CN113929772A - SARS-CoV-2 neutralizing antibody and its preparation method and use - Google Patents
SARS-CoV-2 neutralizing antibody and its preparation method and use Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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Abstract
The present invention provides SARS-CoV-2 neutralizing antibodies that are highly neutralizing active antibodies that bind SD sequences against the RBD region of a novel virus. The neutralization inhibition rates of the screened SARS-CoV-2 neutralizing antibodies 7H9, 7C4, 8D1 and 6D11 are respectively as low as 0.145, 9.33, 0.170 and 0.121ug/ml, and the binding antibody activities are respectively as low as more than 0.001 ug/ml, more than 0.01 ug/ml and more than 0.001 ug/ml, so the antibody can be applied to the preparation of therapeutic drugs for treating diseases caused by SARS-CoV-2; or in the quality control for the research and development of new corona vaccines, and has good application prospect.
Description
Technical Field
The invention relates to the fields of medicine and biotechnology, in particular to the field of antibody engineering, and more particularly relates to a SARS-CoV-2 neutralizing antibody and a preparation method and application thereof.
Background
The novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is caused by a novel beta coronavirus, a novel coronavirus (SARS-CoV-2 Virus). The novel coronavirus belongs to a novel coronavirus of beta genus, and has envelope, round or elliptical particle, usually polymorphism, and diameter of 60-140 nm. The novel coronavirus (SARS-CoV-2) has four major structural proteins: spike protein (S protein), Nucleocapsid protein (N protein), Membrane protein (M protein), Envelope protein (E protein).
Spike protein (S protein) on the mantle of coronaviruses is a key protein for recognizing host cell receptors during virus infection, and S protein is composed of two subunits, S1 and S2, wherein the S1 subunit is composed of four domains, NTD (N-terminal domain), RBD (receptor domain), SD1 and SD 2. The S2 subunit contains essential elements required for membrane fusion processes, including a fusion peptide, 2 peptide repeat sequences HR (HR), and a transmembrane region. Neutralizing antibodies are a class of antibodies that protect the body against antigenic stimuli, and may be a useful treatment even in the relatively advanced stages of the disease. As a supplement to vaccines and chemotherapy, antibody-mediated measures for preventing and treating viral infections have shown good results and have promising application prospects. The neutralizing antibody can be used for developing therapeutic neutralizing antibody medicines and can also be applied to quality control of development of new corona vaccines. When a vaccine is developed, the in vitro activity of the vaccine must be evaluated by using neutralizing antibodies, namely the neutralizing antibodies are necessary tools for quality analysis during vaccine development. For SARS-CoV-2, only the neutralizing antibody directed against pathogenic sequences in the S protein sequence can neutralize the virulence of the virus and prevent infection of the body by the virus. In most of the detection kits developed previously, the SARS-CoV-2 neutralizing antibody has a target of S protein in most and N protein (nucleocapsid protein) in less part.
Disclosure of Invention
Binding of the RBD region to the SD sequence, which is capable of producing highly neutralizing antibodies, has not been a precedent for successful study. Therefore, the inventor obtains the SARS-CoV-2 neutralizing antibody through research and test, and also provides the preparation method and the application of the neutralizing antibody.
The invention provides a SARS-CoV-2 neutralizing antibody with good neutralizing activity, which is characterized in that the CDR sequences of the light chain and the heavy chain are selected from one of the following groups:
the CDR1 of the light chain is: SSVSY; CDR2 is: DTS; CDR3 is: QQWSSNPFT and its heavy chain CDR1 are: GYTFTEYT; CDR2 is: INPNNGDT; CDR3 is: ARDGYYVTYAMDY, respectively;
light chain CDR1 is: QNVGIN; CDR2 is: an SAS; CDR3 is: QQYNSYPCT, respectively; and heavy chain CDR1 is: GFNIKDTY; CDR2 is: IDPANGNT; CDR3 is: AGGYYDYDAWFAY, respectively;
light chain CDR1 is: SSVSFRY; CDR2 is: GTS; CDR3 is: QQYHSDPFTF and heavy chain CDR1 are: GYTFTDYY; CDR2 is: INPNNGGT; CDR3 is: ARGLTGAMDYWGMDY, respectively;
light chain CDR1 is: QNVGTN; CDR2 is: an SAS; CDR3 is: QQYNSYPWT and heavy chain CDR1 are: GYTFTEYT; CDR2 is: INPYNGDT; CDR3 is: ARDGYYVTYAMDY are provided.
Preferably, it is selected from one of the following groups of light and heavy chain amino acid sequences:
the amino acid sequence of the light chain is: ELDIVITQTTAIMSTSPGEKVTMTCSASSSVSYMHWYQQKSGTSP KRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPFTFGSGTKLEIKRADAAP TVSAC, respectively; and the heavy chain amino acid sequence: EFEVKLQQSGPELVKPGASVKISCKTSGYTFTEYTMYWVKQS HGKSLEWIGGINPNNGDTLYNQKFKGKATLTVDKSSSTAYMALRSLTSEDSAVYYCARDGYYVTYAMDY WGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKRS, respectively;
light chain amino acid sequence: ELDIVMTQTPKFMSTSVGDRVSVTCKASQNVGINVAWYQQKPGQSPKAL IYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPCTFGGGTKLEIKRADAAPTVS AC, respectively; and the heavy chain amino acid sequence: EFEVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIYWVKQRPEQ GLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCAGGYYDYDAWFAYWGQ GTLVTVSAAKTTAPSVYPLAPRS, respectively;
light chain amino acid sequence: ELDIVMTQSPAIMSASPGEKVTMTCSSVSFRYLHWYQQKSGASPKLWIY GTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYHSDPFTFFGSGTKLEIKRADAAPTVSA C, respectively; and the heavy chain amino acid sequence: EFQVKLEQSGPELVKPGASVKIKASGYTFTDYYMNWVKQSHGKSLE WIGDINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGLTGAMDYWGMDYWGQG TSVTVSSAKTTPPSVYPLAPRS, respectively;
light chain amino acid sequence: ELDIVITQSTKFMSTSVGDRVTVTCKASQNVGTNVAWYQQKPGQSPKAL IYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPWTFGGGTKLEIKRADAAPTVS AC, respectively; and the heavy chain amino acid sequence: EFEVKLEQSGPELVKPGASVKISCKTSGYTFTEYTMYWVKQSHGK SLEWIGGINPYNGDTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARDGYYVTYAMDYWGQ GTSVTVSSAKTTPPSVYPLVPRS are provided.
