Novel coronavirus SARS-Cov-2 resistant neutralizing single-domain antibody and application thereof

A sars-cov-2, single-domain antibody technology, used in antiviral agents, antiviral immunoglobulins, applications, etc.

Active Publication Date: 2020-09-11
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Currently, there is no SARS-CoV-2 virus-specific vaccine and neutralizing antibody for clinical application

Method used

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  • Novel coronavirus SARS-Cov-2 resistant neutralizing single-domain antibody and application thereof
  • Novel coronavirus SARS-Cov-2 resistant neutralizing single-domain antibody and application thereof
  • Novel coronavirus SARS-Cov-2 resistant neutralizing single-domain antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Screening of fully human single domain antibodies targeting SARS-Cov-2-RBD

[0039] Using Tomlinson I&J phage library (Genservice Ltd., Cambridge, UK, the library size is 1.47×10 8 ) antibody sequence as a template, and the antibody heavy chain sequence of the library was amplified by PCR to construct a single domain antibody phage library.

[0040] Phage display technology was used to screen SARS-Cov-2-RBD his (Arg330-Val524) protein as positive antigen and SARS-Cov-2-RBD mut-hFC as negative antigen.

[0041] Use 50 μg / ml of the above-mentioned SARS-Cov-2-RBD his antigen and SARS-Cov-2-RBD mut-hFc to coat the immunoplate overnight at 4°C; use 5% skimmed milk powder, 0.1% Tween- 20 PBS solution at room temperature to block the immunoplate for 1 hour; the single domain antibody phage library obtained above was diluted with 10 12 Mix pfu with 10% skimmed milk powder PBS solution 1:1, incubate at room temperature for 2 hours, add the blocked SARS-Cov-2-RBD mut...

Embodiment 2

[0068] Example 2, Antigen specificity analysis of single domain antibody

[0069] In this example, ELISA was used to detect the binding status of the phages 4A12, 4D5, 4A10 and 4C5 in Example 1 to the SARS-Cov-2-RBD-hFc protein.

[0070] The specific process is as follows: use 5 μg / ml of SARS-Cov-1-RBD-hFc, SARS-Cov-2-RBD-hFc, and SARS-Cov-2-RBD mut-hFc to coat the immune plate overnight at 4°C; The PBS solution containing 3% skimmed milk powder and 0.05% Tween-20 blocked the immune plate at room temperature for 1 hour; respectively added 4A12, 4D5, 4A10, and 4C5 phages to each blocked immune plate (50 μl / well), and incubated at room temperature for 1 hour ;Wash the immunoplate 3 times with 0.05% Tween-20 in PBS solution (340ul / well); Mix HRP / Anti-M13 Monoclonal conjugate with 5% skimmed milk powder and 0.05% Tween-20 in PBS solution at a ratio of 1:4000 , added to the washed immune plate (50 μl / well), incubated at room temperature for 1 hour; washed the immune plate with 0.0...

Embodiment 3

[0072] Example 3, Expression and Purification of Single Domain Antibody

[0073] Construct the eukaryotic cell expression vector pFUSE-4A12-hFC, pFUSE-4D5-hFC, pFUSE-4A10-hFC, pFUSE-4C5-hFC (Invivigen, San Diego, CA) of each antibody of Example 1, such as image 3 shown. After transfection in 293T cells, the supernatant was collected and purified with a protein A-Agarose separation column, and the purity of the antibody was detected by SDS-PAGE, as shown in Figure 4 shown.

[0074] The specific process is: Cloning the VH sequence of antibodies 4A12, 4D5, 4A10, and 4C5 into expression vectors pFUSE-4A12-hFc, pFUSE-4D5-hFc, pFUSE-4A10-hFc, pFUSE-4C5-hFc (Invivigen, San Diego, CA), plasmids were prepared. 5 million HEK293T cells were planted in a cell culture dish with DMEM medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin, and cultured in a 5% CO2, 37°C incubator. When the cell density reached 60-80%, 10 μg of pFUSE-4A12-hFc, p...

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Abstract

The invention relates to a novel coronavirus SARS-Cov-2 resistant neutralizing single-domain antibody and application thereof. The antibody at least has one of heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and can be used for preparing diagnostic reagent or diagnostic kit, an antibody medicine or medicinal composition against COVID-19. The novel coronavirus SARS-Cov-2 resistant neutralizing single-domain antibody is obtained through a phage display technology, can stop combination of the SARS-Cov-2-RBD and ACE2 positive cells, has a remarkable virus neutralizing effect on SARS-Cov-2pseudovirion, and provides an efficient backup antibody medicine for COVID-19 prevention and treatment.

Description

technical field [0001] The invention relates to a neutralizing single-domain antibody against novel coronavirus SARS-Cov-2 and its application, belonging to the technical field of biomedicine. Background technique [0002] The number of infected cases of the novel coronavirus infectious disease (COVID-19) in 2019 has exceeded 2 million worldwide, with a fatality rate of 6% ( https: / / covid19.who.int / ), seriously endangering human health. COVID-19 is induced by infection with novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2). SARS-CoV-2 is a positive-sense single-stranded RNA virus with a diameter of about 80-120nm and a member of the genus Betacoronavirus [1] , which belong to the Sarbecovirus subgenus with SARS-CoV, with a nucleic acid homology of 79.5% [2] . At present, the source and natural host of the SARS-CoV-2 virus have not been clarified, and effective measures cannot be taken to prevent and stop the spread of the virus. [0003] E...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/569A61K39/42A61P31/14
CPCA61K39/42A61P31/14C07K16/10C07K2317/565C07K2317/569C07K2317/76G01N33/56983G01N2333/165
Inventor 高威高芳刘晓宇苟黎明
Owner NANJING MEDICAL UNIV
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