Monoclonal antibody specific to omicron strain of novel coronavirus and application thereof

Abstract

The application relates to the field of immunology, and particularly relates to a monoclonal antibody specific to a novel coronavirus of an Omicron strain and application thereof. The monoclonal antibody is prepared by immunizing a mouse with a purified inactivated novel coronavirus of the Omicron strain, wherein three CDR regions of a heavy chain variable region are as follows: a CDR1 sequence is GYTFTDYS; a CDR2 sequence is INTETGEP; and a CDR3 sequence is ARGHYYGYGGY; three CDR regions of a light chain variable region are as follows: a CDR1 sequence is QSVDSDGDSF; a CDR2 sequence is AAS; and a CDR3 sequence is QQSNEDPYT. The monoclonal antibody can specifically bind to the novel coronavirus of the Omicron strain, and cannot bind to a prototype strain, a variant strain A, a variant strain B and other novel coronaviruses, is an ideal antigen detection antibody for the novel coronavirus of the Omicron strain, and is beneficial to accelerating the research and development process of a new generation of vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology, and in particular to a monoclonal antibody specific to the Omicron strain of the novel coronavirus and an application thereof. BACKGROUND

[0002] The Omicron mutant strain is a variant of the novel coronavirus in 2019, and the spike protein of the Omicron mutant strain is characterized by at least 30 amino acid changes, 3 small deletions and 1 small insertion, and among the 30 amino acid changes, 15 are located in the receptor binding domain (RBD), which has a significantly higher transmission and immune evasion capacity than the Delta mutant strain, and can evade many current vaccines against the novel coronavirus.

[0003] At present, many biological companies have carried out research and development of monovalent vaccines and multivalent vaccines against the Omicron mutant strain.

[0004] In order to promote the research and development of monovalent vaccines and multivalent vaccines against the Omicron mutant strain, convenient and efficient vaccine development tools play an important role, and at present, the antigen detection of the novel coronavirus vaccine is a general antibody, which cannot specifically detect the Omicron strain of the novel coronavirus antigen. SUMMARY

[0005] Based on the above analysis, the present application uses the Omicron strain of the novel coronavirus inactivated vaccine stock solution as an immunogen to immunize Balb / C mice, and through fusion and screening and three subcloning, a hybridoma cell strain capable of secreting a monoclonal antibody is obtained, and further experiments verify that the monoclonal antibody can specifically recognize the Omicron strain of the novel coronavirus antigen.

[0006] One of the purposes of the present application is to provide a monoclonal antibody specific to the Omicron strain of the novel coronavirus, wherein the three CDR regions of the heavy chain variable region of the monoclonal antibody are: the CDR1 sequence is GYTFTDYS;

[0007] the CDR2 sequence is INTETGEP;

[0008] and the CDR3 sequence is ARGHYYGYGGY.

[0009] The three CDR regions of the light chain variable region of the monoclonal antibody are: the CDR1 sequence is QSVDSDGDSF;

[0010] the CDR2 sequence is AAS;

[0011] and the CDR3 sequence is QQSNEDPYT.

[0012] Further, the heavy chain variable region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 1; and the light chain variable region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 2.

[0013] Further, the monoclonal antibody is an IgG2b type antibody.

[0014] The second object of the present application is to provide a nucleic acid molecule encoding the above monoclonal antibody.

[0015] Further, the nucleic acid molecule encoding the heavy chain is any one of the following:

[0016] a. has a nucleotide sequence as shown in SEQ ID NO: 3;

[0017] b. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 3;

[0018] c. a nucleotide sequence different from the nucleotide sequences of a and b, but encoding the same protein due to the degeneracy of the genetic code;

[0019] and / or

[0020] The nucleic acid molecule encoding the light chain is any one of the following:

[0021] e. has a nucleotide sequence as shown in SEQ ID NO: 4;

[0022] f. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 4;

[0023] g. a nucleotide sequence different from the nucleotide sequences of e and f, but encoding the same protein due to the degeneracy of the genetic code.

[0024] The third object of the present application is to provide an expression vector containing the nucleic acid molecule encoding the above monoclonal antibody.

[0025] The fourth object of the present application is to provide a host cell containing the above nucleic acid molecule, or containing the above expression vector.

[0026] The fifth object of the present application is to provide an antibody conjugate comprising the above monoclonal antibody or antigen binding fragment thereof and a label selected from one or more of an enzyme label, a biotin label, a chemiluminescent dye label.

