CN113429478B - Monoclonal antibody H9 against novel coronavirus SARS-CoV-2 - Google Patents

Monoclonal antibody H9 against novel coronavirus SARS-CoV-2 Download PDF

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CN113429478B
CN113429478B CN202110747989.2A CN202110747989A CN113429478B CN 113429478 B CN113429478 B CN 113429478B CN 202110747989 A CN202110747989 A CN 202110747989A CN 113429478 B CN113429478 B CN 113429478B
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单喜军
董金华
董航
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Shandong Kuanhezheng Bio Medicine Co ltd
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Abstract

The application discloses a phage display antibody library and screens five antibodies capable of combining with S protein of new coronavirus SARS-CoV-2, which is based on synthetic biology and phage display technology, introduces mutation into hypervariable region of antibody variable region, and transfers the gene into colibacillus, thus constructing a recombinant DNA construct containing 10 8 A synthetic antibody repertoire of germline antibodies; the phage display antibody library can screen and obtain antibodies with specificity and detection function, and powerful resources of biological research and medical diagnosis are enlarged; the application also screens five antibodies capable of combining with S protein of the new coronavirus, can be used for detecting the virus, and part of the antibodies can block the combination of the virus and cells, have the capability of neutralizing the infectious capacity of the new coronavirus, can be used for preparing new coronavirus detection products, preparing medicines for inhibiting the new coronavirus and preparing pharmaceutical preparations for preventing or treating diseases caused by the new coronavirus, and have wide application prospects.

Description

Monoclonal antibody H9 against novel coronavirus SARS-CoV-2
The application is application number 202010499744.8, the application name: phage display antibody library and the separated application of monoclonal antibody against SARS-CoV-2 based on its screening, and the application date of the mother case is 2020, 06 and 04.
Technical Field
The application relates to the technical fields of biomedicine and molecular biology, in particular to a phage display antibody library and a monoclonal antibody aiming at novel coronavirus SARS-CoV-2, which is obtained based on a phage display antibody library panning.
Background
Compared with the original coronavirus, SARS-CoV-2 has transmissibility for severe patients, has slight symptoms and strong transmissibility even for patients in a latent period, has a plurality of transmission ways, can be transmitted through spray and contact, and brings great difficulty to prevention.
Various treatment schemes are proposed for treating the new coronatine by various countries, mainly including the use of small molecular medicines of Ruidexivir, chloroquine and hydroxychloroquine, lopinavir and ritonavir and the use of lopinavir, ritonavir and interferon, and unfortunately, no obvious treatment effect exists and serious toxic and side effects exist in some cases. On the other hand, antibody drugs play an important role in the treatment of infectious diseases, autoimmune diseases, tumors, and the like. Under the conditions of high mutation rate of SARS-CoV-2, difficult development of new coronavirus vaccines, long period, large side effect or even no effect of traditional medicines, the serum of a rehabilitation patient is used for treating a severe patient, which is a relatively effective treatment scheme and plays a certain role in actual treatment, so that the antibody of the new coronavirus can effectively weaken the infectious capability of the SARS-CoV-2 virus, plays a certain role in the treatment of the new coronavirus patient and provides a new way for medical research and development personnel.
The SARS-CoV-2 virus has a diameter of 75-160 nm, the genome of the SARS-CoV-2 virus is continuous linear single-stranded RNA, and the genome of the SARS-CoV-2 virus sequentially encodes nucleoprotein (nucleoprotein), envelope protein (envelope protein), membrane protein (membrane protein) and spike protein (spike protein), wherein the spike protein is the most important protein on the surface of the SARS-CoV-2 virus, and the main function of the spike protein is to determine the host range and specificity of the virus, and the spike protein is combined and fused with a host cell membrane receptor to realize the infection of cells. The spinous process protein has two subunits S1 and S2, the receptor binding region (receptor binding domain, RBD) in S1 interacts with human SARS-CoV receptor angiotensin converting enzyme II (ACE 2) 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 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 an important glycoprotein molecule in the mammalian immune system. The antibody molecule consists of two Heavy chains (heavies chain) consisting of a variable region (Variable region of Heavy chain, VH) and three constant regions (Constant region of Heavy chain; CH1, CH2, CH 3) and two Light chains (Light chain) consisting of a variable region (VL) and a constant region (CL). The variable region has a function of binding to an antigen, and varies depending on the individual antibody, while the constant region of an antibody varies depending on the species and subtype of the antibody. The variable region of the antibody is further divided into 4 Framework regions (FR 1, FR2, FR3, FR 4) and three Complementarity determining regions (Complementarity-determining regions; CDR1, CDR2, CDR 3), wherein the structure of the heavy chain variable region of the antibody is HFR1-CDRH1-HFR2-CDRH2-HFR3-CDRH3-HFR4, and the structure of the light chain variable region of the antibody is LFR1-CDRL1-LFR2-CDRL2-LFR 3-LFR4, wherein the Complementarity determining regions are also known as hypervariable regions, and bind directly to an epitope of an antigen.
Disclosure of Invention
To solve the above problems, the present application aims to provide a phage display antibody library and a monoclonal antibody against the novel coronavirus SARS-CoV-2 obtained based on a phage display antibody library panning.