More specifically, the monoclonal antibody is obtained by secretion of a monoclonal antibody cell strain with the preservation number of CGMCC NO.23001, CGMCC NO.23002, CGMCC NO.23003 or CGMCC NO.23004 respectively.
The invention also provides the coding nucleotide of the neutralizing antibody. Preferably, it is selected from one of the following groups of light and heavy chain nucleotide sequences:
nucleotide sequence of the light chain: GAGCTCGACATTGTGATCACACAGACTACAGCAATCATGTCTACATC TCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCA GAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCG CTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGC CACTTATTACTGCCAGCAGTGGAGTAGTAACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAA ACGGGCTGATGCTGCACCAACTGTATCCGCATGC, respectively; and GAATTCGAAGTAAAGCTGCAGCAGTCTGG ACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGA ATACACCATGTACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTAA CAATGGTGATACTCTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAG CACAGCCTACATGGCGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTGCCAGAGATGG TTACTACGTCACTTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAAC GACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACTCTAGG ATGCCTGGTCAAGAGATCT;
the light chain nucleotide sequence: GAGCTCGATATTGTGATGACACAGACTCCAAAATTCATGTCCACATCAG TTGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTATTAATGTAGCCTGGTATCAAC AGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATC GCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGG CAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTGCACGTTCGGAGGGGGGACCAAGCTGGAAATAA AACGGGCTGATGCTGCACCAACTGTATCCGCATGC, respectively; and a heavy chain nucleotide sequence: GAATTCGAAGTG CAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCT GGCTTCAACATTAAAGACACCTATATATACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATT GGAAGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACA GCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCATCTAT TACTGTGCCGGGGGGTACTATGATTACGACGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACT GTCTCTGCAGCCAAAACAACAGCCCCATCAGTCTATCCACTGGCCCCTAGATCT, respectively;
the light chain nucleotide sequence: GAGCTCGACATTGTGATGACACAATCTCCAGCAATCATGTCTGCATCTC CAGGGGAGAAGGTCACCATGACCTGCTCAAGTGTAAGTTTCAGGTACTTGCACTGGTACCAGCAGAAGT CAGGAGCCTCCCCCAAACTCTGGATTTATGGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCA GTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCGTGGAGGCTGAAGATGCTGCCACTT ATTACTGCCAGCAGTATCATAGTGACCCATTCACGTTCTTCGGCTCGGGGACAAAGTTGGAAATAAAAC GGGCTGATGCTGCACCAACTGTATCCGCATGC, respectively; and a heavy chain nucleotide sequence: GAATTCCAGGTCAAG CTGGAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATAAAGGCTTCTGGATACACG TTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATT AATCCTAACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAG TCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCA AGAGGACTAACTGGGGCTATGGACTACTGGGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTC TCCTCAGCCAAAACGACACCCCCATCAGTCTATCCACTGGCCCCTAGATCT, respectively;
the light chain nucleotide sequence: GAGCTCGATATTGTGATCACACAGTCTACAAAATTCATGTCCACATCAG TAGGAGACAGGGTCACCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAAC AGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATC GCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGG CAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCA AACGGGCTGATGCTGCACCAACTGTATCCGCATGC and the heavy chain nucleotide sequence: GAATTCGAGGTAAA GCTGGAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGG ATACACATTCACTGAATACACTATGTACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGG AGGTATTAATCCTTACAATGGTGATACTAGCTATAATCAGAAGTTCAAGGGCAAGGCCACATTGACTGT AGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTA CTGTGCCAGAGATGGTTACTACGTCACTTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGT CTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCCTTGGTCCCTAGATCT are provided.
The invention provides SARS-CoV-2 neutralizing antibodies 7H9, 7C4, 8D1 and 6D11 with good neutralizing activity, corresponding monoclonal antibody cell strains CoV S-RBD 7H9, CoV S-RBD 7C4, CoV S-RBD 8D1 and CoV S-RBD 6D11 are preserved in the China general microbiological culture Collection center (address: No. 3 of Xilu No. 1 North Chen of the south China area of the Beijing city) on 7-13 days in 2021, and the preservation numbers are CGMCC NO.23001, CGMCC NO.23002, CGMCC NO.23003 and CGMCC NO.23004 respectively.