[0027] The sixth objective of this invention is to provide the application of the above-mentioned antibody-drug conjugate in the identification of novel coronavirus vaccines, the quality control of novel coronavirus vaccines, the preparation of novel coronavirus detection reagents, or the preparation of anti-novel coronavirus drugs.

[0028] Specifically, in the identification of novel coronavirus vaccines, this monoclonal antibody is used to determine whether a novel coronavirus vaccine contains the Omicron strain of novel coronavirus antigen and its content level, or to determine the authenticity of a novel coronavirus vaccine, i.e., whether the vaccine is specifically designed to target the Omicron strain of novel coronavirus. Identification methods can include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, and other detection methods.

[0029] In the quality control of novel coronavirus vaccines, this monoclonal antibody is specifically used to detect whether the quality, content, and stability of the antigens in the novel coronavirus vaccine meet the requirements. It can be used as a coating antibody for antigen detection methods to detect antigen quality, content, and stability.

[0030] The novel coronavirus test kit contains the antibody and a detectable marker, and may also include a second antibody carrying the detectable marker to detect the monoclonal antibody of the present invention.

[0031] Anti-novel coronavirus drugs are drugs for the prevention and / or treatment of diseases caused by the novel coronavirus, and these drugs also include medically acceptable excipients.

[0032] The seventh objective of this invention is to provide a diagnostic kit for diagnosing the Omicron strain of novel coronavirus, the kit comprising the aforementioned monoclonal antibody or antibody-drug conjugate.

[0033] The monoclonal antibody provided by this invention binds only to the Omicron strain of the novel coronavirus, without binding to the antigens of the original strain, variant A, or variant B. It is an ideal antibody for detecting the Omicron strain of the novel coronavirus antigen and can be used for the detection and identification of Omicron strain of the novel coronavirus vaccine or multivalent vaccines. It can also quantitatively detect the antigen content and serve as a convenient and efficient vaccine development tool, which is conducive to accelerating the research and development process of next-generation vaccines. Attached Figure Description

[0034] Figure 1 The above are the SDS-PAGE electrophoresis results of monoclonal antibody 1G8 in Example 1 of this invention, wherein lane 1 is for non-reducing SDS-PAGE electrophoresis and lane 2 is for reducing SDS-PAGE electrophoresis.

[0035] Figure 2 This is the ELISA specific binding analysis result of monoclonal antibody 1G8 in Example 2 of the present invention;

[0036] Figure 3 The results of antigen detection analysis are based on the antigen detection kit established using monoclonal antibody 1G8 in Example 3 of this invention. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0038] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

[0039] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0040] The "monoclonal antibody" mentioned in this invention refers to a preparation of an antibody molecule having a single molecular composition.

[0041] The “CDR region” or “CDR” mentioned in this invention refers to the hypervariable regions of the heavy and light chains of immunoglobulins, which contain most of the amino acid residues responsible for binding to antigens or their recognition epitopes through the affinity of antibodies.

[0042] The Omicron strain of novel coronavirus, the prototype strain, variant A, and variant B are all publicly disclosed strains in the existing technology and are all recorded in the NCBI database. The accession number for the Omicron strain of novel coronavirus is EPI_ISL_7138045, the accession number for the prototype strain is MN996528, the accession number for variant A is EPI_ISL_2536954, and the accession number for variant B is EPI_ISL_13858924. All were isolated from patients with COVID-19. After successful isolation of the original samples, the strains were passaged and continuously transferred using Vero cells. Purification was performed using the limiting dilution method, and further expanded culture in Vero cells yielded the final product.

[0043] Example 1: Preparation and Analysis of Monoclonal Antibodies

[0044] This invention provides a method for the preparation and analysis of a monoclonal antibody specific to the Omicron strain of the novel coronavirus, as detailed below:

[0045] (1) Immunization: Mice were immunized with purified inactivated Omicron strain novel coronavirus antigen using Freund's adjuvant. After four intraperitoneal immunizations at 14-day intervals, serum antibody titers were measured by blood tests. Before fusion, a tail vein pulse immunization was performed, followed by fusion of splenic lymphocytes and myeloma cells.

[0046] (2) Screening: The supernatant of fusion cells was screened using ELISA to select cells that specifically bound the Omicron strain of the novel coronavirus, but did not bind to the antigens of the prototype strain, variant A, or variant B. Specifically:

[0047] The original strain, variant A, variant B, and Omicron whole virus inactivated antigen were diluted with 0.05 M carbonate buffer and coated with 96-well microplates at a concentration of 2 μg / mL. The plates were incubated overnight at 4 °C and then blocked at 37 °C for 1 h with blocking buffer (PBST-1% BSA).

