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Anti-H1N1 influenza virus hemagglutinin protein monoclonal antibody ZJU-A1 with neutralizing activity

A ZJU-A1, monoclonal antibody technology, applied in antiviral immunoglobulins, antiviral agents, antibodies, etc., can solve problems such as fever and allergic reactions

Active Publication Date: 2021-01-01
THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, convalescent plasma has the risk of causing fever and allergic reactions, transfusion-related acute lung injury, etc.

Method used

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  • Anti-H1N1 influenza virus hemagglutinin protein monoclonal antibody ZJU-A1 with neutralizing activity
  • Anti-H1N1 influenza virus hemagglutinin protein monoclonal antibody ZJU-A1 with neutralizing activity
  • Anti-H1N1 influenza virus hemagglutinin protein monoclonal antibody ZJU-A1 with neutralizing activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Preparation method of monoclonal antibody against H1N1 influenza virus hemagglutinin protein

[0051] (1) Immunization of mice: For the first immunization, the purified H1N1 influenza virus hemagglutinin whole protein and the adjuvant were uniformly mixed in equal volumes. Each BALB / C mouse 0.1 ml (containing 30 micrograms of H1N1 influenza virus hemagglutinin protein) was intramuscularly injected into the inner thigh. On the 21st day, a dose of booster immunization was given in the same way. On the 35th day, a small amount of tail vein blood was collected for enzyme-linked immunosorbent assay, and the antibody titer reached 1:256,000. Immediately, a booster immunization was injected into the tail vein, and cell fusion was carried out 3 days later.

[0052] (2) Cultivation of mouse myeloma cell SP2 / 0: the SP2 / 0 myeloma cell line from BALB / C mouse was cultured and subcultured in DMEM medium containing 10% bovine serum, and incubated at 37°C containing 5% carb...

Embodiment 2

[0061] Example 2. Antiviral effect of monoclonal antibody ZJU-A1 against H1N1 influenza virus HA protein

[0062] (1) Microneutralization experiment: ① H1N1 influenza virus (A / Michigan / 45 / 2015) was titrated with TCID50 (half tissue culture infectious dose); ② MDCK cells were seeded in 96-well plates, 2×10 4Each hole was cultured in a 5% CO2 incubator at 37°C for one day; ③ Dilute the virus to 100 TCID50 per 50 microliters with virus culture solution containing 0.2% trypsin; ④ Put 10 micrograms per milliliter of monoclonal antibody ZJU in 96-well plate -A1 was diluted with virus culture solution to different concentrations (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1 :521), 50 microliters per hole; ⑤ add 50 microliters of 100TCID50 virus solution per 50 microliters to the wells with antibodies, mix well, and make 4 duplicate holes for each dilution; do the penultimate column Virus back titration, the virus was diluted from 100TCID50 per 100 microliters (1:1, 1:2, 1:4...

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Abstract

The invention provides an anti-H1N1 influenza virus hemagglutinin protein monoclonal antibody ZJU-A1 with neutralizing activity. The antibody can specifically recognize the hemagglutinin protein of the H1N1 influenza virus, and exert antiviral effect through neutralization. The hybridoma cells producing the monoclonal antibody were obtained after fusion of spleen lymphocytes of BALB / C mice immunized with purified hemagglutinin protein and mouse myeloma cells SP2 / 0, screening, cloning and passage screening. Finally, the hybridoma cells could stably secrete antibody ZJU-A1. The heavy-chain aminoacid sequence of the antibody is shown as SEQ ID No.2, and the light-chain amino acid sequence is shown as SEQ ID No.4. ZJU-A1 can be effectively bined, neutralize and treat H1N1 influenza virus, andcan be used in combination with other antibodies or drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the preparation and application of an anti-H1N1 influenza virus hemagglutinin protein monoclonal antibody. It uses cell engineering and antibody engineering technology to obtain a hybridoma cell line that secretes an anti-hemagglutinin protein monoclonal antibody. Ascites was induced from the same strain of mice to prepare the anti-hemagglutinin monoclonal antibody ZJU-A1, which was identified as IgG1 and κ type, and then the application of the antibody was realized through affinity purification and immunization techniques. Background technique [0002] In 1918, the "Spanish flu" caused by H1N1 swept the world, causing tens of millions of illnesses and deaths. It was the most serious influenza pandemic known to mankind and had detailed records. The speed of transmission is unprecedented; the "Mexican flu" in 2009 spread rapidly in the summer of the northern hemisphere and caused a large ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C07K1/22C12N5/20A61K39/42A61P31/16C12R1/91
CPCC07K16/1018A61P31/16C07K2317/76A61K2039/505Y02A50/30
Inventor 吴海波吴南屏姚航平杨帆王莉彦
Owner THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE