Anti-H1N1 influenza virus hemagglutinin protein monoclonal antibody ZCMU-H1N1 with neutralizing activity and application thereof

A ZCMU-H1N1, monoclonal antibody technology, applied in antiviral immunoglobulins, antiviral agents, antibodies, etc., can solve the problems of decreased antiviral treatment efficacy, oseltamivir resistance, and influenza virus resistance mutations.

Inactive Publication Date: 2022-05-27
浙江省中医院、浙江中医药大学附属第一医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, antiviral drug treatment should be taken within 48 hours after the onset of symptoms, otherwise the efficacy of antiviral treatment will be significantly reduced
In addition, in clinical studies, it was found that influenza virus can develop resistance mutations during drug exposure, resulting in resistance to oseltamivir

Method used

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  • Anti-H1N1 influenza virus hemagglutinin protein monoclonal antibody ZCMU-H1N1 with neutralizing activity and application thereof
  • Anti-H1N1 influenza virus hemagglutinin protein monoclonal antibody ZCMU-H1N1 with neutralizing activity and application thereof
  • Anti-H1N1 influenza virus hemagglutinin protein monoclonal antibody ZCMU-H1N1 with neutralizing activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Preparation method of monoclonal antibody against H1N1 influenza virus hemagglutinin protein

[0044] (1) Immunization of mice: For the first immunization, the H1N1 influenza virus hemagglutinin whole protein and adjuvant were mixed uniformly in equal volume, and the total volume was 600 microliters. Each BALB / C mouse 0.1 ml (containing 30 micrograms of H1N1 influenza virus hemagglutinin whole protein antigen) was intramuscularly injected into the inner thigh. On the 21st day, boost the immunization in the same way. On the 35th day, a small amount of tail blood was collected for enzyme-linked immunosorbent assay, and the antibody titer reached 1:100,000, followed by a booster immunization by tail vein injection, and cell fusion was performed 3 days later.

[0045] (2) Culture of mouse myeloma cell SP2 / 0: The SP2 / 0 myeloma cell line from BALB / C mice was cultured and passaged in DMEM medium containing 10% bovine serum, and incubated at 37°C saturated with 5% c...

Embodiment 2

[0055] Example 2. Antiviral effect of monoclonal antibody ZCMU-H1N1 against HA protein of H1N1 influenza virus

[0056] (1) Microneutralization experiment: ①H1N1 influenza virus (A / Michigan / 45 / 2015) was titrated by half of the tissue cells infective dose; ②MDCK cells were inoculated in 96-well culture plates, 2×10 per well 4 Cells were cultured in a 37°C incubator saturated with 5% carbon dioxide for 24 hours; ③ The virus was diluted with 0.2% trypsin-containing virus culture medium to 100 times the infection dose of half the tissue cells per 50 microliters; ④ In a 96-well culture plate 10 micrograms per milliliter of the monoclonal antibody ZCMU-H1N1 were diluted to different concentrations (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:16, 1:32, and 64, 1:128, 1:256, 1:521), 50 microliters per well; ⑤ Add 50 microliters of 100-fold half histiocytic infection dose per 50 microliters of virus solution to the wells with antibodies, and mix For each dilution, 4 replicate wells were made; t...

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Abstract

The invention provides a neutralizing monoclonal antibody ZCMU-H1N1 for resisting H1N1 influenza virus hemagglutinin protein and application of the neutralizing monoclonal antibody ZCMU-H1N1. The invention relates to an anti-H1N1 influenza virus hemagglutinin protein neutralizing monoclonal antibody ZCMU-H1N1, the subtype of the monoclonal antibody is IgG1, kappa type, and the monoclonal antibody can be specifically combined with an H1N1 influenza virus hemagglutinin protein antigen. The heavy chain amino acid sequence of the antibody is shown as SEQ ID No.2, and the light chain amino acid sequence of the antibody is shown as SEQ ID No.4. The monoclonal antibody is subjected to further physical and chemical property analysis and functional identification, and the monoclonal antibody can effectively neutralize and treat H1N1 seasonal influenza virus infection. The invention provides an effective tool for treating H1N1 seasonal influenza virus infection, and can be popularized and applied.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the preparation and application of anti-H1N1 influenza virus hemagglutinin protein neutralizing monoclonal antibodies. Cell line, through the induction of ascites in mice of the same strain, the anti-hemagglutinin protein monoclonal antibody ZCMU-H1N1 was prepared, identified as IgG1, κ type, and then the application of the antibody was realized through affinity purification and other technologies. Background technique [0002] Seasonal influenza viruses remain a global public health threat, and their prevalence has serious public health and socioeconomic impacts. Influenza viruses are estimated to kill more than 300,000 people each year, and the most common cause of human influenza infection is influenza A viruses (including H1N1 and H3N2 subtypes). In 2009-2010, influenza A virus (H1N1pdm09) first appeared in Mexico, Canada and the United States and spread rapidly around the world, ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N5/20C07K1/22A61K39/42A61P31/16
CPCC07K16/1018A61K39/42A61P31/16C07K2317/565C07K2317/567
Inventor 陆如凤吴海波杨帆
Owner 浙江省中医院、浙江中医药大学附属第一医院
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