[0048] Add 100 μL of the test sample to each well and incubate at 37 ℃ for 1 h. Add 100 μL of HRP-labeled goat anti-mouse IgG (1:20000 dilution, purchased from Wuhan Boster Biological Engineering Co., Ltd.) to each well and incubate at 37 ℃ for 1 h. After washing the plate, add 50 μL of substrate solutions A and B (both purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) to each well, and incubate at 37 ℃ in the dark for 15 min. Add 50 μL of stop solution to each well and read the values ​​at 450 nm using a microplate reader. 450nm value.

[0049] (3) Subcloning: The positive maternal clone was subcloned using the limiting dilution method, and the subcloning was screened using the ELISA method.

[0050] (4) Hybridoma cell antibody gene sequencing, the results are as follows:

[0051] The amino acid sequences of the three CDR regions of the heavy chain variable region, CDR1, CDR2, and CDR3, are shown below:

[0052] The sequence information for CDR1 is: GYTFTDYS;

[0053] The sequence information for CDR2 is: INTETGEP;

[0054] The sequence information for CDR3 is: ARGHYYGYGGY.

[0055] The amino acid sequences of the three CDR regions of the light chain variable region, namely CDR1, CDR2, and CDR3, are shown below:

[0056] The sequence information for CDR1 is: QSVDSDGDSF;

[0057] The sequence information for CDR2 is: AAS;

[0058] The sequence information for CDR3 is: QQSNEDPYT.

[0059] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:3.

[0060] The sequence information of SEQ ID NO:1 is: QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWINTETGEPIYADDFKGRVAFSLETSASTAYLQINNPKNEDTATYFCARGHYYGYGGYWGQGTLVTVSA;

[0061] The sequence information of SEQ ID NO:3 is: CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCA AGGCTTCTGGTTATAACCTTCACAGACTACTCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTT AAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAATATATGCAGATGACTTCAAGGGACGG GTCGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCCCAAAAATGAGGACACGGCTACATATTTCTGTGCTAGGGGTCATTACTACGGCTACGTGGTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA;

[0062] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:2, and the nucleotide sequence is shown in SEQ ID NO:4.

[0063] The sequence information of SEQ ID NO:2 is: DIVLTQSPASLAVSLGQRATISCKASQSVDSDGDSFLNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK;

[0064] SEQ ID The sequence information of NO:4 is: GATATCGTGCTCACTCAGTCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTCTGATGGTGATAGTTTTTTGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGC TGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAAATAAAA;

[0065] (5) Antibody production and purification: Female BALB / c mice aged 6-8 weeks were selected and injected intraperitoneally with 0.5 ml of liquid paraffin per mouse. Seven to ten days after the liquid paraffin injection, hybridoma cells were injected intraperitoneally. Mice were euthanized by cervical dislocation when their abdomens were distended and they were near death. Ascites fluid was aseptically collected in a clean bench. Protein A affinity chromatography was used to purify the monoclonal antibody in the ascites fluid. Antibody purity was analyzed by SDS-PAGE, and the monoclonal antibody subtype was identified using a mouse antibody subtype detection kit.

[0066] Results analysis: SDS-PAGE results are shown below. Figure 1 The results showed that under non-reducing conditions, the monoclonal antibody 1G8 exhibited a band with a molecular weight between approximately 130-250 kDa; under reducing conditions, it showed two bands with molecular weights of approximately 50 kDa and 25 kDa, corresponding to the heavy and light chains of the antibody, respectively. The isotype identification results for monoclonal antibody 1G8 showed that the isotype was IgG2b, and it was named 1G8.

[0067] Example 2: Specificity analysis of monoclonal 1G8

[0068] The monoclonal antibody 1G8 obtained in Example 1 was specifically analyzed using an ELISA method, as follows:

[0069] (1) Dilute the original strain, mutant strain A, mutant strain B and Omicron whole virus inactivated antigen with 0.05 M carbonate buffer, coat 96-well microplates with a coating concentration of 2 μg / ml, and incubate overnight at 4 ℃.

[0070] (2) Block with blocking buffer (PBST-1% BSA) at 37 °C for 1 h; add serially diluted 1G8 antibody and incubate at 37 °C for 1 h;

[0071] (3) Add 100 μL / well of HRP-labeled goat anti-mouse IgG (1:20000 dilution, purchased from Wuhan Boster Biological Engineering Co., Ltd.) and incubate at 37 ℃ for 1 h. After washing the plate, add substrate solutions A and B (both purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd.), 50 μL / well each, and develop color at 37 ℃ in the dark for 15 min; add stop solution, 50 μL / well;

[0072] (4) Place it in an ELISA reader and read A at a wavelength of 450 nm. 450 nm value.