The invention further provides a preparation method of the SARS-CoV-2 neutralizing antibody, which comprises the following steps:
1. preparation of hybridoma cell lines for monoclonal antibodies: the RBD-SD1-hFc antigen is used for immunizing a mouse, serum of the immunized mouse is collected and detected by an ELISA indirect method, S protein RBD-SD1-hFc protein is coated, the serum of the immunized mouse diluted in a gradient manner is added, a goat anti-mouse secondary antibody is added for detection, the titer of the serum against immunogen is preliminarily evaluated, and the immunized mouse with the titer larger than 36 ten thousand is selected; collecting spleen cells of the cells, and fusing the spleen cells with SP2/0 cells; screening the cultured cells by an indirect ELISA method to obtain positive clones;
2. detection of the antigen recognition domain of monoclonal antibodies: respectively coating RBD-SD1-hFc, RBD-hFc and RBD-HSA, adding monoclonal cell culture supernatant, and adding goat anti-mouse secondary antibody for detection; selecting an antibody that recognizes RBD, preferably both a south african mutant and a HuB01 strain;
3. detecting the binding activity of the monoclonal antibody, and selecting the monoclonal antibody with the binding antibody activity more than 0.1 mug/ml;
4. and (3) detecting the neutralizing activity of the monoclonal antibody, namely determining the titer of the neutralizing antibody of the monoclonal antibody screened in the step 3 by adopting a novel coronavirus pseudovirus neutralizing method, and selecting the monoclonal antibody with the IC50 of less than 10 mu g/ml, namely the candidate SARS-CoV-2 neutralizing antibody.
Further, the present invention also provides the use of neutralizing antibodies against SARS-CoV-2, comprising: 1) the application in preparing the medicine for treating the diseases caused by SARS-CoV-2 has good effect on the measures for preventing and treating virus infection mediated by the antibody, and the application prospect is accepted by experts; 2) the application of the method in quality control for developing the new corona vaccine can be used for identification and quantification of the vaccine, detection of in vitro efficacy and the like, and the in vitro efficacy detection needs a neutralizing antibody to participate in pairing.
Furthermore, the invention also provides the application of the SARS-CoV-2 neutralizing antibody 7H9, 7C4, 8D1 as enzyme-labeled antibody.
The invention firstly researches and obtains the high-efficiency neutralizing active antibody aiming at the combination of the RBD region of the novel virus and the SD sequence. The neutralization inhibition rate of the SARS-CoV-2 neutralizing antibodies 7H9, 7C4, 8D1 and 6D11 is respectively as low as 0.145, 9.33, 0.170 and 0.121ug/ml, and the binding antibody activity is respectively as low as more than 0.001 ug/ml, more than 0.01 ug/ml and more than 0.001 ug/ml. Therefore, the compound can be applied to the preparation of therapeutic drugs for treating diseases caused by SARS-CoV-2; or in the quality control for the research and development of new corona vaccines, and has good application prospect.
Drawings
FIG. 1 ELISA two-antibody sandwich standard curve (6D11(2ug/ml) +7H9-HRP (0.5ug/ml) paired groups).
FIG. 2 ELISA two-antibody sandwich standard curve (8D1(1ug/ml) +7C4-HRP (0.5ug/ml) paired groups).
Detailed Description
The invention is further illustrated by the following specific examples in order to provide a better understanding of the invention, which are not to be construed as limiting the invention.
EXAMPLE I development of hybridoma cell lines for monoclonal antibodies
1. Mouse immunization and detection of serum titer after immunization: RBD-SD1-hFc antigen (see Chinese patent 202011454175.1) and Freund's complete adjuvant are mixed in equal volume and fully emulsified, and injected subcutaneously by several points to immunize 3 Balb/c mice, with the injection amount of 10 μ g per mouse. The boosting immunity is performed once every two weeks, and the boosting immunity adopts emulsion of antigen and Freund's incomplete adjuvant. 3 days before cell fusion, injecting a physiological saline solution containing 15ug of antigen into an abdominal cavity, collecting serum of an immune mouse, detecting by using an ELISA indirect method, coating S protein RBD-SD1-hFc protein, adding the serum of the immune mouse diluted in a gradient manner, adding a goat anti-mouse secondary antibody for detection, and primarily evaluating the titer of the serum against the immunogen.
TABLE 1 post-immunization mouse serum test results
Note: NC is the negative control of the detection system (preimmune serum), and PC is the positive control of the detection system (previously prepared positive serum of an immunized mouse).
The serum binding antibody is detected, and the No. 1 mouse has relatively highest immune titer, the titer is more than 36 ten thousand, and the immune response is strong. Therefore, mouse No. 1 was selected for fusion and subsequent hybridoma cell preparation.
2. Preparation of hybridoma cells and establishment of cell lines
Spleen cells from the pooled mice were fused with SP2/0 cells at a 10:1 ratio with 500g/L PEG 4000. Selectively culturing in HAT semi-solid culture solution (IMDM culture solution containing 1-3% methylcellulose, 1 × HAT and 10-20% fetal calf serum) for 10-20 days, selecting 1200 clones, culturing in 96-well plate, and culturing in liquid DMEM culture medium. And continuously culturing for 5-10 days, and after the cells grow up, sucking half of the culture medium to perform screening and cloning by an indirect ELISA method. 13 of these positive clones were finally retained. The results of the experiments are shown in the table below.
The indirect ELISA method was performed as follows:
1) the antigen was dissolved in the coating solution at a concentration of 2. mu.g/ml.
2) Add 100. mu.l of antigen to the corresponding wells and incubate at room temperature for 2h or overnight at 4 ℃.
3) The liquid was emptied and the residual liquid was patted dry and washed twice with 300. mu.l of wash solution.
4) Mu.l of blocking solution was added to each well and incubated for 1h.
5) The liquid was emptied and the residual liquid was patted dry and washed twice with 300. mu.l of wash solution.
6) Mu.l of primary antibody was added to each well and incubated at 37 ℃ for 1h or at room temperature for 3h.
7) Emptying the liquid and patting dry the residual liquid, filling each hole with washing liquid, emptying the liquid and patting dry the residual liquid, and repeating for 3 times.
8) Mu.l of goat anti-mouse secondary antibody was added to each well and incubated for 1h at room temperature.
