Human SARS-CoV-2 monoclonal antibody and preparation method and application thereof

A monoclonal antibody, sars-cov-2 technology, applied in the direction of botanical equipment and methods, biochemical equipment and methods, applications, etc., to achieve the effect of good specificity and high affinity

Pending Publication Date: 2020-05-15
BEIJING BIOSYNTHESIS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And there is no commercial diagnostic kit with SARS-CoV-

Method used

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  • Human SARS-CoV-2 monoclonal antibody and preparation method and application thereof
  • Human SARS-CoV-2 monoclonal antibody and preparation method and application thereof
  • Human SARS-CoV-2 monoclonal antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of embodiment 1 human SARS-CoV Nucleocapsid recombinant protein

[0036] (1) Construction of human SARS-CoV Nucleocapsid prokaryotic expression vector

[0037] Human SARS-CoV Nucleocapsid gene sequence SEQ ID NO.2 was artificially synthesized, inserted into the prokaryotic expression plasmid pET2a digested with NdeI and XhoI, and transformed into competent Escherichia coli BL21(DE3). 0.1g / L kanamycin resistance plate screens positive clones, and induces them with a final concentration of 1mM IPTG at 16°C, 25°C, 30°C, and 37°C respectively, preferably at 25°C, shaking at 200rpm Cultured for 14 hours to induce human SARS-CoV Nucleocapsid recombinant protein expression. Finally identified by SDS-PAGE, the results are as follows figure 1 shown.

[0038] (2) Expression of pET28a-SARS-CoV-N fusion protein

[0039] The correctly identified pET28a-SARS-CoV-N strains were transferred to LB medium containing 100ug / ml kanamycin for overnight culture, and then t...

Embodiment 2

[0050] The preparation of embodiment 2 anti-human SARS-CoV-2 N monoclonal antibodies

[0051] (1) Animal immunity

[0052] Using purified human SARS-CoV-N recombinant protein as immunogen, immunize 8-12 week-old BALB / c female mice by conventional methods, emulsify with complete Freund's adjuvant and antigen for the first time, and immunize subcutaneously and intraperitoneally at multiple points. The dose is 100 μg / mouse; booster immunization once every two weeks, a total of three times, the adjuvant is Freund’s incomplete adjuvant, the dose is 100 μg / mouse / time; the tail blood titer of mice is detected by indirect ELISA method>1:10 4 , Three days before the fusion, intraperitoneal injection of 100 μg of human SARS-CoV-2 N antigen was carried out.

[0053] (2) cell fusion

[0054] Mix immune spleen cells and myeloma cells (SP2 / 0) at a ratio of 5:1, centrifuge at 1000rpm for 10min, discard the supernatant; resuspend the cells in RPMI-1640 medium, centrifuge at 1000rpm for 10min,...

Embodiment 3

[0070] Monoclonal antibody purification of embodiment 3 anti-human SARS-CoV-2 N recombinant protein

[0071] (1) Purification of monoclonal antibodies

[0072] The monoclonal antibody ascites obtained in Example 2 was purified by Protein A affinity chromatography, and the specific steps were as follows:

[0073] The basic steps are: equilibration-loading-equilibrium-elution-equilibrium-preservation.

[0074] Equilibrium solution: 20mM PB (pH7.2) buffer;

[0075] Eluent: 0.1M pH 3.0 glycine-HCl buffer;

[0076] Neutralization buffer: 1M, pH9.0, Tris-HCl buffer;

[0077] Preservation solution: 20% absolute ethanol.

[0078] Take 3-5ml of ascitic fluid, precipitate with 50% saturated ammonium sulfate for more than 2 hours, centrifuge at 10,000rmp for 20 minutes, discard the supernatant; dissolve the precipitate with equilibrium solution, filter it with a 0.45μm filter membrane, and prepare for loading; Protein A affinity column Pre-equilibrated with 5 times the column volume...

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Abstract

The invention discloses a human SARS-CoV-2 monoclonal antibody. The preparation method of the human SARS-CoV-2 monoclonal antibody comprises the steps: adopting SARS-CoV Nucleocapsid recombinant protein as immunogen, immunizing BALB/c mice, performing fusion and subcloning on spleen cells and myeloma cells of mice, then performing a large amount of repeated screening and domestication of cell lines through commercialized products SARS-CoV-2 Nucleocapsid and MERS Nucleocapsid so as to obtain a hybridoma cell line capable of secreting the SARS-CoV-2-resistant N monoclonal antibody with high affinity and high specificity finally and successfully, and finally performing ascites preparation and purification so as to obtain the monoclonal antibody, wherein the amino acid sequence of the SARS-CoVNucleocapsid recombinant protein is shown in SEQ ID No. 1. The invention also discloses application of the monoclonal antibody in preparation of SARS-CoV-2 virus detection products and preparation ofdrugs for inhibiting the SARS-CoV-2 viruses. The monoclonal antibody can be used for detecting the SARS-CoV-2 in human throat swabs/pulmonary secretions and other samples by using a double-antibody sandwich method, and can be applied to diagnosis and prevention and control of SARS-CoV-2 virus infection and scientific researches of viruses and other study.

Description

technical field [0001] The invention relates to a human SARS-CoV-2 monoclonal antibody and its preparation method and application, belonging to the technical field of monoclonal antibody preparation. Background technique [0002] On January 9, 2020, scientists observed under an electron microscope that the pathogen that caused the pneumonia presented an enveloped, corona-like shape typical of a coronavirus. At the same time, the sequencing results of the pathogenic genome showed that its nucleic acid sequence was not completely consistent with the six previously discovered coronaviruses (such as SARS, MERS, etc.). Therefore, on January 12, 2020, the World Health Organization (WHO) named the new virus: 2019 Novel Coronavirus (2019 Novel Coronavirus, 2019-nCoV), and on February 11, the International Committee on Taxonomy of Viruses (ICTV) named the new coronavirus Named SARS-CoV-2. The virus has a long incubation period and is highly contagious. As of 24:00 on February 18, 2...

Claims

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Application Information

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IPC IPC(8): C07K16/10C07K14/165C12N15/50C12N15/70G01N33/577G01N33/569A61K39/42A61P31/14
CPCA61K2039/505A61P31/14C07K14/005C07K16/10C07K2317/33C12N15/70C12N2770/20022G01N33/56983G01N33/577
Inventor 赵风强秦李娜吴炬王咚咚董兵张凤英邬晓乐
Owner BEIJING BIOSYNTHESIS BIOTECH
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