The application aims to achieve the aim, and the aim is achieved by the following technical scheme:
a phage display antibody library comprising DNA sequences corresponding to the resulting antibody heavy chain variable region by mutating amino acids corresponding to all mutation positions of the antibody heavy chain variable region to arbitrary amino acids using NNK codons; and mutating amino acids corresponding to all mutation positions of the antibody light chain variable region into any amino acid, and correspondingly obtaining a DNA sequence of the antibody light chain variable region; the antibody heavy chain variable region gene and the antibody light chain variable region gene are recombined with a phage vector to construct plasmids, and then the plasmids are transformed into escherichia coli, so that a phage display antibody library is constructed;
wherein the mutation position of the heavy chain variable region of the antibody is 50, 52a,52b,53, 55, 56, 58, 97, 98, 99, 100, 101, 102, 103, 104, 105; the mutation positions of the antibody light chain variable regions are 50, 51, 52, 53, 89, 90, 91, 92, 93, 94, 95, 96, 97;
wherein the DNA sequence of the heavy chain variable region of the antibody library is SEQ ID NO:53;
the DNA sequence of the antibody library light chain variable region is SEQ ID NO:54.
the application also includes a method of constructing a phage display antibody library, comprising the steps of:
(1) amplifying the HFR1-CDHR1-HFR2 gene fragment VH1 of the antibody heavy chain variable region VH by polymerase chain reaction using the antibody heavy chain variable region DNA as a template and a primer SfiVHback and a primer VHFR2For, amplifying the gene of CDRH2-HFR3-CDRH3-HFR4, namely VH2, by using a degenerate primer CDRH2 library and a degenerate primer CDRH3 library 9For, performing agarose electrophoresis, and confirming the amplified VH1 and VH2; cutting and purifying the two gene fragments, and performing overlap PCR by using the primers SfiVHback and XhoVHfor by taking the two gene sequences as templates; performing agarose electrophoresis, confirming the amplified DNA fragment, purifying the DNA fragment, and using the DNA fragment as a heavy chain variable region gene of a phage display antibody library;
wherein the heavy chain variable region template DNA sequence of the antibody is SEQ ID NO:1, a step of;
the base sequence of the primer SfiVHBack is SEQ ID NO:2;
the base sequence of the primer VHFR2For is SEQ ID NO:3, a step of;
the base sequence of the degenerate primer CDRH2 liback is SEQ ID NO:4, a step of;
the base sequence of the degenerate primer CDRH3lib9for is SEQ ID NO:5, a step of;
the base sequence of XhoVHfor is SEQ ID NO:6, preparing a base material;
the DNA sequence of the heavy chain variable region of the antibody repertoire is SEQ ID NO:53;
(2) amplifying LFR1-CDRL1-LFR2 gene fragment VL1 of an antibody light chain VL by polymerase chain reaction by using an antibody light chain variable region DNA as a template and using a primer SalVLback and a primer VLFR2For, amplifying a gene fragment VL2 of CDRL2-LFR3-CDRL3-LFR4 by using a degenerate primer CDRL2 library and a degenerate primer CDRL3libfor, performing agarose electrophoresis, confirming the amplified VL1, VL2, cutting and purifying the two gene fragments, and performing overlap PCR by using two gene sequences as templates and using the primers SalVLback and NotVLfor; performing agarose electrophoresis, confirming the amplified DNA fragment, purifying the DNA fragment, and using the DNA fragment as a light chain variable region gene of a phage display antibody library;
wherein the light chain variable region template DNA sequence of the antibody is SEQ ID NO:7, preparing a base material;
the base sequence of the primer SalVLback is SEQ ID NO:8, 8;
the base sequence of the primer VLFR2For is SEQ ID NO:9, a step of performing the process;
the base sequence of the degenerate primer CDRL2 liback is SEQ ID NO:10;
the base sequence of the degenerate primer CDRL3libfor is SEQ ID NO:11;
the base sequence of NotVLfor is SEQ ID NO:12;
the DNA sequence of the antibody library light chain variable region is SEQ ID NO:54;
(3) treating the light chain variable region gene by using restriction enzymes SalI and NotI, cloning the light chain variable region gene into a phage display vector pDONG1 after purification, treating the light chain variable region gene by using restriction enzymes SfiI and XhoI after amplification, and connecting the light chain variable region gene with the heavy chain variable region gene by using T4 DNA ligase after the heavy chain variable region gene is treated by using restriction enzymes SfiI and XhoI to transform escherichia coli TG-1;
(4) culturing and transforming escherichia coli TG-1, adding helper phage KM13 for infection, and obtaining phage display antibody library after post-treatment.
The application also includes monoclonal antibody A9 screened by phage display antibody library,
the amino acid sequence of the heavy chain variable region of the A9 antibody is SEQ ID NO:13, cdrh1 amino acid sequence is SEQ ID NO:14; CDRH2 amino acid sequence is SEQ ID NO:15; CDRH3 amino acid sequence is SEQ ID NO:16;
the amino acid sequence of the light chain variable region of the A9 antibody is SEQ ID NO:17, cdrl1 amino acid sequence is SEQ ID NO:18; the amino acid sequence of CDRL2 is SEQ ID NO:19; the amino acid sequence of CDRL3 is SEQ ID NO:20.
the application also includes monoclonal antibody E11 screened by phage display antibody library,
the amino acid sequence of the heavy chain variable region of the E11 antibody is SEQ ID NO:21, cdrh1 amino acid sequence is SEQ ID NO:22; CDRH2 amino acid sequence is SEQ ID NO:23; CDRH3 amino acid sequence is SEQ ID NO:24, a step of detecting the position of the base;
the amino acid sequence of the variable region of the light chain of the E11 antibody is SEQ ID NO:25, cdrl1 amino acid sequence is SEQ ID NO:26; the amino acid sequence of CDRL2 is SEQ ID NO:27; the amino acid sequence of CDRL3 is SEQ ID NO:28.
