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A kind of b-type influenza virus broad-spectrum neutralizing antibody, its preparation method and application

An influenza virus and antibody technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of being unable to prevent bird flu and swine flu, and not keeping up with the speed of seasonal influenza virus mutation

Active Publication Date: 2020-09-01
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Candidate strains of influenza vaccines will be updated as the prevailing strains change, but they still cannot catch up with the speed of seasonal influenza virus mutation, let alone prevent cross-species spread of bird flu and swine flu

Method used

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  • A kind of b-type influenza virus broad-spectrum neutralizing antibody, its preparation method and application
  • A kind of b-type influenza virus broad-spectrum neutralizing antibody, its preparation method and application
  • A kind of b-type influenza virus broad-spectrum neutralizing antibody, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Preparation of Antibody

[0069] The preparation method of broad-spectrum neutralizing antibody of type B influenza virus comprises the following steps:

[0070] (1) PBMCs in peripheral blood were collected on day 0 and day 7 after volunteers were inoculated with seasonal influenza vaccine, RNA was extracted, and cDNA was reverse transcribed;

[0071] (2) Amplify the hypervariable region sequences of the heavy chain and light chain, sequence the amplified target fragments using Miseq 2 X 300bp, and analyze the sequencing results;

[0072] (3) Using CDR abundance as the main parameter to select the high-frequency variable region sequences of immunized persons 7 days after immunization, and calculate the probability of natural pairing through the heavy chain and light chain pairing algorithm, and then select the high frequency occurrence of CDR1, CDR2 and CDR3 The VH and VL sequences plus their respective constant regions are synthesized;

[0073] The nucleoti...

Embodiment 2

[0080] Example 2 Antigen-antibody affinity test

[0081] Using surface plasmon resonance technology, the Fab (10 μg / mL) form of 0809 was fixed on the CM5 chip through amino coupling, and the fixed value reached 500RU. The mobile phase antigen protein influenza virus type B HA was diluted by 2 times (concentration range 1 μM-100 μM). Select the KINJECT mode for the determination of kinetic parameters, the flow rate of the machine is 30 μL / min, the injection time is 1-2 minutes, and the dissociation time is 2-6 minutes. Then, the affinity was calculated using BIA evaluation software, and Table 1 shows the affinity measurement results between 0809 Fab and various strains of influenza B virus HA. It can be seen from Table 1 that the HA binding and dissociation modes of 0809 and the strains of influenza B viruses Yamagata and Victoria are almost all fast binding and fast dissociation, with affinities of 4.11nM and 7.63nM, respectively.

[0082] Table 1. Affinity determination of ...

Embodiment 3

[0084] Example 3 Hemagglutination inhibition (HI) test and neutralization test of antibodies

[0085] Hemagglutination inhibition test: using the method of fixing the virus diluted antibody, the 0809 IgG antibody dilution was reacted with 4 hemagglutination units of influenza B virus at room temperature for 30 minutes, and then 1% chicken red blood cells of the same volume as the virus solution were added, Table 2 The hemagglutination inhibitory activity of 0809 IgG to various strains of influenza B viruses is shown. It was found that 0809 IgG is a hemagglutination-inhibiting antibody, which has high hemagglutination inhibitory activity against different strains of influenza B virus, and the HI titer is between 0.24μg / ml-4.7μg / ml.

[0086] Neutralization test: using the method of fixing virus diluted antibody, dilute 200TCID of the same volume of 0809IgG antibody dilution 50 Different influenza B virus strains were mixed together and left at room temperature for 1 hour. Then...

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Abstract

The invention relates to a broad-spectrum neutralizing antibody for influenza virus B. The broad-spectrum neutralizing antibody is a human monoclonal antibody and is named as 0809. The amino acid sequences of a light chain variable region and a heavy chain variable region of the antibody are respectively shown as in SEQ ID No.1 and SEQ ID No.2. The antibody provided by the invention can well neutralize the influenza virus B of a Yamagata strain, a Victoria strain and ancient B / Lee / 1940 strains; the antibody can inhibit the ability of the influenza virus B agglutinating chicken erythrocyte, and is a hemagglutination inhibition antibody; meanwhile, the antibody can inhibit replication of the influenza virus B in mice, inhibits weight loss of the mice due to infection of the influenza virus B, protects the mice to still have a survival rate of 100% after infection of the influenza virus B, and has important economic and social significances.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an influenza virus antibody, its preparation method and application, and in particular to a type B influenza virus broad-spectrum neutralizing antibody, its preparation method and application. Background technique [0002] Influenza virus (Influenza virus) is a zoonotic infectious disease pathogen that causes influenza, belongs to Orthomyxoviridae (Orthomyxoviridae), and belongs to the genus Influenza virus. According to the antigenicity of influenza virus nucleoprotein and matrix protein, influenza virus can be divided into three types: A, B, and C. The bovine influenza virus discovered in recent years is classified as type D. [0003] Influenza A virus can cause infection and disease in humans, poultry, mammals and bats, and is the most harmful. According to the difference of hemagglutinin protein (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA) on the surface of virus pa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10A61K39/395A61P31/16
CPCA61K2039/505C07K16/1018C07K2317/21C07K2317/565C07K2317/567C07K2317/76C07K2317/92
Inventor 高福校海霞冯俊霞严景华陈维之洪媛媛孙中平
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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