Neutralizing single-domain antibody with function of resisting novel coronavirus SARS-Cov-2 and application of neutralizing single-domain antibody

A sars-cov-2, single-domain antibody technology, applied in antiviral agents, antiviral immunoglobulins, applications, etc., can solve the problems of limited and difficult changes in the structure of S protein

Active Publication Date: 2020-12-11
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limited structural changes of the S protein of SARS-CoV-2 caused by a single point mutation, it is difficult to obtain neutralizing antibodies against the mutant virus

Method used

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  • Neutralizing single-domain antibody with function of resisting novel coronavirus SARS-Cov-2 and application of neutralizing single-domain antibody
  • Neutralizing single-domain antibody with function of resisting novel coronavirus SARS-Cov-2 and application of neutralizing single-domain antibody
  • Neutralizing single-domain antibody with function of resisting novel coronavirus SARS-Cov-2 and application of neutralizing single-domain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] This example is to screen all human single domain antibodies targeting SARS-CoV-2-RBD.

[0032] Tomlinson I&J phage library (Genservice Ltd, Cambridge, UK, the library size is 1.47×10) 8 ) antibody sequence as a template, clone the antibody heavy chain sequence of the library, and construct a single-domain antibody library. Using phage display technology, SARS-CoV-2-RBD-HIS (ARG330-VAL524) protein as antigen was screened in single domain antibody library.

[0033] The specific process is as follows:

[0034] The ELISA plate was coated with 50μg / ml SARS-CoV-2-RBD his antigen at 4℃ overnight, 50ul / well; Using PBS solution (PBS STM) containing 5% skim milk powder and 0.1% tween-20 to block the enzyme-labeled plate at room temperature for 1 hour; Single domain antibody phage library with 10 12 Pfu was mixed with 10% skim milk powder PBS solution (PBSM) 1:1, then incubated at room temperature. After 2 hours, it was transferred to a sealed ELISA plate of SARS-CoV-2-RBD his antig...

Embodiment 2

[0045] This example is the specific binding of phage 4D8 clone to SARS-CoV-2-RBD protein.

preparation Embodiment 1

[0046] The 4D8 phage of Example 1 was prepared, and the binding specificity of 4D8 clone with SARS-CoV-2-RBD protein was detected by Phage ELISA.

[0047] The specific process is:

[0048] The ELISA plate was coated with 5μg / ml of SARS-CoV-RBD-hFc, SARS-CoV-2-RBD-his and SARS-CoV-2-RBD-hFc at 4℃ overnight. Seal that enzyme labeled plate with 3% pbstm at room temperature for 1 hour; 4D8 phage was incubated with 6% PBSM at the ratio of 1:1 at room temperature for 2 hours, then added to the sealed enzyme-labeled plate (50μl / well), and incubated at room temperature for 1 hour; The enzyme-labeled plate was washed with 0.05％PBST for 3 times (340ul / well); The HRP / anti-M13 monoclonal conjugate was mixed with 5% pbstm at the ratio of 1: 4000, added into the washed enzyme-labeled plate (50μl / well), and incubated at room temperature for 1 hour; Wash the enzyme-labeled plate with 0.05％PBST for 5 times; Add TMB chromogenic solution into enzyme-labeled plate (100μl / well), develop color at r...

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Abstract

The invention relates to a neutralizing single-domain antibody with a function of resisting novel coronavirus SARSCov2 and application of the neutralizing single-domain antibody. The antibody has at least one of a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3. The antibody can be used for preparing a diagnostic reagent or a diagnostic kit, an antibody medicine or a medicinal composition against COVID-19. An SARS-CoV-2 specific human monoclonal single-domain antibody is obtained by a phage display technology, has an SARS-CoV-2 pseudo-virus neutralizing effect, and has a synergistic neutralizing effect on D614G mutant strain pseudovirus when being combined with an IgG type SARS-CoV-2 neutralizing antibody 4A3 for use. Therefore, the antibody provides an efficient alternative antibody medicine for prevention and treatment of COVID-19.

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. technical background [0002] The outbreak of novel coronavirus (COVID-19) at the end of 2019 is an acute respiratory infectious disease caused by novel coronavirus (SARS-CoV-2) infection. [1] . SARS-CoV-2 is a positive stranded RNA virus with a diameter of about 80-120nm, and it is a β coronavirus. [2] , which is very similar to the structure of SARS-CoV virus, and its genome homology is as high as 79.5%. [3] . Studies have confirmed that both SARS-Cov-2 and SARS-CoV host receptors are angiotensin converting enzyme 2(ACE2). [3] . ACE2 is a carboxypeptidase and a protein receptor, which is mainly expressed in alveolar cells and intestinal epithelial cells. [4] . SARS-CoV-2 binds to ACE2 through the receptor binding domain,RBD) of spike protein S1 subunit on the virus coat, which induces the...

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
CPCA61K2039/505A61P31/14C07K16/10C07K2317/565C07K2317/569C07K2317/76C07K2317/92G01N33/56983G01N2333/165
Inventor 高威马素娟叶伟敖磊刘晓宇
Owner NANJING MEDICAL UNIV
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