Monoclonal antibody of anti-SARS-CoV-2 nucleocapsid protein and application

A monoclonal antibody, nucleocapsid protein technology, applied in anti-viral immunoglobulin, application, immunoglobulin and other directions, can solve the problem of not actually preparing antibody, not providing antibody, etc., to achieve strong affinity, high sensitivity and stability good effect

Active Publication Date: 2021-01-15
ZHEJIANG MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Monoclonal antibody of anti-SARS-CoV-2 nucleocapsid protein and application
  • Monoclonal antibody of anti-SARS-CoV-2 nucleocapsid protein and application
  • Monoclonal antibody of anti-SARS-CoV-2 nucleocapsid protein and application

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Experimental program
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Embodiment 1

[0024] Embodiment 1: Preparation of SARS-CoV-2-N recombinant protein

[0025] The N protein gene sequence is shown in SEQ ID NO: 1, and the designed primers are as follows:

[0026] N-F: 5'-GCCGGATCCATGTCTGATAATGGACCCCAAAA-3' (with BamHI restriction site);

[0027] N-R: 5'-GCCGTCGACAGGCCTGAGTTGAGTCAGCAC-3' (with SalI restriction site),

[0028] Using the above primers, PCR was carried out using the artificially synthesized N protein gene as a template, and the product was double-digested with BamHI and SalI restriction endonucleases, and then inserted into pET-28a(+) prokaryotic expression treated with the same two restriction enzymes Vector, the recombinant plasmid pET28a-SARS-CoV-2-N inserted with the N protein gene sequence was obtained.

[0029] Transform the correct pET28a(+)-SARS-CoV-2-N expression vector plasmid verified by sequencing into Escherichia coli BL21, spread it on an LB plate containing 100 μg / ml kanamycin sulfate, culture overnight at 37°C, and pick Take ...

Embodiment 2

[0032]Embodiment 2: Preparation of SARS-CoV-2-N monoclonal antibody

[0033] Using the recombinant antigen SARS-CoV-2-N protein as the immunogen, for the first immunization, emulsify 50-100 μg / one of the recombinant antigen SARS-CoV-2-N protein with an equal volume of complete Freund’s adjuvant, and put it in BALB / c Mice were immunized by subcutaneous injection at multiple points on the back. For the second and third times, 50-100 μg / only recombinant antigen SARS-CoV-2-N protein was emulsified with an equal volume of incomplete Freund's adjuvant, and the immunization interval was 15 days. After three times of immunization, blood was collected from the tail vein of the mice, the serum was separated and the titer of the mouse serum anti-recombinant antigen SARS-CoV-2-N protein was detected by indirect ELISA.

[0034] 7 days before cell fusion, mice were intraperitoneally injected with 50-100 μg / only recombinant antigen SARS-CoV-2-N protein for booster immunization. The feeder...

Embodiment 3

[0036] Embodiment 3: Indirect method ELISA method detects monoclonal antibody titer

[0037] The SARS-CoV-2-N protein purified in Example 1 was coated with a microtiter plate at 1 μg / ml, 100 μl / well, and incubated overnight at 4°C. Wash the ELISA plate with PBST, add 200 μl / well of 5% skimmed milk powder, and block at 37°C for 2 hours. After the plate was washed, the monoclonal antibody in Example 2 was added at a doubling dilution concentration of 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1 :64000, 1:128000, 1:256000, 1:512000, 1:1024000, 100μl / well, and incubated at 37°C for 1h. After washing the plate, horseradish peroxidase-labeled goat anti-mouse antibody at appropriate dilution was added and incubated at 37°C for 1 hour. Add 100 μl / well of TMB chromogenic substrate to react for 10 min, add 50 μl 2mol / L sulfuric acid stop solution to terminate the reaction, and read the OD value of each well at 450 nm with a microplate reader. Test results such as Figu...

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Abstract

The invention discloses a monoclonal antibody of an anti-SARS-CoV-2 nucleocapsid protein and an application. The monoclonal antibody comprises a light chain and a heavy chain, and is characterized inthat the amino acid sequences of variable regions CDR1, CDR2 and CDR3 of the light chain are respectively RASENIYSSLA, VATDLA and QHFWGTPWT; and the amino acid sequences of the variable regions CDR1,CDR2 and CDR3 of the heavy chain are respectively TDHYII, EIYPGNGHTYYNERFKG and SRYYGPFAYWG. The monoclonal antibody of the anti-SARS-CoV-2 nucleocapsid protein is an IgG1 subtype, the affinity of theantibody can reach 5.26 * 10 <11>, the immunocompetence is strong, the monoclonal antibody has the advantages of good stability, high activity, strong affinity and the like, the detection specificityis strong, the sensitivity is high, the accuracy is good, and the monoclonal antibody can be used in a kit of a full-automatic immunoassay analyzer. The kit is also applied to detection of the SARS-CoV-2 nucleocapsid protein by an immunochromatographic test strip, and can effectively promote popularization and application of SARS-CoV-2 detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a monoclonal antibody against SARS-CoV-2 nucleocapsid protein and its application. Background technique [0002] Novel coronavirus pneumonia is a novel respiratory disease caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). SARS-CoV-2 is the seventh enveloped positive-strand RNA virus capable of infecting humans. Genomic identification of SARS-CoV-2 showed that it is a binuclear virus with 96% homology to bat coronavirus RaTG13, but different from SARS-CoV. SARS-CoV-2 has a receptor-binding domain (RBD) structure similar to SARS-CoV. The functional open reading frame (ORF) of SARS-CoV-2 includes ORF1a and ORF1b, and the main structural proteins include spike protein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein (N ). According to existing research, M and E proteins are necessary for virus assembly. The S protein is associated w...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13G01N33/577G01N33/569
CPCC07K16/10G01N33/577G01N33/56983C07K2317/565C07K2317/56C07K2317/94C07K2317/92G01N2333/165G01N2469/10
Inventor 陆绍红孔庆明丁豪杰丁建祖高孟卓洵辉谢承作袁亚杰
Owner ZHEJIANG MEDICAL COLLEGE
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