9) Emptying liquid and beating to dry residual liquid, filling each hole with washing liquid, soaking for 5min with washing liquid, emptying liquid and beating to dry residual liquid, and repeating for 5 times.
11) And adding 100 mu l of substrate into each hole, developing for 5-20 min, and immediately reading at OD.450nm in an enzyme labeling instrument.
TABLE 2 ELISA method for detecting 4 batches of protein (Note: reading in the table is OD)450)
The remaining clones, 13 strains in total, were able to recognize 4 batches of antigenic protein, and since the immunogenic protein was RBD-SD1-hFc, these clones were able to recognize either RBD or SD1 of the novel coronavirus, or hFc, and therefore the detection of the antigen recognition domain was performed below.
EXAMPLE II detection of antigen recognition Domain of monoclonal antibody
The recognition domain of the monoclonal antibody was detected by indirect ELISA. Respectively coating proteins such as HuB01 RBD-SD1-hFc (see Chinese patent 202011454175.1), south Africa mutant RBD-SD1-Fc (see Chinese patent application 202110659137.8), HuB01 RBD-hFc, HuB01 RBD-HSA and the like, adding monoclonal cell culture supernatant, and adding goat anti-mouse secondary antibody for detection.
TABLE 3 summary of antigen recognition region detection (note: the reading in the Table is OD)450)
The above results indicate that 6D11,7C4,7H9, 8D1, 7H1, 5B9, 8B5, 4C4, 1G1 are able to recognize RBD-SD1-hFc, RBD-hFc and RBD-HSA, and thus are antibodies recognizing RBD, and all antibodies recognize a south africa mutant strain and a HuB01 strain, indicating that they are broad-spectrum antibodies. On the other hand, 5C10, 6A10, 6E8 and 8E4 can recognize RBD-SD1-hFc and RBD-hFc, but cannot recognize RBD-HSA, so that these four antibodies are antibodies recognizing hFc.
EXAMPLE III detection of the binding Activity of monoclonal antibodies
100. mu.L of RBD-SD1-hFc protein with a concentration of 5. mu.g/mL was added to each well of a 96-well microplate and placed in a refrigerator at 4 ℃ overnight. Blocking was added at 250. mu.L per well with 2% BSA-PBST and incubated for 1.5 hours at room temperature. Monoclonal purified antibody was diluted with 0.1% BSA-PBST in a 10-fold stepwise gradient starting at 1. mu.g/mL. mu.L of monoclonal antibody purified antibody diluted in a gradient was added to each well and incubated with 0.1% BSA-PBST as a negative control for 1.5 hours at room temperature. Commercial goat anti-mouse secondary antibody was diluted 1:2000 with 0.5% BSA-PBST and 100. mu.L was added to each well and incubated for 1 hour at room temperature. Add 300. mu.L PBST wash plates 5 times per well. Commercial TMB substrate was added and developed in the dark.
The 450nm light absorption was read with a microplate reader. And taking the reading value of the corresponding hole of the sample diluent as a negative control hole, and defining the average value of the negative control which is more than 2 times as positive. Calculating the antibody titer of the monoclonal antibody purified antibody to be positive. The experimental results are as follows:
table 4 results of antibody binding activity
As can be seen from the results of the tables, the binding activity of 6A10, 6E8, 7H1 to the antibody was more than 0.0001. mu.g/ml; the binding antibody activity of 5B9, 5C10, 6D11,7C4,7H9 and 8E4 is more than 0.001 mu g/ml; the activity of the 8D1 binding antibody is more than 0.01 mu g/ml; 1G1, 4C4, 8B5 bound antibody activity of about 0.1. mu.g/ml.
EXAMPLE four detection of neutralizing Activity of monoclonal antibodies
The titer of the neutralizing antibody is detected by adopting a novel coronavirus pseudovirus neutralizing method, and the experimental method comprises the following steps:
(1) cell preparation: the day before the experiment, will be about 1x104Inoculum size of individual cells/well cells to be infected were seeded in 96-well cell culture plates.
(2) Pseudovirus infection: taking out the frozen pseudovirus, thawing, sucking the pseudovirus with required amount and antibody with different amount, mixing, adding into cell culture system to infect target cell, and culturing continuously after 6-8H after virus infection.
(3) Infection detection: after the cells are infected with pseudovirus 48-72H, the infection efficiency is judged by observing the activity of the detected luciferase.
(4) According to the result of the neutralization inhibition rate, the IC50 of the antibody to be detected is calculated by using a Reed-Muench method. The results are given in the table below.
TABLE 5 results of neutralization inhibition
The results showed that 6D11,7C4,7H9 and 8D1 had good neutralizing activity, while the remaining antibodies, which did not achieve 50% inhibition at 10. mu.g/ml, were not considered neutralizing activity.
EXAMPLE V development of double antibody Sandwich ELISA
The developed 4 strains of neutralizing antibodies 6D11,7C4,7H9 and 8D1 are used for double antibody ELISA sandwich development.
1. Horseradish peroxidase labeled antibody
2mg of each purified antibody was labeled with horseradish peroxidase (HRP) using the sodium periodate method.
1) Dissolving 2mg HRP in 0.2mL of 1mM sodium acetate solution with pH 4.4, mixing, and dropwise adding new 0.1M NaIO40.08mL, react for 30min at 4 ℃ in the dark and shake up every 5 minutes.
2) A new 0.2mL of 0.16M ethylene glycol was added dropwise and shaken on a shaker for 30 min.
3) The treated HRP was added dropwise to the dialyzed antibody and dialyzed overnight at 4 ℃ in 0.2M carbonate buffer pH 9.6.
