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Anti-COVID-19 virus neutralizing antibody mhC3 as well as humanized antibody and application thereof

An antibody, consistent technology, applied in antiviral agents, antiviral immunoglobulins, applications, etc., can solve the problem of no new coronary pneumonia-specific treatment drugs have been approved.

Active Publication Date: 2020-11-27
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, many institutions in the world are conducting research on antibodies against COVID-19, but no specific therapeutic drug against COVID-19 has been approved

Method used

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  • Anti-COVID-19 virus neutralizing antibody mhC3 as well as humanized antibody and application thereof
  • Anti-COVID-19 virus neutralizing antibody mhC3 as well as humanized antibody and application thereof
  • Anti-COVID-19 virus neutralizing antibody mhC3 as well as humanized antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, the discovery of antibody

[0058] 1. Mouse immunization and preparation of immune antibody library

[0059] Take 6-8 week-old Balb / C mice, and collect blood from the tail vein of the mice before immunization to keep the background serum. For the first immunization, the recombinant protein of the RBD domain of the new coronavirus S protein (Shenzhou Yiqiao, 40592-V08H5) was emulsified with complete Freund's adjuvant, and 100 μg of the recombinant protein was injected intraperitoneally into each mouse. Boost immunization at intervals of 1 week, take recombinant RBD protein (Shenzhou Yiqiao, 40592-V08H5) and emulsify it with Fischer’s incomplete adjuvant, inject 100 μg of recombinant protein intraperitoneally into each mouse, and collect blood by docking the tail before injection, and perform two booster immunizations in total . The fourth immunization was changed to impulse immunization, and the recombinant RBD protein without adjuvant (prepared in step 1...

Embodiment 2

[0063] Embodiment 2, preparation of anti-new coronavirus chimeric antibody

[0064] The mouse single-chain antibody clone C3 was picked to prepare a human-mouse chimeric whole antibody, named mhC3 (the constant region of the heavy chain is IgG1, and the light chain is Kappa). The preparation process is as follows:

[0065] 1. Construction of recombinant plasmids

[0066] 1. The sequence of SEQ ID No.11 is cloned into pcDNA3.1 (+) (Invitrogen, V79020) using conventional molecular biology techniques, and inserted between HindIII and BamH I restriction sites, and the resulting recombinant plasmid is passed through After sequencing and verification, it was named pcDNA3.1-mhC3H, which is the heavy chain expression vector of antibody mhC3.

[0067] 2. The sequence of SEQ ID No.12 is cloned into pcDNA3.1 (+) (Invitrogen, V79020) using conventional molecular biology techniques, inserted between the two restriction sites of Hind III and BamH I, and the resulting recombinant plasmid ...

Embodiment 3

[0083] Example 3, Detection of specific binding ability of mhC3 antibody

[0084] 1. Preparation of RBD recombinant protein

[0085] 1. Yeast expression and preparation of recombinant RBD glycoprotein

[0086] 1.1 Construction of recombinant protein expression vector

[0087] According to the SARS-CoV-2 gene sequence (NC_045512.2) published by GENEBANK, the S protein RBD region (SEQID No.15) was determined, and Shanghai Sangon Bioengineering Co., Ltd. was entrusted to carry out the whole gene synthesis, and enzyme cleavage sites were added at the upper and lower ends. XhoI and NotI were digested and inserted into the yeast expression vector pPICZαA (Invitrogen) which was digested with the same restriction enzyme, and named as pPICZαA-RBD.

[0088] 1.2 Yeast Competent Cell Preparation

[0089] Pick Pichia pastoris GJK0601 bacteria (the basic bacterial strain used in the present invention is the GJK0601 bacterial strain constructed earlier, the preservation number is CGMCC No...

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Abstract

The invention discloses an anti-COVID-19 virus neutralizing antibody mhC3 as well as a humanized antibody and application thereof. The amino acid sequences of HCDR1, HCDR2 and HCDR3 in a heavy chain variable region of the antibody provided by the invention are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in sequence; amino acid sequences of LCDR1, LCDR2 and LCDR3 in a light chain variable region of the antibody are shown as SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 in sequence. The antibody disclosed by the invention can be specifically combined with novel coronavirus RBD protein and can neutralize novel coronavirus (SARS-CoV-2). The antibody provided by the invention can be used for preventing and treating coronavirus infection, and has important biological and medical significance.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to anti-COVID-19 virus neutralizing antibody mhC3 and its humanized antibody and application. Background technique [0002] The novel coronavirus (SARS-CoV-2) is widely distributed in humans and other mammals in nature, and can infect wild animals, pets, herds, and humans, but most coronaviruses that can infect humans cause only mild infections. The novel coronavirus SARS-CoV-2 belongs to the subfamily Coronaviridae of the family Coronaviridae of the order Reticuloviridae. The subfamily of coronaviruses is divided into 4 genera: α, β, γ, and δ, among which SARS-CoV-2 is a β coronavirus. Before this outbreak, six coronaviruses were known to cause respiratory diseases in humans, four of which - 229E, OC43, NL63 and HKU1 - were ubiquitous in nature and usually caused only common cold symptoms in healthy people, only the 2002 outbreak The Severe Acute Respiratory Syndrome Coronavirus (SAR...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14G01N33/569G01N33/68
CPCC07K16/10A61P31/14G01N33/56983G01N33/6854C07K2317/622C07K2317/565C07K2317/56C07K2317/24C07K2317/76C07K2317/92A61K2039/505G01N2333/165G01N2469/10
Inventor 杨志新王荣杜鹏陆健昇陈蕾刘波吴军王波周权余云舟
Owner ACADEMY OF MILITARY MEDICAL SCI
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