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Method for preparing anti-H1N1 specific immunoglobulin of egg yolk

An influenza virus, specific technology, applied in the field of immunology, can solve problems such as side effects of the central nervous system, insanity, etc., to achieve high-yield effects

Inactive Publication Date: 2010-07-28
GUANGDONG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, oseltamivir and zanamivir are already on the market, both of which have obvious antiviral activity. This new type of H1N1 influenza virus that is currently popular is sensitive to neuraminidase inhibitors, but This type of drug has obvious side effects of the central nervous system. In Japan, there have been adverse reactions of oseltamivir patients with mental confusion; and it may lead to mutation pressure to further produce drug-resistant strains

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0008] Embodiment 1: Preparation of anti-H1N1 influenza virus-specific chicken yolk antibody

[0009] (1) Preparation of Influenza A H1N1 Antigen

[0010] The HA amino acid sequence of H1N1 in GenBank was found to be ACP41934, and the antigen against influenza A (H1N1) was obtained by gene expression of the HA fragment of influenza A (H1N1) virus. The lyophilized antigen was diluted to 1 mg / ml with 0.01 mol / L phosphate buffer saline PBS (pH=7.2), and fully emulsified with Freund's adjuvant according to 1:1 (V / V) to prepare the immune antigen.

[0011] (2) Immune hens with H1N1 influenza virus antigen

[0012] Take 10 20-week-old laying hens, and inject the emulsified H1N1 antigen at multiple points through the wings or breast muscle of the chicken; the first immunization uses Freund's complete adjuvant, and the rest uses Freund's incomplete adjuvant; before the first immunization, collect unimmunized Eggs were used as a negative control substance, immunized for the second ti...

Embodiment approach 2

[0018] Embodiment 2: Detection of anti-H1N1 influenza virus-specific IgY

[0019] Application of SDS-PAGE (sodium dodecylsulfonate-polyacrylamide gel) electrophoresis assay, the crude extract of anti-H1N1 influenza virus specific IgY produced by the above process is detected, using 5% concentration Gel, 15% separating gel, the result contained IgY 45-56%. The crude IgY extract was purified by ammonium sulfate salting out with a saturation of 33%, and the purity reached 90-95% by SDS-PAGE analysis.

Embodiment approach 3

[0020] Embodiment 3: Titer detection of anti-influenza A (H1N1) virus-specific IgY

[0021] "ELISA method" (enzyme-linked immunosorbent assay) is used to detect the potency of the anti-H1N1 influenza virus-specific IgY prepared by the process of the present invention. The test results show that the anti-H1N1 influenza virus-specific IgY titer reaches 1:2560.

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PUM

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Abstract

The invention discloses methods for preparing and isolating anti-H1N1 specific immunoglobulin of egg yolk (IgY). The IgY is prepared by the following steps: 1. expressing HA segments of H1N1 by genes to obtain the antigen of H1N1; 2. carrying out injection immersion on the laying hens with the prepared H1N1 antigen to obtain anti-H1N1 immune eggs; and 3. extracting the crude products of the anti-H1N1 specific IgY and purifying and filtering the crude products, thus obtaining the finished anti-H1N1 IgY. The obtained anti-H1N1 IgY has low cost, high yield and high specificity and can be used for preparing various preparations having the functions of preventing and treating H1N1.

Description

technical field [0001] The invention belongs to the technical field of immunology, and relates to a preparation method of anti-type A H1N1 influenza virus-specific chicken egg yolk antibody IgY. Background technique [0002] In late April 2009, WHO announced the emergence of a new influenza virus that had not circulated in the population before, that is, influenza A (H1N1) virus. Influenza A (H1N1) is an acute respiratory infectious disease caused by a mutated new type of influenza A (H1N1) virus. After genetic analysis of the virus isolated from a patient in California, it is known that the virus strain contains swine flu, isolated Influenza and Human Influenza Genetic segments of three influenza viruses. [Ministry of Health of the People's Republic of China. Influenza A H1N1 Influenza Diagnosis and Treatment Program (Trial Version 1st Edition in 2009)]. H1N1 is a virus of the Orthomyxoviridae series. It is mainly transmitted through the respiratory tract through droplets...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C07K16/02
Inventor 赵肃清刘端苏娟朱屋彪张焜
Owner GUANGDONG UNIV OF TECH
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