4) NaBH was added dropwise in a reaction volume of 1/104(4mg/mL), mixed well and left to stand at 4 ℃ for 2 hours. Equal volume of saturated ammonium sulfate was added dropwise and overnight at 4 ℃.
5) Centrifuging at 13000rpm for 15-20 min at 4 ℃, discarding the supernatant, washing the precipitate with 1mL of half-saturated ammonium sulfate, centrifuging at 13000rpm for 15-20 min, and discarding the supernatant.
6) Dissolving the precipitate in 0.5mL of 0.01M PBS with pH of 7.4, carrying out centrifugation at 13000rpm for 5-10 min, and discarding the precipitate.
7) Adding 1/3 glycerol, mixing, and storing at-20 deg.C.
TABLE 6 summary of enzyme-labeled antibody data
The HRP-labeled antibody 6D11 showed an antibody recovery rate of only 29.4% and a concentration of only 0.453mg/ml, and thus, it was not suitable as an enzyme-labeled antibody. The recovery rate and concentration of the other three strains of the antibody can meet the requirement of subsequent ELISA pairing, and the antibody can be used as an enzyme-labeled antibody.
ELISA double antibody Sandwich method antibody pairing screening
The ELISA double antibody sandwich method is used for carrying out the mutual pairing experiment of the antibodies.
ELISA double antibody sandwich: the coated antibody is diluted to 10 mu g/mL by 0.05mol/L carbonate buffer solution with pH 9.6, 100 mu L of the coated antibody is added into each hole of an enzyme label plate, the coated antibody is coated overnight at 4 ℃, the coating solution is poured out, washed for 2 times by PBST and dried by beating, then 200 mu L of 3 percent calf serum albumin (BSA) is added into each hole, the hole is put into a 37 ℃ thermostat and sealed for 2 hours, washed for 1 time by PBS, added with 10 percent sucrose aqueous solution and protected for 1 hour at room temperature, and the dried coated antibody is dried, packaged in an aluminum foil bag and vacuumized and stored at 4 ℃.
Adding protein samples into the plate respectively, incubating for 1.5 hours at 37 ℃, washing the plate, adding 100 mu L of enzyme-labeled antibody (0.5ug/ml), incubating for 1 hour at 37 ℃, washing, drying, adding a color developing agent for color development, incubating for 10min at 37 ℃, adding 50 mu L of stop solution/hole, and reading by an enzyme-labeling instrument at a wavelength of 450 nm.
Two batches of the HuB01 strain proteins (1:0.59mg/ml and 2:5mg/ml) with different concentrations are respectively used for establishing a standard curve, detecting the protein with the known concentration of 1.2mg/ml, and comparing the detected value with a theoretical value to calculate the accuracy. The results are shown in the table. The preferable confidence interval OD value range is reasonable (the difference of high and low values is large, the high value is more than 1.5, the low value is less than 0.6), the negative control value is less than 0.15, and the accuracy is in the pairing group of 80% -120%. According to the matching optimization conditions, only two groups of matching in the 7-group pair respectively correspond to the 6D11+7H9-HRP and 8D1+7C4-HRP, and the other matching respectively have the condition that the detection items do not meet the optimization conditions.
TABLE 7 results of different pairing combinations
3, establishment of standard curve of ELISA double antibody sandwich method
The standard curve is established by using two groups of screened 6D11+7H9-HRP and 8D1+7C4-HRP pairs according to the principles that the upper platform and the lower platform of the standard curve are obvious, the concentration range of a confidence interval is wide (at least 6 points), the span of a signal value is large, and the background value is low.
TABLE 86D 11+7H9-HRP pair groups
EXAMPLE sixthly, determination of antibody variable region sequences
The neutralizing antibodies 6D11,7C4,7H9, 8D1 were sequenced, and the results are as follows.
1. Sequencing results of 6D11 antibody
1)6D 11-light chain
The nucleotide sequence is as follows: GAGCTCGACATTGTGATCACACAGACTACAGCAATCATGTCTACATCTCCAGGGGAG AAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGC ACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGC AGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTAC TGCCAGCAGTGGAGTAGTAACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGAT GCTGCACCAACTGTATCCGCATGC
Amino acid sequence: ELDIVITQTTAIMSTSPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPFTFGSGTKLEIKRADAAPTVSAC
Its light chain CDR1 is: SSVSY; CDR2 is: DTS; CDR3 is: QQWSSNPFT are provided.
2)6D 11-heavy chain
The nucleotide sequence is as follows: GAATTCGAAGTAAAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCA GTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATACACCATGTACTGGGTGAAGCAGAGC CATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTAACAATGGTGATACTCTCTACAACCAGAAG TTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGCGCTCCGCAGCCTG ACATCTGAGGATTCTGCAGTCTATTACTGTGCCAGAGATGGTTACTACGTCACTTATGCTATGGACTAC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCC CCTGGATCTGCTGCCCAAACTAACTCCATGGTGACTCTAGGATGCCTGGTCAAGAGATCT
Amino acid sequence: EFEVKLQQSGPELVKPGASVKISCKTSGYTFTEYTMYWVKQSHGKSLEWIGGINPNN GDTLYNQKFKGKATLTVDKSSSTAYMALRSLTSEDSAVYYCARDGYYVTYAMDYWGQGTSVTVSSAKTT PPSVYPLAPGSAAQTNSMVTLGCLVKRS
Its heavy chain CDR1 is: GYTFTEYT; CDR2 is: INPNNGDT; CDR3 is: ARDGYYVTYAMDY are provided.
