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Neutralizing human monoclonal antibody aiming at novel coronavirus and application thereof

A coronavirus, cloned antibody technology, applied in applications, antibodies, antiviral agents, etc., can solve the problems of no effective preventive vaccines, no specific antiviral drugs, etc.

Active Publication Date: 2021-09-14
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For the new coronavirus, there is currently no specific antiviral drug, nor is there an effective preventive vaccine

Method used

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  • Neutralizing human monoclonal antibody aiming at novel coronavirus and application thereof
  • Neutralizing human monoclonal antibody aiming at novel coronavirus and application thereof
  • Neutralizing human monoclonal antibody aiming at novel coronavirus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Expression and purification of novel coronavirus receptor binding domain (RBD) protein

[0043]The amino acid sequence (319-541) of the extracellular domain of the spike glycoprotein (S protein) gene sequence (GenBank: QHR63250.2) of the new coronavirus SARS-CoV-2, the signal peptide from albumin at the N-terminal, and the thrombin site at the C-terminal The point and the Fc fragment tag of human IgG1 were constructed on the pSectag2A vector. The specific process was to convert the receptor binding domain (RBD, amino acid from 319 After fusion of 541) (WH-RBD), thrombin site (T) and the Fc fragment (Fc) of human IgG1, the enzyme was constructed into the pSecTag2A vector (ThermoFisherScientific, catalog number: V90020) by homologous recombination At the cutting site SfiI, the vector pSectag2A-WH-RBD-TFc was successfully obtained. Expressed in mammalian cell 293F expression system. 1 day before transfection, 293F cells (control cell density was 5×10 5 cells / ...

Embodiment 2

[0046] Example 2: Screening using phage Fab display library

[0047] Using the antibody Fab phage display library, the receptor binding domain of the novel coronavirus spike glycoprotein (S) expressed in the mammalian cell 293F expression system was used as the antigen, and the phage Fab library was panned by the immunomagnetic bead method, and the specific phage was screened. Antigen capture, washed with PBS+0.05% Tween-20, after 4 rounds of screening, and monoclonal identification and sequencing, a clone with neutralizing activity on live virus in vitro was obtained, named Fab-F1-A3.

[0048] Sequence listing of Fab-F1-A3

[0049]

Embodiment 3

[0050] Example 3: Expression and purification of Fab-F1-A3

[0051] Fab-F1-A3 was expressed and purified according to existing literature (Zhu Z, Dimitrov DS. Methods Mol Biol. 2009; 525:129-4). The Fab-F1-A3 prokaryotic expression vector was constructed and transformed into E.coli HB2151. Re-inoculate the strains in SB medium containing 100 μg / ml ampicillin (1L medium contains 30g tryptone, 20g yeast extract and 10g MOPS, the pH value is adjusted to 7.0 with NaOH), and when the OD600 reaches 0.7-1.0, add The final concentration of IPTG was 200 μg / ml, and the expression was induced for 14-16 hours at 37° C. and 220 rpm. The cells were collected by centrifugation at 4°C, 6000 rpm, and 15 min, the medium was discarded, the pellet was resuspended in 1×PBS, and the supernatant was collected by centrifugation after being treated with polymyxin B for 45 min. Purified with Ni-NTA filler and verified its purity by SDS-PAGE. The replacement buffer was then ultrafiltered using an ult...

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Abstract

The invention discloses a neutralizing human monoclonal antibody aiming at a novel coronavirus. The antibody can specifically recognize a novel coronavirus antigen and has better neutralizing activity on a live virus. The epitope directed by the gene is mainly a receptor binding domain of a novel coronavirus. In addition, the antibody disclosed by the invention can be prepared into an antibody drug for preventing and treating the novel coronavirus, so that the antibody drug is clinically used for preventing and treating diseases caused by infection of the novel coronavirus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a neutralizing human monoclonal antibody against novel coronavirus and its application. Background technique [0002] Coronaviruses belong to the Coronaviridae family of the order Viridae, which are enveloped single-stranded positive-sense RNA viruses. Coronaviruses can cause diseases of different severity in the respiratory tract, intestinal tract, liver, kidney and nervous system in different species. As of March 1, 2020, the novel coronavirus (2019-nCoV, SARS-CoV-2) has spread in more than 60 countries, with more than 87,000 confirmed cases and 2,980 deaths. [0003] During the virus invasion process of coronavirus, the spike glycoprotein (S protein) on the surface of the virus is the target for the production of monoclonal antibodies. The S protein is functionally divided into two regions, the S1 subunit containing the receptor binding domain (RBD) is responsible for binding to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14
CPCC07K16/10A61P31/14C07K2317/565C07K2317/24A61K2039/505Y02A50/30
Inventor 龚睿赵少娟张化俊张晓晴张哲张海伟詹焱程彭诚高晓霄
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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