[0073] Results: The binding activity of 1G8 antibody with the prototype strain, variant A, variant B, and Omicron whole virus antigen is shown in the results below. Figure 2 It can be seen that monoclonal antibody 1G8 only reacts with the novel coronavirus antigen of Omicron strain, and does not react with the novel coronavirus antigen of the prototype strain, variant A, and variant B.

[0074] Example 3: Application of Monoclonal 1G8

[0075] This embodiment provides a method for detecting the novel coronavirus using a double-antibody sandwich ELISA, as detailed below:

[0076] (1) The purified SARS-CoV-2 rabbit polyclonal antibody (obtained by immunizing Japanese white rabbits with inactivated Omicron strain of novel coronavirus as antigen under conventional methods) was diluted to 1:1000 with 0.05 mol / L carbonate buffer (pH 9.6), coated microplates, 100 μL / well, and incubated overnight at 4 ℃.

[0077] (2) After washing the plate 3 times with PBST, add blocking buffer (PBST containing 1% BSA), 200 μL / well, and block at 37 ℃ for 1 h;

[0078] (3) Discard the blocking solution and dry at 37 °C for 2 h to prepare coated microplates;

[0079] (4) Add serially diluted prototype strain, mutant strain A, mutant strain B and Omicron whole virus antigen, 100 μL / well, and incubate at 37 ℃ for 1 h; wash the plate 5 times, add 1G 8-HRP (1:1000 dilution), 100 μL / well, and incubate at 37 ℃ for 1 h; add substrate solutions A and B (both purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd.), 50 μL / well, and develop color at 37 ℃ in the dark for 15 min; add stop solution, 50 μL / well.

[0080] (5) Place it in an ELISA reader and read A at a wavelength of 450 nm. 450 nm value.

[0081] Results: The results of the double-antibody sandwich ELISA detection of the novel coronavirus are shown in the figure. Figure 3 It can be seen that monoclonal antibody 1G8 can be used to detect and identify Omicron strain novel coronavirus vaccines or multivalent vaccines using a double antibody sandwich ELISA, and to quantitatively detect the antigen content.

[0082] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A monoclonal antibody specific to the Omicron strain of novel coronavirus, characterized in that, The three CDR regions of the heavy chain variable region of the monoclonal antibody: CDR1 sequence is GYTFTDYS; The CDR2 sequence is INTETGEP; The CDR3 sequence is ARGHYYGYGGY; The monoclonal antibody has three CDR regions in its light chain variable region: the CDR1 sequence is QSVDSDGDSF; The CDR2 sequence is AAS; The CDR3 sequence is QQSNEDPYT.

2. The novel coronavirus-specific monoclonal antibody of Omicron strain according to claim 1, characterized in that, The heavy chain variable region of the monoclonal antibody contains an amino acid sequence as shown in SEQ ID NO:1; The light chain variable region of the monoclonal antibody contains an amino acid sequence as shown in SEQ ID NO:

2.

3. The novel coronavirus-specific monoclonal antibody of Omicron strain according to claim 1 or 2, characterized in that, The monoclonal antibody is an IgG2b type antibody.

4. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleic acid molecule encoding the heavy chain is any one of the following: a. Has a nucleotide sequence as shown in SEQ ID NO:3; b. A complementary nucleotide sequence to the nucleotide sequence shown in SEQ ID NO:3; c. A nucleotide sequence that encodes the same protein as a and b, but is different from them due to the degeneracy of the genetic code; and / or The nucleic acid molecule encoding the light chain is any one of the following: e. Has a nucleotide sequence as shown in SEQ ID NO:4; f. A nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO:4; g and e encode the same protein as the nucleotide sequences of f, but are different nucleotide sequences due to the degeneracy of the genetic code.

6. An expression carrier, characterized in that, A nucleic acid molecule containing the encoding of any one of the monoclonal antibodies according to claims 1 to 3.

7. A host cell, characterized in that, It contains the nucleic acid molecule as described in claim 4 or 5, or the expression vector as described in claim 6.

8. The use of the monoclonal antibody according to any one of claims 1 to 3 in the identification of Omicron strain novel coronavirus vaccine or in the quality control or preparation of Omicron strain novel coronavirus vaccine for the detection of Omicron strain novel coronavirus.

9. A diagnostic kit for diagnosing the Omicron strain of novel coronavirus, characterized in that, It includes the monoclonal antibody according to any one of claims 1 to 3.

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