2. Sequencing results of 7C4 antibody
1)7C 4-light chain
The nucleotide sequence is as follows: GAGCTCGATATTGTGATGACACAGACTCCAAAATTCATGTCCACATCAGTTGGAGAC AGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTATTAATGTAGCCTGGTATCAACAGAAACCA GGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACA GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTAT TTCTGTCAGCAATATAACAGCTATCCGTGCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCT GATGCTGCACCAACTGTATCCGCATGC
Amino acid sequence: ELDIVMTQTPKFMSTSVGDRVSVTCKASQNVGINVAWYQQKPGQSPKALIYSASYRY SGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPCTFGGGTKLEIKRADAAPTVSAC its light chain CDR1 is: QNVGIN; CDR2 is: an SAS; CDR3 is: QQYNSYPCT are provided.
2)7C 4-heavy chain
The nucleotide sequence is as follows: GAATTCGAAGTGCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCA GTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATATATACTGGGTGAAGCAGAGG CCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAAG TTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTG ACATCTGAGGACACTGCCATCTATTACTGTGCCGGGGGGTACTATGATTACGACGCCTGGTTTGCTTAC TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACAACAGCCCCATCAGTCTATCCACTGGCC CCTAGATCT
Amino acid sequence: EFEVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIYWVKQRPEQGLEWIGRIDPAN GNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCAGGYYDYDAWFAYWGQGTLVTVSAAKTT APSVYPLAPRS
Its heavy chain CDR1 is: GFNIKDTY; CDR2 is: IDPANGNT; CDR3 is: AGGYYDYDAWFAY are provided.
3. Sequencing results of 7H9 antibody
1)7H 9-light chain
The nucleotide sequence is as follows: GAGCTCGACATTGTGATGACACAATCTCCAGCAATCATGTCTGCATCTCCAGGGGAG AAGGTCACCATGACCTGCTCAAGTGTAAGTTTCAGGTACTTGCACTGGTACCAGCAGAAGTCAGGAGCC TCCCCCAAACTCTGGATTTATGGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGT GGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCGTGGAGGCTGAAGATGCTGCCACTTATTACTGC CAGCAGTATCATAGTGACCCATTCACGTTCTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGAT GCTGCACCAACTGTATCCGCATGC
Amino acid sequence: ELDIVMTQSPAIMSASPGEKVTMTCSSVSFRYLHWYQQKSGASPKLWIYGTSNLASG VPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYHSDPFTFFGSGTKLEIKRADAAPTVSAC
Its light chain CDR1 is: SSVSFRY; CDR2 is: GTS; CDR3 is: QQYHSDPFTF are provided.
2)7H 9-heavy chain
The nucleotide sequence is as follows: GAATTCCAGGTCAAGCTGGAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCA GTGAAGATAAAGGCTTCTGGATACACGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGA AAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGGTACTAGCTACAACCAGAAGTTCAAG GGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCT GAGGACTCTGCAGTCTATTACTGTGCAAGAGGACTAACTGGGGCTATGGACTACTGGGGTATGGACTAC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCAGTCTATCCACTGGCC CCTAGATCT
Amino acid sequence: EFQVKLEQSGPELVKPGASVKIKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGG TSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGLTGAMDYWGMDYWGQGTSVTVSSAKTT PPSVYPLAPRS
Its heavy chain CDR1 is: GYTFTDYY; CDR2 is: INPNNGGT; CDR3 is: ARGLTGAMDYWGMDY are provided.
4. 8D1 antibody sequencing results
1)8D 1-light chain
The nucleotide sequence is as follows: GAGCTCGATATTGTGATCACACAGTCTACAAAATTCATGTCCACATCAGTAGGAGAC AGGGTCACCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCA GGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACA GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTAT TTCTGTCAGCAATATAACAGCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCT GATGCTGCACCAACTGTATCCGCATGC
Amino acid sequence: ELDIVITQSTKFMSTSVGDRVTVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRY SGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPWTFGGGTKLEIKRADAAPTVSAC its light chain CDR1 is: QNVGTN; CDR2 is: an SAS; CDR3 is: QQYNSYPWT are provided.
2)8D 1-heavy chain
The nucleotide sequence is as follows: GAATTCGAGGTAAAGCTGGAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCA GTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATACACTATGTACTGGGTGAAGCAGAGC CATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTTACAATGGTGATACTAGCTATAATCAGAAG TTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTG ACATCTGAGGATTCTGCAGTCTATTACTGTGCCAGAGATGGTTACTACGTCACTTATGCTATGGACTAC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCCTTGGTC CCTAGATCT
Amino acid sequence: EFEVKLEQSGPELVKPGASVKISCKTSGYTFTEYTMYWVKQSHGKSLEWIGGINPYN GDTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARDGYYVTYAMDYWGQGTSVTVSSAKTT PPSVYPLVPRS
Its heavy chain CDR1 is: GYTFTEYT; CDR2 is: INPYNGDT; CDR3 is: ARDGYYVTYAMDY are provided.
