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Influenza virus antibody and preparation method and application thereof

An influenza virus and antibody technology, applied in the biological field, can solve problems such as the preparation of antibodies that are not suitable for influenza virus, achieve important economic and social significance, and inhibit replication.

Active Publication Date: 2017-07-07
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

However, this method requires the establishment of an antibody library and is not suitable for the preparation of antibodies against influenza virus

Method used

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  • Influenza virus antibody and preparation method and application thereof
  • Influenza virus antibody and preparation method and application thereof
  • Influenza virus antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Preparation and purification of embodiment 1 antibody

[0053] The preparation method of described influenza virus antibody, comprises the steps:

[0054] (1) Isolate PBMCs in the peripheral blood of infected patients, extract RNA, and reverse transcribe cDNA;

[0055] (2) Amplify the hypervariable region sequences of the heavy chain and light chain, sequence the amplified target fragments using Miseq 2X300bp, and analyze the sequencing results;

[0056] (3) Use CDR abundance as the main parameter to select the high-frequency variable region sequences of infected patients, and calculate the probability of natural pairing through the heavy chain and light chain pairing algorithm, and then select the high-frequency VH and CDR1, CDR2 and CDR3 VL sequences plus their respective constant regions are synthesized;

[0057] 所述VL的核苷酸序列为:gacatcgtgatgacacagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcagctacttagcctggtaccagcagaaacctggccaggctcccaggctcc...

Embodiment 2

[0063] Example 2 Antigen-antibody affinity test

[0064] Using surface plasmon resonance technology, the Fab (10 μg / mL) form of AF4H1K1 was immobilized on the CM5 chip through amino coupling, the fixed value reached 500RU, and the mobile phase antigen protein HA was diluted by 2 times. Select the KINJECT mode for kinetic parameter determination, 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. The combination or dissociation of two proteins causes the difference in molecular mass on the metal surface to cause a change in the refractive index. Change, calculate the size of the affinity, the results are shown in Table 1.

[0065] Table 1 Affinity determination of AF4H1K1Fab and various subtypes of influenza virus HA

[0066]

[0067] It can be seen from Table 1 that the binding and dissociation modes of AF4H1K1 and each subtype of HA in group 2 are almost slow binding and slow dissociation, with an affini...

Embodiment 3

[0068] The neutralization experiment of embodiment 3 antibody

[0069] Prepare antibodies and viruses after preparing MDCK cells in a 96-well plate with a confluence of 90%. First, the antibody was diluted 2-fold, then an equal volume of 200 TCID was added 50 Influenza virus, the influenza virus and antibody were mixed and placed at 37°C for 1 hour. Then, the antibody-virus mixture was added to MDCK cells in a 96-well plate washed twice with PBS at an amount of 100 μL per well, and three replicate wells were set for each antibody dilution. Put the 96-well plate with antibody-virus mixture in 37°C, 5% CO 2Incubate in the incubator for 1 h, wash once with PBS, add 100 μL of serum-free DMEM medium containing 2.5 μg / mL final concentration of trypsin to each well, 37 ° C, 5% CO 2 Culture, observe the cytopathic changes every day, and measure the hemagglutination activity of each well after 3 days of infection, and determine whether there is virus infection by combining the cytop...

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Abstract

The invention relates to the biotechnology field, in particular to an influenza virus antibody. A light-chain variable region of the influenza virus antibody has an amino acid sequence shown as the SEQ ID No.1; a heavy-chain variable region of the influenza virus antibody has an amino acid sequence shown as the SEQ ID No.2. The antibody can well neutralize H3N2, H4N6 and H14N5 subtype influenza viruses, combine HA proteins of all subtype influenza viruses in a group 2, neutralize H3-subclade viruses and inhibit copying of the H3 subtype influenza viruses in a mouse body and has the important economic and social significance.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an influenza virus 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. Influenza type A has the most frequent antigenic changes and is ubiquitous in humans and poultry, causing the greatest harm. Influenza type A can be divided into multiple subtypes according to the difference of hemagglutinin (HA) and neuraminidase (Neuraminidase, NA) on the surface of virus particles, and 18 subtypes (H1-H18) of HA have been found so far. ), NA has 11 subtypes. The 18 HA subtypes can be further divided into two groups, group 1 and group 2....

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/85A61K39/42A61P31/16
CPCA61K2039/505C07K16/1018C07K2317/14
Inventor 高福校海霞郭天玲陈维之洪媛媛孙中平
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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