the application also includes monoclonal antibody F5 screened by phage display antibody library,
the amino acid sequence of the heavy chain variable region of the F5 antibody is SEQ ID NO:29, cdrh1 amino acid sequence is SEQ ID NO:30; CDRH2 amino acid sequence is SEQ ID NO:31; CDRH3 amino acid sequence is SEQ ID NO:32;
the amino acid sequence of the light chain variable region of the F5 antibody is SEQ ID NO:33, cdrl1 amino acid sequence is SEQ ID NO:34; the amino acid sequence of CDRL2 is SEQ ID NO:35; the amino acid sequence of CDRL3 is SEQ ID NO:36.
the application also includes monoclonal antibody F10 screened by phage display antibody library,
the amino acid sequence of the heavy chain variable region of the F10 antibody is SEQ ID NO:37, cdrh1 amino acid sequence is SEQ ID NO:38, a step of carrying out the process; CDRH2 amino acid sequence is SEQ ID NO:39; CDRH3 amino acid sequence is SEQ ID NO:40, a step of performing a;
the amino acid sequence of the light chain variable region of the F10 antibody is SEQ ID NO:41, cdrl1 amino acid sequence is SEQ ID NO:42; the amino acid sequence of CDRL2 is SEQ ID NO: 43. The amino acid sequence of CDRL3 is SEQ ID NO:44.
the application also includes monoclonal antibody H9 screened by phage display antibody library,
the amino acid sequence of the H9 antibody heavy chain variable region is SEQ ID NO:45, cdrh1 amino acid sequence is SEQ ID NO:46; CDRH2 amino acid sequence is SEQ ID NO:47; CDRH3 amino acid sequence is SEQ ID NO:48;
the amino acid sequence of the H9 antibody light chain variable region is SEQ ID NO:49, cdrl1 amino acid sequence is SEQ ID NO:50; the amino acid sequence of CDRL2 is SEQ ID NO:51; the amino acid sequence of CDRL3 is SEQ ID NO:52.
the application also comprises the application of any monoclonal antibody (A9, E11, F5, F10, H9) screened by phage display antibody library in preparing a novel coronavirus SARS-CoV-2 detection product; for example, the kit for detecting the novel coronavirus SARS-CoV-2 can be prepared, and the kit contains any one or more of the antibodies A9, E11, F5, F10 and H9 and derivatives thereof, such as full-length antibody containing antibody variable region, scFv, or Fab fragment or other fusion protein containing variable region and other auxiliary materials for assembling the kit.
The application also comprises the application of any monoclonal antibody (A9, E11, F5, F10 and H9) screened by phage display antibody library in preparing medicines for inhibiting novel coronavirus SARS-CoV-2.
The application also comprises the application of any monoclonal antibody (A9, E11, F5, F10, H9) screened by phage display antibody library in preparing pharmaceutical preparations for preventing or treating diseases caused by novel coronavirus SARS-CoV-2.
The application also includes any engineered monoclonal antibody, the antigen binding fragment of which is an antibody fragment selected from any monoclonal antibody A9, E11, F5, F10, H9, including Fab, fab '-SH, fv, scFv, or (Fab') 2 fragments.
Antibodies are divided into variable regions, which determine the antigen binding properties of the antibody, and constant regions. The antibody variable region sequences according to the present application can be used to make Fab, fab '-SH, fv, scFv or (Fab') 2 fragments with the same antigen binding specificity and use.
The application also includes any monoclonal antibody, the amino acid sequences of the hypervariable regions of the heavy chain and the light chain of which have more than 80% identity with the corresponding sequences of any monoclonal antibody A9, E11, F5, F10 or H9.
The hypervariable region sequence of the antibody is subjected to further mutation and evolution, and under the condition of keeping 80% of identity, the antigen binding specificity of the antibody is unchanged, and the antigen binding capability can be obviously improved.
Compared with the prior art, the application has the following advantages:
the application is based on synthetic biology and phage display technology, introduces mutation into specific position of antibody including super variable region, and transfers the gene into colibacillus, thus constructing a 10-containing gene 8 A synthetic antibody repertoire of diverse species capable of efficient expression in E.coli; the phage display antibody library of the present application can be used for screeningThe antibody with specificity and detection function is obtained, and powerful resources of biological research and medical diagnosis are expanded.
Five antibodies capable of combining with S protein of a novel coronavirus SARS-CoV-2 are screened from the phage display antibody library, and the hypervariable sequence of the antibodies is a novel sequence, so that the antibodies are novel humanized monoclonal antibodies; the antibodies can block the combination of viruses and cells, have the capability of neutralizing the infectious force of the new coronavirus, can be used for preparing new coronavirus SARS-CoV-2 detection products, preparing medicines for inhibiting the new coronavirus SARS-CoV-2 and preparing medicinal preparations for preventing or treating diseases caused by the new coronavirus SARS-CoV-2, and have wide application prospects.
Drawings
FIG. 1 is an agarose gel electrophoresis of a gene fragment obtained by PCR;
FIG. 2 shows the results of an ELISA for the antigen binding energy of phage obtained from a panning antibody library;
FIG. 3 shows the results of an ELISA test of monoclonal antibody antigen specificity;
FIG. 4 is a schematic diagram of the principle of detection of a novel coronavirus using antibodies from a panning screen;
FIG. 5 shows the results of an ELISA assay for detecting viral S protein using Fab fragments of the A9 antibody in combination with F5, F10 or H9;
FIG. 6 shows the results of an ELISA assay for detecting viral S protein using Fab fragments of E11 antibodies in combination with F5, F10 or H9.
Detailed Description
The application aims to provide a phage display antibody library and a monoclonal antibody aiming at novel coronavirus SARS-CoV-2 obtained based on phage display antibody library panning, which are realized by the following technical scheme:
the application is further described below in connection with specific embodiments.
The S protein and the viral S-RBD protein of the novel coronavirus SARS-CoV-2 used in the examples were purchased from Beijing Yiqiao Shenzhou Biotech Co.
