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Neutralizing antibody for resisting novel coronavirus SARS-Cov-2 and application thereof

A sars-cov-2, coronavirus technology, applied in the direction of antiviral agents, antiviral immunoglobulins, applications, etc., can solve problems such as affinity differences

Active Publication Date: 2020-08-28
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Subtle differences in the contact interface between the two RBDs and ACE2 lead to differences in the affinity of SARS-Cov-1 and SARS-Cov-2 to ACE2, which may benefit follow-up studies
[0005] Currently, there is no SARS-CoV-2 virus-specific vaccine and neutralizing antibody for clinical treatment

Method used

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  • Neutralizing antibody for resisting novel coronavirus SARS-Cov-2 and application thereof
  • Neutralizing antibody for resisting novel coronavirus SARS-Cov-2 and application thereof
  • Neutralizing antibody for resisting novel coronavirus SARS-Cov-2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Screening of fully human antibodies targeting SARS-Cov-2-RBD

[0038] Using phage display technology, the SARS-Cov-2-RBD-his protein was used as the positive antigen, and the SARS-Cov-1-RBD-hFc was used as the negative antigen. is 1.47x10 8 ) for differential screening.

[0039] Immunoplates were coated with 50 μg / ml SARS-Cov-2-RBD his antigen and SARS-Cov-1-RBD hFc antigen at 4°C overnight; blocked with PBS solution containing 5% nonfat dry milk and 0.1% Tween-20 at room temperature Immunoplate for 1 hour; 12 pfu was mixed 1:1 with 10% nonfat dry milk PBS solution, incubated at room temperature for 2 hours, added to the blocked SARS-Cov-1-RBD hFc antigen immunoplate (100 μl / well), incubated at room temperature for 1 hour, and pre-adsorbed with negative antigen ; After pre-adsorption, the supernatant was transferred to the SARS-Cov-2-RBD his antigen immune plate (100 μl / well), which was sealed, and incubated at room temperature for 1 hour. The immune plat...

Embodiment 2

[0050] Example 2. Antigen specificity analysis of antibodies

[0051] In this example, ELISA was used to detect the binding of the 4A3 phage of Example 1 to the SARS-Cov-2-RBD protein.

[0052] The specific process is as follows: use 5 μg / ml SARS-Cov-1-RBD-hFc and SARS-Cov-2-RBD-hFc to coat the immune plate overnight at 4°C; The PBS solution of 0.05% Tween-20 was used to block the immune plate for 1 hour at room temperature; the phage of 4A3 was added to the blocked immune plate (50 μl / well), and incubated at room temperature for 1 hour; the immune plate was washed 3 times with 0.05% Tween-20 in PBS (340 μl / well). ); Mix HRP / Anti-M13 Monoclonal conjugate with PBS solution containing 5% nonfat milk powder and 0.05% Tween-20 at a ratio of 1:4000, add it to the washed immune plate (50μl / well), and incubate at room temperature for 1 hour; Wash the immune plate 5 times with 0.05% Tween-20 in PBS; add TMB color development solution to the immune plate (100 μl / well), and after 3 min...

Embodiment 3

[0054] Example 3. Expression and purification of antibodies

[0055] The heavy chain variable region sequence and light chain variable region sequence of the 4A3 scFv sequence were inserted into the pFUSE-CHIg-HG1 and pFUSE2-CLIg-hk vectors (Invivogen, San Diego, CA), respectively, to construct an adult IgG1 expression plasmid, such as image 3 , Figure 4 shown. The above plasmids were co-transfected in 293T cells, and the supernatant was collected and purified with a protein A-Agarose separation column. The purity of the antibody was detected by SDS-PAGE. Figure 5 shown.

[0056] The specific process is as follows: insert the heavy chain variable region sequence and light chain variable region sequence of the 4A3 scFv sequence into pFUSE-CHIg-HG1 and pFUSE2-CLIg-hk (Invivogen, San Diego, CA), respectively, to construct an adult IgG expression plasmid. 5 million HEK293T cells were seeded in a cell culture dish with DMEM medium supplemented with 10% fetal bovine serum, 100...

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Abstract

The invention relates to a neutralizing antibody for resisting a novel coronavirus SARS-Cov-2 and an application of the neutralizing antibody. The antibody at least comprises one of a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3. The antibody can be used for preparing a diagnostic reagent or a diagnostic kit, a drug or a pharmaceutical composition for detecting, preventing and treating a COVID-19. According to the neutralizing antibody, differential antibody screening is carried out through a phage display technology ina manner of targeting SARS-Cov-2-RBD and SARS-Cov-1-RBD; the neutralizing antibody for resisting the novel coronavirus SARS-Cov-2 is obtained; binding of the SARS-Cov-2-RBD and ACE2 positive cells can be blocked; and the neutralizing antibody has a remarkable virus neutralizing effect on an SARS-Cov-2 pseudo virus and provides an effective alternative antibody drug for prevention and treatment ofthe COVID-19.

Description

technical field [0001] The invention relates to a neutralizing antibody against novel coronavirus SARS-Cov-2 and its application, belonging to the technical field of biomedicine. Background technique [0002] The 2019 novel coronavirus pneumonia (COVID-19) is caused by the infection of the new coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2). At present, more than 2 million people have been infected worldwide, with a fatality rate of more than 6% ( https: / / covid19.who.int / ), is a major infectious disease endangering human health. SARS-CoV-2 is a positive-stranded single-stranded RNA virus belonging to the beta coronavirus [1] , 79.5% nucleic acid homology with SARS-Cov-1 [2] , its natural host is unknown. Both SARS-CoV-2 and SARS-Cov-1 infect host cells by binding to angiotensin-converting enzyme 2 (ACE2) on the host cell membrane [3] . [0003] ACE2 is an angiotensin-converting enzyme, and its substrates are type I and type II angiotensin. Un...

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
CPCA61K2039/505A61P31/14C07K16/10C07K2317/33C07K2317/565C07K2317/76C07K2317/92G01N33/569G01N2333/165G01N2469/10
Inventor 高威刘晓宇高芳苟黎明
Owner NANJING MEDICAL UNIV
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