Monoclonal antibody for detecting SARS-CoV-2 virus nucleocapsid protein (N protein) and application thereof

A monoclonal antibody, sars-cov-2 technology, applied in the field of biomedicine, can solve the problems of high environmental, transportation, operation requirements, weak sensitivity, etc.

Pending Publication Date: 2020-12-15
BEIJING PROTEIN INNOVATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Nucleic acid detection is the etiological "gold standard" for detection of viral infection, but this detection method has high requirements on the environment,

Method used

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  • Monoclonal antibody for detecting SARS-CoV-2 virus nucleocapsid protein (N protein) and application thereof
  • Monoclonal antibody for detecting SARS-CoV-2 virus nucleocapsid protein (N protein) and application thereof
  • Monoclonal antibody for detecting SARS-CoV-2 virus nucleocapsid protein (N protein) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 Recombinant SARS-Cov-2 virus N protein immunogen preparation

[0038] The complete open reading frame region was synthesized with reference to the nucleotide sequence encoding the N protein in the novel coronavirus genome sequence numbered MN908947.3 in GenBank, and cloned into the plasmid vector pUC57 (Nanjing KingScript Biotechnology Co., Ltd. company), designed the upstream primer n-Cov-NF: 5'- GGATCGAATTCGATGTCTGATAATG-3' and the downstream primer n-Cov-NR: 5'-ACGCTCGAGTTAGTGGTGGTGGTGGTGGTGGGCCTGAGTTGA GTCAGC-3', introduced the EcoRI cutting point through the upstream primer, and introduced the group through the downstream primer Amino acid purification tag and XhoI restriction site, primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. According to the standard nucleic acid amplification process, the target gene was amplified from the plasmid vector, and cloned into the eukaryotic expression vector pcDNA3.4 (purchased from Invitrogen ...

Embodiment 2

[0039] Example 2 Establishment of Hybridoma Cell Lines and Antibody Screening

[0040] 1. Animal immunity

[0041] The recombinant N protein in Example 1 was inactivated at 60°C and emulsified with complete Freund's adjuvant (purchased from Sigma), and six 4-6-week-old female Balb / c mice (purchased from Beijing Weitongli Co., Ltd.) were immunized. China Experimental Animal Technology Co., Ltd.), serially numbered 64360#A, 64360#B, 64360#C, 64360#D, 64360#E and 64360#F. The dose of each mouse was injected subcutaneously in the abdomen at a dose of 60 μg / only. Immunization was boosted every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Company) at a dose of 30 μg per mouse. 7 days after the third booster immunization, indirect ELISA (wavelength 450nm) was used to detect the polyantibody titer of the anti-immunogen in the mouse serum. The mouse with the highest titer was injected into the tail vein for shock immunization. 50μg / only.

[0042]...

Embodiment 3

[0051] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method

[0052] Wash and suspend the cells in the logarithmic growth phase with serum-free medium, and count about 5×10 5 , 1ml of suspended cells were injected intraperitoneally into mice sensitized with paraffin oil in advance. Ascites collection was started 7 days later. The removed ascites was centrifuged at 4000rpm at 4°C, and the ascites in the middle was carefully sucked out for 10 minutes and collected in a centrifuge tube for purification. Purification of the antibody was carried out according to the instructions of HiTrap rProtein AFF (purchased from GE, catalog number 17-5079-02).

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Abstract

The invention discloses a monoclonal antibody 64360-52D1 capable of specifically combining with SARS-Cov-2 virus structural nucleocapsid protein (N protein), a preparation method thereof, and 6 complementarity-determining regions (CDR) of heavy chain and light chain variable regions; and more specifically, the monoclonal antibody is secreted by a hybridoma strain 64360#52D1, and can specifically recognize the SARS-Cov-2 virus N protein rather than SARS virus N protein. Thus, the monoclonal antibody can be used for identifying the two coronaviruses with high similarity. The invention further provides an enzyme-linked immunosorbent assay (ELISA) and an immune colloidal gold test strip detection method for specifically detecting the SARS-Cov-2 virus N protein by preparing the 64360#52D1 antibody. The antigen of the antibody is SARS-Cov-2 virus N protein subjected to heat treatment and expressed in mammalian cells; the finally obtained antibody belongs to an IgG1 subtype; and a sequence for encoding the variable region of the antibody is obtained in a gene cloning mode.

Description

technical field [0001] The present invention relates to the field of biomedicine. More specifically, the present invention relates to a monoclonal antibody that can specifically recognize the N protein of SARS-CoV-2 virus and its application in the detection of new coronavirus, the antibody of the present invention can be used in the screening of new coronavirus infection, It has important application value in detection and prognosis evaluation. Background technique [0002] SARS-CoV-2 is mainly transmitted through droplets, direct contact and infected objects, and its incubation time is 1 to 14 days, and it is also transmitted by asymptomatic infected persons. Most infected people show only mild to moderate respiratory symptoms, or no symptoms at all. Only 5-10% of those infected show a full blown severe respiratory syndrome known as coronavirus disease (COVID)-19. Furthermore, the mortality rate from COVID-19 was 0.2% among young, healthy individuals, but increased with...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N5/20G01N33/569G01N33/577
CPCC07K16/10G01N33/56983G01N33/577G01N2333/165C07K2317/56C07K2317/565
Inventor 马晓飞孔双泉张立帆金丽珠武雷尹长城
Owner BEIJING PROTEIN INNOVATION
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