The synthetic method references for phage display vector pDong1 are: dong, et al, anal biochem 2009, 386 (1): 36-44.
Example 1
1. Construction of phage display antibody libraries
Amplifying the HFR1-CDRH1-HFR2 gene fragment VH1 of the heavy chain variable region VH of the antibody by polymerase chain reaction using the synthesized heavy chain variable region DNA of the antibody (base sequence SEQ ID NO: 1) as a template, using primers SfiVHBack (base sequence SEQ ID NO: 2) and VHFR2For (base sequence SEQ ID NO: 3), amplifying the genes of the CDRH2-HFR 3-HFR4 using degenerate primers CDRH2 liback (base sequence SEQ ID NO: 4) and CDRH3lib9For (base sequence SEQ ID NO: 5), performing agarose electrophoresis, confirming the amplified VH1 and VH2, cutting and purifying the two gene fragments, and performing overlap PCR using the primers SfiVHback (base sequence SEQ ID NO: 2) and XhoVHfor (base sequence SEQ ID NO: 6) as templates;
the PCR reactions above all used KOD-plus-neo (TOYOBO Bio), the reaction system was 50. Mu.L, the DNA template was 50ng, the concentration of the primer was 1. Mu.M, the reaction conditions were 94℃for 2 minutes, 55℃for 30 seconds, the elongation reaction of the DNA was run at 68℃for 1 minute, the total reaction was 30 cycles, agarose electrophoresis was performed, the amplified DNA fragment was confirmed, and the DNA fragment was excised and purified as a heavy chain variable region gene of a phage display antibody library, the base sequence of which was SEQ ID NO:53.
the synthesized antibody light chain DNA (SEQ ID NO: 7) was used as a template, primers SalVLback (SEQ ID NO: 8) and VLFR2For (SEQ ID NO: 9) were used to amplify the LFR1-CDRL1-LFR2 gene fragment VL1 of the antibody light chain VL by polymerase chain reaction, degenerate primers CDRL2Libback (SEQ ID NO: 10) and degenerate primers CDRL3 Libffor (SEQ ID NO: 11) were used to amplify the gene fragment VL2 of CDRL2-LFR3-CDRL3-LFR4, agarose electrophoresis was performed to confirm the amplified VL1, VL2, the two gene fragments were cut and purified, and overlapping PCR was performed using the primers SalVLback (SEQ ID NO: 8) and Notfor (SEQ ID NO: 12) as templates.
The PCR reactions were carried out using KOD-plus-neo (TOYOBO Bio), 50. Mu.L of the reaction system, 50ng of the DNA template, 1. Mu.M of the primer concentration, 94℃denaturation for 2 minutes, 55℃quenching for 30 seconds, and 1 minute of DNA elongation reaction at 68℃for 30 cycles. Agarose electrophoresis was performed to purify the PCR-amplified DNA fragments, which were used as light chain genes for phage display antibody libraries, whose base sequences were SEQ ID NOs: 54.
the light chain variable region gene was treated with 10 units of restriction enzymes SalI and NotI at 37℃for 3 hours, and cloned into phage display vector pDONG1 after purification. Amplifying the successfully cloned plasmid (containing the light chain variable region gene) and further treating with 10 units of SfiI and XhoI, and connecting the plasmid with the heavy chain variable region gene of the antibody treated with the same enzyme (SfiI and XhoI) at 16 ℃ by using T4 DNA ligase to transform escherichia coli TG-1;
culturing and transforming E.coli at 37 ℃ to OD from 25mL 600 To 0.5, helper phage KM13 was added, after 1 hour of infection at 37℃and centrifugation was performed for 30 minutes at 3000g, the supernatant was discarded, 50mL (1.6%Tryptone,1%Yeast Extract,0.5%NaCl) of the suspension was used in a 2YT medium containing 100. Mu.g/mL ampicillin, 50. Mu.g/mL kanamycin and 0.1% glucose, 30℃and 250rpm were used, shaking was performed for 16 hours, 5000g was performed for the next day, 30 minutes of the centrifugation medium was separated and 40mL of the supernatant was recovered, 10mL of PEG/NaCl solution was added to the supernatant, and after mixing, the mixture was left on ice for 30 minutes, 5000g and 30 minutes of centrifugation were performed, and 2mL of sterilized PBS solution was added to dissolve the pellet, thereby obtaining a phage display antibody library solution.
Amplified agarose electrophoresis diagram of the synthetic antibody library variable region gene is shown in figure 1, VL1 and VL2 are two fragments of the synthetic antibody library light chain variable region gene, and VL is the gene of the antibody library light chain variable region; VH1 and VH2 are two fragments of a synthetic antibody repertoire heavy chain variable region gene, VH is an antibody repertoire heavy chain variable region gene; lane M is DNA marker; the antibody variable region gene is successfully amplified, wherein the length of the heavy chain variable region gene is about 400bp, the length of the light chain variable region gene is about 370bp, the gene fragments are cloned to a phage display vector pDONG1, phage is prepared by using escherichia coli TG-1, and finally a phage containing about 10 is obtained 8 Phage display antibody library of species antibodies。
The obtained phage display antibody library comprises DNA sequences of the heavy chain variable region of the antibody, wherein the NNK codon is utilized to mutate the amino acid corresponding to the mutation position of the heavy chain variable region of the antibody into any amino acid; and mutating an amino acid corresponding to a mutation position of the antibody light chain variable region to an arbitrary amino acid, a DNA sequence corresponding to the obtained antibody light chain variable region; the antibody heavy chain variable region gene and the antibody light chain variable region gene are recombined with a phage vector to construct plasmids, and then the plasmids are transformed into escherichia coli, so that a phage display antibody library is constructed;
wherein the mutation position of the heavy chain variable region of the antibody is 50, 52a,52b,53, 55, 56, 58, 97, 98, 99, 100, 101, 102, 103, 104, 105; the mutation positions of the antibody light chain variable regions are 50, 51, 52, 53, 89, 90, 91, 92, 93, 94, 95, 96, 97;
wherein the DNA sequence of the antibody heavy chain variable region is SEQ ID NO:53;
the DNA sequence of the antibody light chain variable region is SEQ ID NO:54.
