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Human monoclonal antibody against novel coronavirus and its application

A technology of cloning antibodies and source list, which is applied in the field of medicine, can solve the problems of no specific drugs and achieve good neutralizing activity

Active Publication Date: 2021-12-28
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] For the new coronavirus 2019-nCoV, there is no specific drug

Method used

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  • Human monoclonal antibody against novel coronavirus and its application
  • Human monoclonal antibody against novel coronavirus and its application
  • Human monoclonal antibody against novel coronavirus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142] Example 1: Expression and purification of 2019-nCoV RBD

[0143] At the 3' end of the coding region of the 2019-nCoV RBD protein (the amino acid sequence is shown in SEQ ID NO: 9), the coding sequence of 6 histidine tags (hexa-His-tag) and the translation stop codon are connected, and the EcoRI and XhoI were constructed into pFastBacl vector (purchased from Invitrogen). The ligated product was then transformed into DH10Bac competent cells (purchased from Tiangen) for baculovirus recombination. The recombinant baculovirus was extracted, transfected into sf9 cells (purchased from Invitrogen) for packaging of the baculovirus, and then amplified by the virus, added to Hi5 cells (purchased from Invitrogen) for 2019-nCoV RBD protein synthesis Express.

[0144] After the cell culture solution containing the target protein is purified by nickel ion affinity chromatography (HisTrap TMHP (GE)) and gel filtration chromatography (SuperoseTM 6Increase 10 / 300GL (GE)), a relatively ...

Embodiment 2

[0145] Example 2: Isolation of 2019-nCoV RBD protein-specific memory B cells

[0146] With the informed consent of those discharged from hospital after 2019-nCoV infection, 15 mL of blood was collected to separate PBMCs. Separated PBMCs in 10 7 The density per mL and the final concentration of 400nM 2019-nCoV RBD protein were incubated on ice for half an hour, then washed twice with PBS, and then incubated with the following antibodies (both purchased from BD): anti-human CD3 / PE-Cy5 (anti-human CD3 / PE-Cy5) -human CD3 / PE-Cy5), anti-human CD16 / PE-Cy5 (anti-human CD16 / PE-Cy5), anti-human CD235a / PE-Cy5 (anti-humanCD235a / PE-Cy5), anti-human CD19 / APC- Cy7 (anti-human CD19 / APC-Cy7), anti-human CD27 / Pacific Blue (anti-human CD27 / Pacific Blue), anti-human CD38 / APC (anti-human CD38 / APC), anti-human IgG / FITC (anti- human IgG / FITC), and anti-His / PE (anti-His / PE). After antibody incubation on ice for half an hour, wash PBMCs twice with PBS.

[0147] After washing with PBS, PBMCs were s...

Embodiment 3

[0148] Example 3: Single B cell PCR, sequence analysis and human antibody design

[0149] According to the method described in Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus published in Science Translational Medicine, Volume 8, No. 369 in December 2016 by Qihui Wang et al., the B cells obtained in Example 2 were Reverse transcription was carried out by Superscript III reverse transcriptase (Invitrogen), and the reverse transcription primers were listed in Table 1, and reacted at 55°C for 60 minutes.

[0150] Table 1. Primers for reverse transcription reactions

[0151]

[0152] 3.1 Human antibody CB6

[0153] Using the above reverse transcription product as a template, PCR (PCRa) was performed with HotStar Tap Plus enzyme (QIAgen) to amplify the antibody variable region sequence. Corresponding primers were designed, and the reaction conditions were as follows: 95°C, 5min; 95°C for 30s, 55°C (heavy chain / κ chain) f...

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Abstract

The invention relates to a human monoclonal antibody against a novel coronavirus and its application. The antibody can specifically bind to the receptor binding domain (RBD) of 2019-nCOV, block the binding of RBD of 2019-nCOV to ACE2, and inhibit the infection of 2019-nCOV.

Description

technical field [0001] The invention belongs to the field of medical technology, and in particular relates to a human monoclonal antibody of a novel coronavirus (2019-nCoV, also known as SARS-CoV-2) with high neutralization activity and an application thereof. Background technique [0002] For the new coronavirus 2019-nCoV, there is currently no specific drug. [0003] Therapeutic antibody drugs not only play an important role in tumors and autoimmune diseases, but are also effective in the treatment of infectious diseases. Drugs currently on the market for the treatment and prevention of viral infections include palivizumab (Synagis) for the prevention of pediatric respiratory syncytial virus (RSV) infection, ibalizumab (Trogarzo) for the treatment of HIV infection, and rabies Rabishield for post-exposure prophylaxis. There are also monoclonal antibodies against numerous viruses in various stages of clinical research (https: / / clinicaltrials.gov / ). [0004] 2019-nCoV is a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10A61K39/42A61P31/14G01N33/577G01N33/569
CPCC07K16/10A61P31/14G01N33/577G01N33/56983C07K2317/565C07K2317/24C07K2317/56C07K2317/51C07K2317/515C07K2317/92C07K2317/76A61K2039/505G01N2333/165G01N2469/10C07K2317/21
Inventor 严景华史瑞王奇慧高福马素芳
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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