Sequence listing
<110> Beijing Kangle guard Biotechnology Ltd
<120> SARS-CoV-2 neutralizing antibody, its preparation method and application
<160>16
<170>PatentIn Version 3.1
<210>1
<211>357
<212>DNA
<213>6D 11-light chain-encoding nucleotides
<400> 1
GAGCTCGACATTGTGATCACACAGACTACAGCAATCATGTCTACATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCGCATGC 357
<210>2
<211>119
<212>PRT
<213>6D 11-light chain
<400>2
ELDIVITQTTAIMSTSPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPFTFGSGTKLEIKRADAAPTVSAC 119
<210>3
<211>462
<212>DNA
<213>6D 11-heavy chain encoding nucleotide
<400>3
GAATTCGAAGTAAAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATACACCATGTACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTAACAATGGTGATACTCTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGCGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTGCCAGAGATGGTTACTACGTCACTTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACTCTAGGATGCCTGGTCAAGAGATCT 462
<210>4
<211>154
<212>PRT
<213>6D 11-heavy chain
<400>4
EFEVKLQQSGPELVKPGASVKISCKTSGYTFTEYTMYWVKQSHGKSLEWIGGINPNNGDTLYNQKFKGKATLTVDKSSSTAYMALRSLTSEDSAVYYCARDGYYVTYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKRS 154
<210>5
<211>360
<212>DNA
<213>7C 4-light chain-encoding nucleotides
<400>5
GAGCTCGATATTGTGATGACACAGACTCCAAAATTCATGTCCACATCAGTTGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTATTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTGCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCGCATGC 360
<210>6
<211>120
<212>PRT
<213>7C 4-light chain
<400>6
ELDIVMTQTPKFMSTSVGDRVSVTCKASQNVGINVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPCTFGGGTKLEIKRADAAPTVSAC 120
<210>7
<211>411
<212>DNA
<213>7C 4-heavy chain
<400>7
GAATTCGAAGTGCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATATATACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCATCTATTACTGTGCCGGGGGGTACTATGATTACGACGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACAACAGCCCCATCAGTCTATCCACTGGCCCCTAGATCT 411
<210>8
<211>137
<212>PRT
<213>7C 4-heavy chain
<400>8
EFEVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIYWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCAGGYYDYDAWFAYWGQGTLVTVSAAKTTAPSVYPLAPRS 137
<210>9
<211>357
<212>DNA
<213>7H 9-light chain-encoding nucleotides
<400>9
GAGCTCGACATTGTGATGACACAATCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCTCAAGTGTAAGTTTCAGGTACTTGCACTGGTACCAGCAGAAGTCAGGAGCCTCCCCCAAACTCTGGATTTATGGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTATCATAGTGACCCATTCACGTTCTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCGCATGC 357
<210>10
<211>119
<212>PRT
<213>7H 9-light chain
<400>10
ELDIVMTQSPAIMSASPGEKVTMTCSSVSFRYLHWYQQKSGASPKLWIYGTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYHSDPFTFFGSGTKLEIKRADAAPTVSAC 119
<210>11
<211>411
<212>DNA
<213>7H 9-heavy chain encoding nucleotide
<400>11
GAATTCCAGGTCAAGCTGGAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATAAAGGCTTCTGGATACACGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGACTAACTGGGGCTATGGACTACTGGGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCAGTCTATCCACTGGCCCCTAGATCT 411
<210>12
<211>137
<212>PRT
<213>7H 9-heavy chain
<400>12
EFQVKLEQSGPELVKPGASVKIKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGLTGAMDYWGMDYWGQGTSVTVSSAKTTPPSVYPLAPRS 137
<210>13
<211>360
<212>DNA
<213>8D 1-light chain-encoding nucleotides
<400>13
GAGCTCGATATTGTGATCACACAGTCTACAAAATTCATGTCCACATCAGTAGGAGACAGGGTCACCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCGCATGC 360
<210>14
<211>120
<212>PRT
<213>8D 1-light chain
<400>14
ELDIVITQSTKFMSTSVGDRVTVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPWTFGGGTKLEIKRADAAPTVSAC 120
<210>13
<211>411
<212>DNA
<213>8D 1-heavy chain encoding nucleotide
<400>15
GAATTCGAGGTAAAGCTGGAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATACACTATGTACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTTACAATGGTGATACTAGCTATAATCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTGCCAGAGATGGTTACTACGTCACTTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCCTTGGTCCCTAGATCT 411
<210>16
<211>137
<212>PRT
<213>8D 1-heavy chain
<400>16
EFEVKLEQSGPELVKPGASVKISCKTSGYTFTEYTMYWVKQSHGKSLEWIGGINPYNGDTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARDGYYVTYAMDYWGQGTSVTVSSAKTTPPSVYPLVPRS 137
Claims (9)
1. A SARS-CoV-2 neutralizing antibody, wherein the CDR sequences of the light and heavy chains are selected from one of the following groups:
the CDR1 of the light chain is: SSVSY; CDR2 is: DTS; CDR3 is: QQWSSNPFT and its heavy chain CDR1 are: GYTFTEYT; CDR2 is: INPNNGDT; CDR3 is: ARDGYYVTYAMDY, respectively;
light chain CDR1 is: QNVGIN; CDR2 is: an SAS; CDR3 is: QQYNSYPCT, respectively; and heavy chain CDR1 is: GFNIKDTY; CDR2 is: IDPANGNT; CDR3 is: AGGYYDYDAWFAY, respectively;
light chain CDR1 is: SSVSFRY; CDR2 is: GTS; CDR3 is: QQYHSDPFTF and heavy chain CDR1 are: GYTFTDYY; CDR2 is: INPNNGGT; CDR3 is: ARGLTGAMDYWGMDY, respectively;
light chain CDR1 is: QNVGTN; CDR2 is: an SAS; CDR3 is: QQYNSYPWT and heavy chain CDR1 are: GYTFTEYT; CDR2 is: INPYNGDT; CDR3 is: ARDGYYVTYAMDY are provided.