2. panning of phage display antibody libraries
100. Mu.L of PBS solution containing 10. Mu.g/ml SARS-CoV-2 virus S protein was added to 10 wells of a 96-well microplate, incubated overnight at 4℃and the antigen solution was discarded the next time, 200. Mu.L of PBS solution containing 2% nonfat milk powder was added to each well, incubated at 25℃for 2 hours for blocking, and after 3 washes with PBST, 100. Mu.L of phage solution (R0; 10 per well) was added to each well 9 cfu phage), for 2 hours at room temperature, washed with PBST, and 100 μl trypsin was added per well to elute phage bound to viral S protein;
culturing TG-1 Escherichia coli until OD600 is 0.4, taking 4mL of bacterial liquid, adding 500 mu L of dissolved phage solution into the bacterial liquid, infecting for 30 minutes at 37 ℃, centrifuging for 20 minutes at 5000g, discarding the supernatant, suspending the bacterial cells by using a 2YT culture medium containing 100 mu g/mL ampicillin, 50 mu g/mL kanamycin and 0.1% glucose, shaking at 30 ℃ for 16 hours at 250rpm, centrifuging the culture liquid for 5000g at the next day for 30 minutes, separating and recovering the supernatant, adding 1/5 volume of PEG/NaCl solution into the supernatant, standing for 30 minutes on ice for centrifugation for 5000g for 30 minutes after uniform mixing, discarding the supernatant, adding 200 mu L of sterilized PBS solution, and taking the supernatant as phage solution (R1) after the first enrichment; repeating the steps to obtain phage solutions R2 and R3 respectively; and performing an enzyme-linked immunosorbent assay to verify the binding specificity and binding performance of the phage display antibody library obtained in the panning process and the novel coronavirus SARS-CoV-2.
The enzyme-linked immunosorbent assay was performed as follows: adding 100 μl of PBS solution containing new coronavirus S protein solution (2 μg/mL) or bovine serum albumin BSA (2 μg/mL) to 96-well ELISA plate, standing overnight at 4deg.C, discarding antigen solution the next time, adding 200 μl of solution containing 2% skimmed milk powder, incubating at 25deg.C for 2 hours, blocking ELISA plate, washing ELISA plate 3 times with PBS solution containing 0.1% Tween 20, adding diluted R0, R1, R2 and R3 phage solutions (10 9 cfu/well) for 1 hour at 25℃and washing the ELISA plate with a PBST solution, adding an HRP-labeled mouse anti-M13 phage antibody, after 1 hour incubation, washing the plate with PBST, adding the HRP substrate TMBZ (formulated with a sodium acetate solution pH6.0, containing 30% H diluted 1/10000) 2 O 2 ) After color development, the absorbance at 450nm is measured by an enzyme-labeled instrument, a histogram is drawn, and the binding performance of the phage antibody obtained in each step, S protein and BSA is compared.
As shown in FIG. 2, the results of ELISA adsorption test show that the binding capacity of the phage solution R3 to S protein is significantly increased when the binding capacities of phage libraries R0, R1, R2 and R3 obtained in the phage panning process to S protein are compared, while the binding capacity to BSA is very weak and unchanged, indicating that antibodies against novel coronavirus S protein in the constructed phage display antibody library are enriched.
3. Monoclonal antibody screening
Culturing TG-1 escherichia coli until the OD600 is 0.4, taking 100 mu L of phage solution dissolved out from an R3 phage antibody library of a panning screen, using the phage solution to infect 200 mu L of escherichia coli bacterial liquid, incubating at 37 ℃ for 30 minutes, coating the bacterial liquid on a 2YT medium flat plate containing 100 mu g/mL ampicillin, 50 mu g/mL kanamycin and 1% glucose, culturing overnight at 37 ℃, inoculating 96 colonies on a 96-well culture plate the next day, culturing at 37 ℃ until the OD600 is 0.4, adding M13 phage into each well, centrifuging at 5000g for 20 minutes after infection, removing the supernatant, adding 200 mu L of 2YT medium containing 100 mu g/mL ampicillin, 50 mu g/mL kanamycin and 0.1% glucose into each well, suspending the bacterial cells, and culturing at 30 ℃ at 250rpm for 16 hours; the culture solution is centrifuged for 30 minutes at 5000g every day, the supernatant is separated and recovered, and an ELISA is performed to verify the binding specificity and binding performance of each monoclonal antibody and the novel coronavirus S protein.
The enzyme-linked immunosorbent assay was performed as follows: 100. Mu.L of PBS solution containing virus S protein (1. Mu.g/mL) was added to the 96-well ELISA plate, the antigen solution was discarded the next time at 4℃and 200. Mu.L of solution containing 2% skimmed milk powder was added, and the ELISA plate was blocked by incubation at 25℃for 2 hours. Washing the ELISA plate 3 times with PBS solution containing 0.1% Tween 20, adding phage solution, incubating at 25deg.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 at pH6.0, containing 30% H diluted 1/10000) 2 O 2 ) After color development, absorbance at 450nm was measured by an enzyme-labeled instrument, and binding properties of phage antibodies prepared by each clone with S protein and bovine serum albumin were compared.
