSA is very weak and unchanged, which indicates that the antibodies against the new coronavirus S protein in the constructed phage display antibody library are enriched.

Thirdly, screening of monoclonal antibody

Culturing TG-1 E.coli to OD₆₀₀0.4, 100. mu.L of panned phage solution dissolved out from the R2 phage antibody library was used to infect 200. mu.L of E.coli bacterial solution, incubated at 37 ℃ for 30 minutes, and then coated on a 2YT medium plate containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin and 1% glucose at 37 ℃ overnightCulturing; 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 ℃ and 250rpm for 16 hours; centrifuging the culture solution at 5000g for 30 min the next day, separating and recovering the supernatant, performing enzyme-linked immunosorbent assay, and verifying 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 the development, absorbance at 450nm and at 630nm was measured with a microplate reader, a histogram was plotted, and the binding properties of the phage antibody prepared by each clone to S protein and bovine serum albumin were compared.

The results showed that 6 wells were dark in color, and had absorbances of 1.40, 1.30, 1.35, 1.05, 1.10 and 0.80, respectively, after the cells in the corresponding wells were expanded and cultured, plasmids were extracted and gene-sequenced for antibodies in wells with absorbances of 1.40, the antibodies were named monoclonal antibody a2 based on their position in the wells of the microplate in the experiment (row a, column 2), plasmids were extracted and gene-sequenced for antibodies in wells with absorbances of 1.30, the antibodies were named monoclonal antibody A3 based on their position in the microplate in the experiment (row a, column 3), and the antibodies were novel antibodies because they did not find sequences identical to the antibody genes described in the present invention by comparison with the antibody sequences registered in the antibody gene library. The details of the amino acid sequence of the antibody are as follows.

The heavy chain variable region sequence of the A2 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 A2 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.

the heavy chain variable region sequence of the A3 antibody is SEQ ID NO: 9, CDRH1 sequence as SEQ ID NO: 10; the CDRH2 sequence is SEQ ID NO: 11; the CDRH3 sequence is SEQ ID NO: 12;

the variable region sequence of the light chain of the A3 antibody is SEQ ID NO: 13, CDRL1 sequence of SEQ ID NO: 14; the CDRL2 sequence is SEQ ID NO: 15; the CDRL3 sequence is SEQ ID NO: 16.

the sequence referred to by A2 has the following specific information:

SEQ ID NO：1：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIDASGYYTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDAYTFDYWGQGTLVTVSS；

SEQ ID NO：2：

GFTFSSYA；

SEQ ID NO：3：

IDASGYYT；

SEQ ID NO：4：

AKDAYTFDY；

SEQ ID NO：5：

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQASADPDTFGQGTKVEIKR；

SEQ ID NO：6：

QSISSY；

SEQ ID NO：7：

SAS；

SEQ ID NO：8：

QQASADPDT。

the sequence referred to by A3 has the following specific information:

SEQ ID NO：9：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYITDDGANTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYATFDYWGQGTLVTVSS；

SEQ ID NO：10：

GFTFSSYA；

SEQ ID NO：11：

ITDDGANT；

SEQ ID NO：12：

AKSYATFDY；

SEQ ID NO：13：

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYNASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAAYYPTTFGQGTKVEIKR；

SEQ ID NO：14：

QSISSY；

SEQ ID NO：15：

NAS；

SEQ ID NO：16：

QQAAYYPTT。

fourth, antigen specificity of monoclonal antibody

Adding 100 mu L of new coronavirus S protein, S-RBD protein and BSA protein solution with the concentration of 1 mu g/mL into a 96-hole enzyme label plate, staying overnight at 4 ℃, discarding the protein solution the next day, adding 200 mu L of 2% skimmed milk powder solution, incubating for 2 hours at 25 ℃, and sealing the enzyme label plate; 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) After the development, absorbance at 450nm was measured with a microplate reader, a histogram was plotted, and the binding properties of the phage antibody produced by each clone and the envelope protein were compared.