2. The neutralizing antibody of claim 1, selected from one of the following group of light and heavy chain amino acid sequences:
the amino acid sequence of the light chain is: ELDIVITQTTAIMSTSPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPFTFGSGTKLEIKRADAAPTVSAC, respectively; and the heavy chain amino acid sequence: EFEVKLQQSGPELVKPGASVKISCKTSGYTFTEYTMYWVKQSHGKSLEWIGGINPNNGDTLYNQKFKGKATLTVDKSSSTAYMALRSLTSEDSAVYYCARDGYYVTYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKRS, respectively;
light chain amino acid sequence: ELDIVMTQTPKFMSTSVGDRVSVTCKASQNVGINVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPCTFGGGTKLEIKRADAAPTVSAC, respectively; and the heavy chain amino acid sequence: EFEVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIYWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCAGGYYDYDAWFAYWGQGTLVTVSAAKTTAPSVYPLAPRS, respectively;
light chain amino acid sequence: ELDIVMTQSPAIMSASPGEKVTMTCSSVSFRYLHWYQQKSGASPKLWIYGTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYHSDPFTFFGSGTKLEIKRADAAPTVSAC, respectively; and the heavy chain amino acid sequence: EFQVKLEQSGPELVKPGASVKIKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGLTGAMDYWGMDYWGQGTSVTVSSAKTTPPSVYPLAPRS, respectively;
light chain amino acid sequence: ELDIVITQSTKFMSTSVGDRVTVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPWTFGGGTKLEIKRADAAPTVSAC, respectively; and the heavy chain amino acid sequence: EFEVKLEQSGPELVKPGASVKISCKTSGYTFTEYTMYWVKQSHGKSLEWIGGINPYNGDTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARDGYYVTYAMDYWGQGTSVTVSSAKTTPPSVYPLVPRS are provided.
3. The neutralizing antibody of claim 2, which is secreted by a monoclonal antibody cell line having a collection number of any one of CGMCC NO.23001, CGMCC NO.23002, CGMCC NO.23003 and CGMCC NO.23004, respectively.
4. The nucleotide sequence encoding the neutralizing antibody of any one of claims 1 to 3.
5. The coding nucleotide of claim 4, selected from one of the following group of light and heavy chain nucleotide sequences:
nucleotide sequence of the light chain: GAGCTCGACATTGTGATCACACAGACTACAGCAATCATGTCTACATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCGCATGC, respectively; and GAATTCGAAGTAAAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATACACCATGTACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTAACAATGGTGATACTCTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGCGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTGCCAGAGATGGTTACTACGTCACTTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACTCTAGGATGCCTGGTCAAGAGATCT;
the light chain nucleotide sequence: GAGCTCGATATTGTGATGACACAGACTCCAAAATTCATGTCCACATCAGTTGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTATTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTGCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCGCATGC, respectively; and a heavy chain nucleotide sequence: GAATTCGAAGTGCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATATATACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCATCTATTACTGTGCCGGGGGGTACTATGATTACGACGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACAACAGCCCCATCAGTCTATCCACTGGCCCCTAGATCT, respectively;
the light chain nucleotide sequence: GAGCTCGACATTGTGATGACACAATCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCTCAAGTGTAAGTTTCAGGTACTTGCACTGGTACCAGCAGAAGTCAGGAGCCTCCCCCAAACTCTGGATTTATGGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTATCATAGTGACCCATTCACGTTCTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCGCATGC, respectively; and a heavy chain nucleotide sequence: GAATTCCAGGTCAAGCTGGAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATAAAGGCTTCTGGATACACGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGACTAACTGGGGCTATGGACTACTGGGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCAGTCTATCCACTGGCCCCTAGATCT, respectively; or
The light chain nucleotide sequence: GAGCTCGATATTGTGATCACACAGTCTACAAAATTCATGTCCACATCAGTAGGAGACAGGGTCACCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCGCATGC and the heavy chain nucleotide sequence: GAATTCGAGGTAAAGCTGGAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATACACTATGTACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTTACAATGGTGATACTAGCTATAATCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTGCCAGAGATGGTTACTACGTCACTTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCCTTGGTCCCTAGATCT are provided.
A monoclonal antibody cell strain of SARS-CoV-2 neutralizing antibody, which is preserved in China general microbiological culture Collection center at 13 months 7 and 2021 with the preservation numbers of CGMCC NO.23001, CGMCC NO.23002, CGMCC NO.23003 and CGMCC NO.23004 respectively.
7. A method for preparing a SARS-CoV-2 neutralizing antibody, which comprises the following steps:
1) preparation of hybridoma cell lines for monoclonal antibodies: immunizing a mouse by an S protein RBD-SD1-hFc antigen, collecting serum of the immunized mouse, detecting by an ELISA indirect method, coating the S protein RBD-SD1-hFc protein, adding the serum of the immunized mouse subjected to gradient dilution, adding a goat anti-mouse secondary antibody for detection, primarily evaluating the titer of the serum against immunogen, and selecting the immunized mouse with the titer of immunity being more than 36 ten thousand; collecting spleen cells of the cells, and fusing the spleen cells with SP2/0 cells; screening the cultured cells by an indirect ELISA method to obtain positive clones;
2) detection of the antigen recognition domain of monoclonal antibodies: respectively coating RBD-SD1-hFc, RBD-hFc and RBD-HSA, adding monoclonal cell culture supernatant, and adding goat anti-mouse secondary antibody for detection; selecting an antibody that recognizes RBD, preferably both a south african mutant and a HuB01 strain;
3) detecting the binding activity of the monoclonal antibody, and selecting the monoclonal antibody with the binding antibody activity more than 0.1 mug/ml;
4) and (3) detecting the neutralizing activity of the monoclonal antibody, wherein the neutralizing antibody titer of the monoclonal antibody screened in the step 3 is determined by a neutralizing method of the new coronavirus pseudovirus, and the monoclonal antibody with the IC50 of less than 10 mu g/ml is selected.
8. Use of SARS-CoV-2 neutralizing antibodies according to any of claims 1 to 3, for the preparation of a medicament for the treatment of a disease caused by SARS-CoV-2; or in quality control for the development of new corona vaccines.
9. Use according to claim 8 as an enzyme-labelled antibody.
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