According to the test results, 5 antibodies A9, E11, F5, F10, H9 and S proteins are combined, plasmids are extracted for gene sequencing, 5 antibody genes with different sequences are obtained, and the sequences identical with the antibody genes of the application are not found by comparison with antibody sequences registered in an antibody gene library, so the sequences are novel antibodies, and the amino acid sequences of the antibodies are as follows:
the variable region sequence of the heavy chain of the A9 antibody is SEQ ID NO:13, cdrh1 sequence is SEQ ID NO:14; CDRH2 sequence is SEQ ID NO:15; CDRH3 sequence is SEQ ID NO:16;
the variable region sequence of the light chain of the A9 antibody is SEQ ID NO:17, cdrl1 sequence is SEQ ID NO:18; CDRL2 sequence is SEQ ID NO:19; CDRL3 sequence is SEQ ID NO:20, a step of;
the variable region sequence of the heavy chain of the E11 antibody is SEQ ID NO:21, cdrh1 sequence is SEQ ID NO:22; CDRH2 sequence is SEQ ID NO:23; CDRH3 sequence is SEQ ID NO:24, a step of detecting the position of the base;
the variable region sequence of the E11 antibody light chain is SEQ ID NO:25, cdrl1 sequence is SEQ ID NO:26; CDRL2 sequence is SEQ ID NO:27; CDRL3 sequence is SEQ ID NO:28;
f5 antibody heavy chain variable region sequence is SEQ ID NO:29, cdrh1 sequence is SEQ ID NO:30; CDRH2 sequence is SEQ ID NO:31; CDRH3 sequence is SEQ ID NO:32;
f5 antibody light chain variable region sequence is SEQ ID NO:33, cdrl1 sequence is SEQ ID NO:34; CDRL2 sequence is SEQ ID NO:35; CDRL3 sequence is SEQ ID NO:36;
the variable region sequence of the F10 antibody heavy chain is SEQ ID NO:37, cdrh1 sequence is SEQ ID NO:38, a step of carrying out the process; CDRH2 sequence is SEQ ID NO:39; CDRH3 sequence is SEQ ID NO:40, a step of performing a;
the variable region sequence of the F10 antibody light chain is SEQ ID NO:41, cdrl1 sequence is SEQ ID NO:42; CDRL2 sequence is SEQ ID NO: 43. CDRL3 sequence is SEQ ID NO:44;
the H9 antibody heavy chain variable region sequence is SEQ ID NO:45, cdrh1 sequence is SEQ ID NO:46; CDRH2 sequence is SEQ ID NO:47; CDRH3 sequence is SEQ ID NO:48;
the variable region sequence of the H9 antibody light chain is SEQ ID NO:49, cdrl1 sequence is SEQ ID NO:50; CDRL2 sequence is SEQ ID NO:51; CDRL3 sequence is SEQ ID NO:52;
4. antigen specificity of monoclonal antibodies
Adding 100 mu L of novel coronavirus S protein, S-RBD protein and BSA with the concentration of 1 mu g/mL into a 96-well ELISA plate, carrying out overnight at 4 ℃, discarding protein solution the next time, adding 200 mu L of 2% skimmed milk powder solution, incubating for 2 hours at 25 ℃, and sealing the ELISA plate; washing the ELISA plate with PBS solution containing 0.1% Tween 20 3 times, adding 100 μl of diluted phage display antibody solution per well, incubating at 25deg.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 at pH6.0 containing 30% H diluted 1/10000) 2 O 2 ) After color development, the absorbance at 450nm is measured by an enzyme-labeled instrument, and a column is drawnThe figure compares the binding properties of phage antibodies made using each clone to the coating protein.
As shown in FIG. 3, the results of ELISA were shown, in which the antibodies A9 and E11 bound to the S protein and the S-RBD protein, and the antibodies F5, F10 and H9 bound to the S protein but were weak in binding to the RBD region, indicating that they bound mainly to other regions of the S protein. And none of the antibodies bound to the coated BSA, indicating that these antibodies did not bind to BSA, it was concluded that antibodies A9, E11, F5, F10, H9 bind specifically to S protein and S-RBD protein.
5. Detection of novel coronavirus S proteins Using A9 antibody Fab fragments with phage displayed F5, F10 or H9 antibodies
The detection principle is shown in figure 4, the A9 protein and F5, F10 and H9 displayed by phage are utilized to detect the S protein of the new coronavirus, the Fab fragment of the A9 antibody is coated in the hole of the 96-hole micro-pore plate, after the micro-pore plate is closed, the S protein or BSA protein is added, after the phage display F5 antibody is added, the anti-phage antibody marked with horseradish peroxidase is added, after the phage display is added, the substrate is added for color development, when the new coronavirus S protein is not present in the sample or the BSA protein is present in the sample, the reaction system does not develop color, the system develops color when the virus S protein is present in the sample, and the more the virus S protein in the sample is present, the more F5 phage of the ELISA plate is captured through the virus S1 protein, the more the corresponding captured anti-phage antibody is present, the color is darker after the color development of the substrate added with enzyme, the method can be used for judging whether the virus S1 protein exists in the sample or not exists in the sample.
The specific operation is as follows: a9 antibody Fab fragment was added to the wells of a 96-well ELISA plate, the wells were discarded overnight at 4℃and 200. Mu.L of a 2% nonfat milk powder solution was added thereto, and the plates were blocked after two hours at room temperature. After washing the plate, 100. Mu.L of a solution containing a novel coronavirus S protein or a Bovine Serum Albumin (BSA) solution was added to the wells, incubated at 25℃for 1 hour, the solution in the wells was removed, the ELISA plate was washed with a PBS solution (PBST solution) containing 0.1% Tween, and a phage-displayed F5 antibody solution (10 9 cfu/mL), incubation at 25℃for 1 hour,after washing the plate, adding an anti-phage antibody solution (1 mug/mL) marked with horseradish peroxidase (HRP), incubating for 1 hour at 25 ℃, washing the plate 3 times by using PBST after removing the solution in the hole, finally adding an HRP substrate 3, 5-tetramethyl biphenyl amine hydrochloride (TMBZ) solution for developing color, measuring the absorbance of the solution in the hole at 450nm, preparing a bar graph, and comparing the binding capacity of the antibody with the novel coronavirus S protein and bovine serum albumin.