The ELISA results of monoclonal antibody A2 are shown in FIG. 2, wherein A2 binds to S protein and S-RBD protein, and A2 antibody is compared with coated BSA as an antibody recognizing RBD region of S protein, indicating that the binding of antibody and S protein has specificity.

The results of ELISA with monoclonal antibody A3 are shown in FIG. 3, in which A3 binds to S protein, but binds to S-RBD protein only weakly and mainly to the region outside the RBD region of S protein, and in which A3 antibody binds to coated BSA with high specificity.

Fifthly, detecting the novel coronavirus S protein by using the Fab fragment of the A2 antibody and the A3 antibody displayed by phage

The detection principle is shown in FIG. 4, the A2 protein and phage-displayed A3 are used for detecting the S protein of the new coronavirus, a Fab fragment of an A2 antibody is coated in the pores of a 96-pore microporous plate (the heavy chain variable region gene and the light chain variable region gene of the A2 antibody are cloned to a pDeng 1 phage display carrier, the Escherichia coli HB2151 is transformed, the transformed Escherichia coli is cultured, then the culture supernatant is taken, a histidine tag is used for purification to obtain the Fab fragment of the A2 antibody), after the microporous plate is sealed, the S protein or BSA protein is added, the phage-displayed A3 antibody is added after the plate is washed, then the anti-bacterial antibody marked with horse radish peroxidase is added, the substrate is added after the plate is washed for color development, when the sample has no S protein of the new coronavirus or the BSA protein, the reaction system does not develop color, and the system develops color when the S protein of the virus is present, and the more virus S protein is present in the sample, the A3 of the virus S, the more the corresponding captured anti-phage antibody, the darker the color of the substrate added with enzyme after color development, and the method can be used for judging whether the virus S1 protein exists in the sample, and the method can be used for qualitative and quantitative detection of SARS-CoV-2 virus in the sample.

The specific operation is as follows: adding A2 antibody Fab fragment into the hole of 96-hole enzyme label plate, staying overnight at 4 ℃, discarding the liquid in the hole, adding 200 μ L of 2% skimmed milk powder solution, standing at room temperature for two hours, and sealing the enzyme label plate. After washing the plate, 100. mu.L of a solution containing the S protein of the novel coronavirus or a Bovine Serum Albumin (BSA) solution was added to the wells, incubated at 25 ℃ for 1 hour, the well-containing solution was removed, the plate was washed with a PBS solution (PBST solution) containing 0.1% Tween, and a phage-displayed A3 antibody solution (10) was added⁹cfu/mL) at 25 ℃ for 1 hour, washing the plate, adding an anti-phage antibody solution (1 mu g/mL) labeled with horseradish peroxidase (HRP), incubating at 25 ℃ for 1 hour, removing the solution in the wells, washing the plate 3 times with PBST, adding a HRP substrate 3, 3, 5, 5-tetramethylbenzidine hydrochloride (TMBZ) solution for color development, and determining the solution in the wellsAbsorbance at 450nm and 630nm, histograms were prepared and the binding capacity of the antibody to neocoronavirus S protein and bovine serum albumin was compared.

As shown in FIG. 5, the results of detecting the virus S protein by the combination of the A2 antibody and phage-displayed A3 were shown in which the horizontal axis represents the name of the detection protein and the vertical axis represents the absorbance of the solution in the corresponding enzyme-labeled well. The absorbance of the enzyme-labeled well plate containing the virus S protein in the solution of the combination of A2 and A3 is 0.46, while the absorbance of the well added with BSA is 0.04, which indicates that the combination of A2 and A3 can be used for qualitatively detecting the new coronavirus S protein in the solution.

