Neutralizing antibody against novel coronavirus, antigen-binding fragment thereof and application thereof

A technology of combining coronavirus and fragments, which is applied in antiviral agents, antiviral immunoglobulins, applications, etc., and can solve the problems of lack of specific and efficient antiviral drugs

Active Publication Date: 2022-03-22
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no specific therapeutic drug against SARS-CoV-2 for clinical use. Clinically, symptomatic and supportive treatment is the main treatment, and there is still a lack of specific and highly effective antiviral drugs.

Method used

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  • Neutralizing antibody against novel coronavirus, antigen-binding fragment thereof and application thereof
  • Neutralizing antibody against novel coronavirus, antigen-binding fragment thereof and application thereof
  • Neutralizing antibody against novel coronavirus, antigen-binding fragment thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Embodiment 1, the discovery of antibody

[0116] 1. Mouse immunization and preparation of immune antibody library

[0117] Female Balb / C mice aged 6-8 weeks were selected, and blood was collected from the tail vein of the mice to keep the background serum. The novel coronavirus S protein RBD domain recombinant protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., product number: 40592-V08H5, referred to as recombinant RBD protein) was emulsified with Freund's complete adjuvant, and each mouse was injected intraperitoneally with 100 μg of recombinant RBD protein. Before the injection, the background serum was taken from the mice as a control. One week after the first immunization, the recombinant RBD protein was emulsified with Fisher's incomplete adjuvant, and each mouse was intraperitoneally injected with 100 μg of the recombinant RBD protein, and the blood was collected by docking the tail before the injection. After three rounds of immunization, shock immunization...

Embodiment 2

[0129] Embodiment 2, preparation of anti-new coronavirus chimeric antibody

[0130] A chimeric antibody is a chimeric antibody assembled from the variable region (V region) of a mouse antibody and the constant region (C region) of a human antibody. These two parts are relatively independent in terms of spatial structure, so their specificity and affinity are maintained. very good. In this example, a human-mouse chimeric whole antibody was prepared based on the murine single-chain antibody A-1F obtained in Example 1, named mhA-1F (the constant region of the heavy chain is IgG1, and the light chain is Kappa). The preparation process is as follows:

[0131] 1. Construction of recombinant plasmids

[0132] In order to express the chimeric antibody mhA-1F, the heavy chain expression vector and the light chain expression vector were prepared respectively:

[0133] 1. Combine the heavy chain variable region (VH) gene (1-354 of SEQ ID No.11) of murine single-chain antibody A-1F wit...

Embodiment 3

[0149] Example 3, Detection of specific binding ability of mhA-1F antibody

[0150] 1. Dilute SARS-CoV-2 RBD recombinant protein (commercialized RBD (Shenzhou, 40592-V08H5)) with carbonate coating buffer (pH 9.6) to 2ng / ul, add to the enzyme at a volume of 100 μL per well In the target plate (Corning, 9018), three replicate wells were set up for each experimental well, and coated overnight at 4°C.

[0151] 2. On the next day, wash the overnight coated ELISA plate with PBST 6 times, add PBS blocking solution containing 2% (mass percentage) skimmed milk powder, and incubate at 37°C for 2 hours.

[0152] 3. After the blocking is completed, discard the blocking solution, add 100 μL of 2-fold diluted mhA-1F antibody solution (initial concentration 1.2 nM) to each well, set up 11 gradients in total, incubate at 37°C for 90 minutes, and then wash the plate with PBST 6 times.

[0153] 4. After completing the above steps, take the ELISA plate, add 100 μL of 1:4000 dilution of HRP-lab...

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Abstract

The invention discloses an anti-novel coronavirus neutralizing antibody, and an antigen binding fragment and application thereof. Specifically disclosed are antibodies or antigen-binding fragments thereof that specifically bind to a novel coronavirus S protein receptor binding domain (RBD). According to the invention, a mouse single-chain antibody A-1F capable of being specifically combined with RBD is obtained through screening of an antibody library, and a human-mouse chimeric antibody mhA-1F and a humanized antibody HSA-1F are developed on the basis of the mouse single-chain antibody A-1F. The antibodies mhA-1F and HSA-1F have higher humanization degree, are not easy to cause immunogenicity reaction, have high affinity, can efficiently neutralize novel coronavirus, can be prepared into specific antibody drugs for clinically preventing and treating novel coronavirus pneumonia, or can be prepared into SARS-CoV-2 diagnostic reagents or kits and the like, and can be used for preparing novel coronavirus pneumonia. The method has very wide prospects and important significance in the fields of medicine application, clinical diagnosis and the like.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to an anti-new coronavirus neutralizing antibody, an antigen-binding fragment thereof and applications thereof. Background technique [0002] The homology of the novel coronavirus (SARS-CoV-2) and the SARS-CoV genome is about 80%, mainly causing novel coronavirus pneumonia. SARS-CoV-2 belongs to the β subgenus of coronaviruses. It is a single-stranded positive-strand RNA virus with a total length of about 30kb. It is composed of protein and RNA. Its membrane surface is mainly composed of three structural proteins: Spike Protein (Spike Protein) , S protein), envelope protein (Envelop Protein, E protein) and membrane protein (Membrane Protein, M protein). Inside the virus is the nucleic acid substance RNA responsible for virus reproduction, which is wrapped and protected by the nucleoprotein (N protein). Among these four proteins, the most important is the spike protein (S pro...

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
CPCC07K16/10G01N33/56983A61P31/14C07K2317/565C07K2317/76C07K2317/92C07K2317/622C07K2317/24G01N2333/165G01N2469/10A61K2039/505
Inventor 王荣杜鹏陆健昇郭家正陈蕾杨志新余云舟
Owner ACADEMY OF MILITARY MEDICAL SCI
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