The phage-displayed F5 antibody solution was changed to F10 or H9, and a histogram was prepared in the same manner as described above, and the binding ability of the antibodies to the novel coronavirus S protein and bovine serum albumin was compared.
As shown in fig. 5, the A9 antibody was combined with phage displayed F5, F10 or H9 to detect viral S protein, respectively, the horizontal axis indicated the name of the antibody combined with A9, and the vertical axis indicated the absorbance of the corresponding solution in the wells of the enzyme label. The absorbance of the wells containing the virus S protease label plate in the solution of the combination of A9 and F5 is 0.45, and the absorbance of the wells containing BSA is 0.03, which indicates that the combination of A9 and F5 can be used for detecting the novel coronavirus S protein in the solution, and the combination of A9 antibody, F10 and H9 obtains similar results, which indicates that the combination of A9 antibody, F5, F10 or H9 antibody can detect whether the novel coronavirus S protein and the novel coronavirus exist in the sample.
6. Detection of novel coronavirus S proteins Using E11 antibody Fab fragments with phage displayed F5, F10 or H9 antibodies
The detection principle is shown in FIG. 4, and the Fab fragment of the E11 antibody and F5, F10 and H9 displayed by phage are used for detecting the S protein of the novel coronavirus. Coating Fab fragments of E11 antibodies in the wells of a 96-well microplate, blocking the microplate, adding S protein, washing the plate, adding phage display F5 antibodies, adding anti-phage antibodies for marking horseradish peroxidase, washing the plate, and adding a substrate for color development. When the sample does not contain the novel coronavirus S protein or is BSA, the reaction system does not develop color; when the virus S protein exists, the system develops color, and the more the virus S protein in the sample is, the more F5 phage is captured by the virus S1 protein, the more the corresponding captured anti-phage antibodies are, and the darker the color is after the color development of the substrate added with the enzyme is. The method can be used for judging whether the sample has the S1 protein of the virus or not, and can also be used for detecting whether the sample contains SARS-CoV-2 virus or not.
The specific operation is as follows:
e11 antibody fragments were added to wells of a 96-well ELISA plate, the liquid in the wells was discarded the next time at 4℃overnight, 200. Mu.L of a 2% nonfat milk powder solution was added, and the plate was blocked by standing at room temperature for two hours. After washing the plate, 100. Mu.L of a solution containing a novel coronavirus S protein or a Bovine Serum Albumin (BSA) solution was added to the wells, incubated at 25℃for 1 hour, the solution in the wells was removed, the ELISA plate was washed with a PBS solution (PBST solution) containing 0.1% Tween, and a phage-displayed F5 antibody solution (10 9 cfu/mL), incubating for 1 hour at 25 ℃, adding an anti-phage antibody solution (1 mug/mL) marked with horseradish peroxidase (HRP) after washing the plate, incubating for 1 hour at 25 ℃, washing the plate 3 times by PBST after removing the solution in the hole, finally adding an HRP substrate 3, 5-tetramethyl biphenyl amine hydrochloride (TMBZ) solution for color development, measuring the absorbance of the solution in the hole at 450nm, preparing a bar graph, and comparing the binding capacity of the antibody with the novel coronavirus S protein and bovine serum albumin. The phage-displayed F5 antibody solution was changed to F10 or H9, and a histogram was prepared as described above, and the binding capacities of the antibodies to the novel coronavirus S protein and bovine serum albumin were compared.
FIG. 6 shows the results of detection of viral S proteins by the E11 antibody Fab fragments in combination with phage displayed F5, F10 and H9, respectively. The horizontal axis represents the antibody name combined with E11, and the vertical axis represents the absorbance for the solution in the wells of the enzyme label. The absorbance of the wells containing the virus S protease label plate in the solution of the combination of E11 and F5 is 0.36, and the absorbance of the wells with BSA added is 0.02, which indicates that the combination of E11 and F5 can be used for detecting the novel coronavirus S protein in the solution. Similarly, similar results were obtained with the combination of the E11 antibody fragment and F10 or H9, indicating that the combination of the E11 antibody and the F5, F10 or H9 antibody can be used to detect whether the sample contains the novel coronavirus S protein or the novel coronavirus.