Sixthly, quantitative detection of novel coronavirus S protein based on A2 and A3 antibodies

The novel coronavirus S protein was quantitatively detected based on the A2 Fab antibody fragment and phage display A3 antibody using the method shown in FIG. 4. A2 Fab antibody fragment is coated in the hole of a 96-hole micropore plate, after the micropore plate is sealed, inactivated new coronavirus S protein is added, after the plate is washed, phage display A3 antibody is added, then anti-phage antibody which is marked with horse radish peroxidase is added, and after the plate is washed, substrate is added for color development. When no virus protein exists in the sample, the reaction system does not develop color; the system develops color in the presence of viral proteins and the more viral proteins in the sample, the more A3 phage are captured by the virus into the microplate, the more anti-phage antibodies are captured accordingly, and the darker the color develops with the addition of the enzyme substrate. The virus content of the solution can be determined by plotting a standard curve of virus concentration versus absorbance.

The specific operation is as follows: adding A2 Fab antibody fragment into the hole of 96-hole enzyme label plate, staying overnight at 4 ℃, discarding the liquid in the hole, adding 200 μ L of 2% skimmed milk powder solution, standing at room temperature for two hours, and sealing the enzyme label plate. After washing the plate, 100 μ L of a new coronavirus S protein solution or Bovine Serum Albumin (BSA) solution containing different concentrations (0, 1.2, 6, 30, 1500ng/mL) was added to the wells, incubated at 25 ℃ for 1 hour, the well solutions were removed, the microplate was washed with a PBS solution containing 0.1% Tween (PBST solution), a phage-displayed A3 antibody solution was added, incubated at 25 ℃ for 1 hour, an anti-phage antibody solution (1 μ g/mL) labeled with horseradish peroxidase (HRP) was added after washing the plate, incubated at 25 ℃ for 1 hour, the well solutions were removed and washed with PBST for 3 times, finally, an HRP substrate 3, 3, 5, 5-tetramethylbenzidine hydrochloride (TMBZ) solution was added for color development, and the absorbances of the well solutions at 450nm and 630nm were measured to prepare a standard curve.

The results are shown in FIG. 6. The horizontal axis represents the concentration of the new coronavirus S protein or BSA solution, the concentration of the virus solution is from 0 to 1500ng/mL, and the vertical axis represents the absorbance of the solution in each concentration corresponding to the enzyme-labeled well. Along with the gradual increase of the virus concentration in the solution, the absorbance of the solution is gradually increased and is in a linear corresponding relation, the absorbance of the solution is not changed due to the increase of the BSA concentration in a hole added with BSA as a negative control, which indicates that the antibodies A2 and A3 have virus S protein specificity, and the content of the new coronavirus S protein in the sample can be determined according to the curve, namely the antibody can be used for detecting the content of the new coronavirus S protein and the content of new coronavirus particles in the sample, and the lowest concentration of the new coronavirus S protein which can be detected by using the method is 10 ng/mL.

Sequence listing

<110> Weifang medical college

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

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<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Gln Gln Ala Ser Ala Asp Pro Asp Thr

1 5

<210> 9

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Ser Tyr Ala Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 10

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 11

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Ile Thr Asp Asp Gly Ala Asn Thr

1 5

<210> 12

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Ala Lys Ser Tyr Ala Thr Phe Asp Tyr

1 5

<210> 13

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Ser Gly Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Ala Tyr Tyr Pro Thr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 14

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Gln Ser Ile Ser Ser Tyr

1 5

<210> 15

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Asn Ala Ser

1

<210> 16

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gln Gln Ala Ala Tyr Tyr Pro Thr Thr

1 5

Claims

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

2. the monoclonal antibody against the RBD region of the spike protein of 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 light chain variable region 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 the novel coronavirus SARS-CoV-2 for non-diagnostic purposes, comprising: the method comprises the following steps:

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

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

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

8. The use of the monoclonal antibody against the spinous process protein RBD region of the neocoronavirus SARS-CoV-2 as claimed in any one of claims 1-2 in the preparation of a medicament for inhibiting the neocoronavirus SARS-CoV-2.

9. The use of the monoclonal antibody against the spinous process protein RBD region of the neocoronavirus SARS-CoV-2 as claimed in any one of claims 1 to 2 in the preparation of a pharmaceutical preparation for preventing or treating pneumonia caused by the neocoronavirus SARS-CoV-2.