Sequence listing
<110> Shandong Width and Zhengbio pharmaceutical Co., ltd
<120> monoclonal antibody H9 against novel coronavirus SARS-CoV-2
<130> 20210701A-2
<141> 2020-06-04
<160> 54
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
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gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcaaat attcatgcga gtggtatgcg tacatcgtac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtga gaaaagtagt 300
ggtacgtttg acttctgggg ccagggaacc ctggtcaccg tctcgagc 348
<210> 2
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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tcgcggccca gccggccatg gccgaggtgc agctgttgga 40
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tgagacccac tccagcccct tccct 25
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<213> Artificial sequence (Artificial Sequence)
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tggagtgggt ctcannkatt nnknnknnkg gtnnknnkac annktacgct gactccgt 58
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ttccctggcc ccamnnmnnm nnmnnmnnmn nmnnmnnmnn acagtaatat acggccgt 58
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<212> DNA
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tgagctcgag acggtgacca gggttccctg gcccca 36
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gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatctt ggatgccatt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtggacag gctggaggac ggggattgag cttcggccaa 300
gggaccaagg tggaaatcaa acgg 324
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gcctgcggcc gcccgtttga tttccacctt ggtcccttgg ccga 44
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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 Gly Ile Thr Asn Ser Gly Ser Ser 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
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Ala Lys Gly Thr Asp Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
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Gly Phe Thr Phe Ser Ser Tyr Ala
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<211> 8
<212> PRT
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Ile Thr Asn Ser Gly Ser Ser Thr
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<210> 16
<211> 9
<212> PRT
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Ala Lys Gly Thr Asp Ala Phe Asp Tyr
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<210> 17
<211> 108
<212> PRT
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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 Ala Ala Ser Thr 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
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Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Cys Ser Pro Ala
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Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
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<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
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Gln Ser Ile Ser Ser Tyr
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<210> 19
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Ala Ala Ser
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<210> 20
<211> 9
<212> PRT
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Gln Gln Ser Asn Cys Ser Pro Ala Thr
1 5
<210> 21
<211> 116
<212> PRT
<213> Artificial sequence (Artificial Sequence)
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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 Ser Ile Asp Ser Ser Gly Tyr Tyr 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 Asn Ser Asp Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 22
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
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Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 23
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 23
Ile Asp Ser Ser Gly Tyr Tyr Thr
1 5
<210> 24
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Ala Lys Asn Ser Asp Ser Phe Asp Tyr
1 5
<210> 25
<211> 108
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 25
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 Ser Ala Ser Tyr 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 Tyr Asp Asp Gly Pro Asn
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 26
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 26
Gln Ser Ile Ser Ser Tyr
1 5
<210> 27
<211> 3
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 27
Ser Ala Ser
1
<210> 28
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 28
Gln Gln Tyr Asp Asp Gly Pro Asn Thr
1 5
<210> 29
<211> 116
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 29
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 Thr Ile Asp Ser Ala Gly Asn Ser Thr Thr 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 Asn Asp Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 30
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 30
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 31
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 31
Ile Asp Ser Ala Gly Asn Ser Thr
1 5
<210> 32
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 32
Ala Lys Asn Asp Ser Ser Phe Asp Tyr
1 5
<210> 33
<211> 108
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 33
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 Ser Ala Ser Trp 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 Tyr Ser Asp Gly Pro Asp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 34
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 34
Gln Ser Ile Ser Ser Tyr
1 5
<210> 35
<211> 3
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 35
Ser Ala Ser
1
<210> 36
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 36
Gln Gln Tyr Ser Asp Gly Pro Asp Thr
1 5
<210> 37
<211> 116
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 37
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 Gly Ile Asp Ser Ala Gly Tyr Tyr Thr Thr 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 Asp Ser Asp Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 38
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 38
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 39
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 39
Ile Asp Ser Ala Gly Tyr Tyr Thr
1 5
<210> 40
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 40
Ala Lys Asp Ser Asp Thr Phe Asp Tyr
1 5
<210> 41
<211> 108
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 41
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 Ala Ala Ser Tyr 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 Tyr Ser Thr Pro Ala
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 42
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 42
Gln Ser Ile Ser Ser Tyr
1 5
<210> 43
<211> 3
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 43
Ala Ala Ser
1
<210> 44
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 44
Gln Gln Ala Tyr Ser Thr Pro Ala Thr
1 5
<210> 45
<211> 116
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 45
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 Asp Ile Thr Asp Asn Gly Ala Ser Thr Gly 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 Thr Asn Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 46
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 46
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 47
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 47
Ile Thr Asp Asn Gly Ala Ser Thr
1 5
<210> 48
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 48
Ala Lys Ser Thr Asn Thr Phe Asp Tyr
1 5
<210> 49
<211> 108
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 49
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
Ile Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Ser 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 Asn Ser Asn Ser Pro Ser
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 50
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 50
Gln Ser Ile Ser Ser Tyr
1 5
<210> 51
<211> 3
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 51
Asp Ala Ser
1
<210> 52
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 52
Gln Gln Asn Ser Asn Ser Pro Ser Thr
1 5
<210> 53
<211> 348
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcannk attnnknnkn nkggtnnknn kacannktac 180
gctgactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtnn knnknnknnk 300
nnknnknnkn nknnktgggg ccagggaacc ctggtcaccg tctcgagc 348
<210> 54
<211> 324
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatnnk nnknnknnkt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtnnknnk nnknnknnkn nknnknnknn kttcggccaa 300
gggaccaagg tggaaatcaa acgg 324

Claims (4)

1. A monoclonal antibody H9 against the novel coronavirus SARS-CoV-2, characterized in that:
the amino acid sequence of the H9 antibody heavy chain variable region is SEQ ID NO:45, cdrh1 amino acid sequence is SEQ ID NO:46; CDRH2 amino acid sequence is SEQ ID NO:47; CDRH3 amino acid sequence is SEQ ID NO:48;
the amino acid sequence of the H9 antibody light chain variable region is SEQ ID NO:49, cdrl1 amino acid sequence is SEQ ID NO:50; the amino acid sequence of CDRL2 is SEQ ID NO:51; the amino acid sequence of CDRL3 is SEQ ID NO:52.
2. use of the monoclonal antibody H9 against the novel coronavirus SARS-CoV-2 according to claim 1 for the preparation of a novel coronavirus SARS-CoV-2 detection product.
3. The use of the monoclonal antibody H9 against the novel coronavirus SARS-CoV-2 according to claim 1 for the preparation of a medicament for inhibiting the novel coronavirus SARS-CoV-2 and for the preparation of a pharmaceutical preparation for preventing or treating diseases caused by the novel coronavirus SARS-CoV-2.
4. An antigen-binding fragment of monoclonal antibody H9 to the novel coronavirus SARS-CoV-2 as claimed in claim 1, selected from Fab, fab '-SH, fv, scFv or (Fab') 2 fragments.
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