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A method for concentrating and purifying avian influenza antigen, and avian influenza antigen

A purification method and avian influenza technology, applied in the field of concentration and purification of avian influenza antigens, can solve the problems of cumbersome and complicated steps of the antigen concentration and purification method, and the removal rate of impurity proteins needs to be further improved, so as to reduce immune side reactions and endotoxin content , Improving the effect of immune effect

Active Publication Date: 2021-04-16
河南后羿生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The steps of the antigen concentration and purification method disclosed in this patent are cumbersome and complicated, and the removal rate of foreign proteins needs to be further improved

Method used

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  • A method for concentrating and purifying avian influenza antigen, and avian influenza antigen
  • A method for concentrating and purifying avian influenza antigen, and avian influenza antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The method for concentrating and purifying the avian influenza antigen of the present embodiment comprises the following steps:

[0036] 1) Virus inoculation and cultivation: After properly diluting the H9 subtype avian influenza virus with sterilized saline, an automatic inoculation machine was used to inoculate chicken embryos. Each embryo was inoculated with 0.1 mL, and placed at 37°C with a humidity of 70% of the incubator incubation for 72h;

[0037] 2) Harvesting of virus liquid: through inspection, discard dead embryos and weak embryos, take out all live embryos, cool at 2°C for 24 hours, and then sterilize with 75% alcohol, use a fully automatic harvester to harvest chicken embryo allantoic fluid, The virus fluid must be harvested, and the hemagglutination value is ≥1:2 7 ;

[0038] 3) Coarse filtration of virus fluid: Centrifuge the harvested virus fluid at a speed of 12,000 rpm for 30 minutes to remove large particles of impurities in the antigen; Filter ho...

Embodiment 2

[0045] The method for concentrating and purifying the avian influenza antigen of the present embodiment comprises the following steps:

[0046] 1) Virus inoculation and cultivation: After properly diluting the H9 subtype avian influenza virus with sterilized physiological saline, an automatic inoculation machine was used to inoculate chicken embryos. Each embryo was inoculated with 0.1 mL, and placed at 37°C with a humidity of 70% of the incubator incubation for 72h;

[0047] 2) Harvesting of virus liquid: through inspection, discard dead embryos and weak embryos, take out all live embryos, cool at 8°C for 4 hours, and then sterilize with 75% alcohol, use a fully automatic harvester to harvest chicken embryo allantoic fluid, The virus fluid must be harvested, and the hemagglutination value is ≥1:2 7 ;

[0048]3) Coarse filtration of virus fluid: Centrifuge the harvested virus fluid at a speed of 10,000 rpm for 40 minutes to remove large particles of impurities in the antigen...

Embodiment 3

[0055] The method for concentrating and purifying the avian influenza antigen of the present embodiment comprises the following steps:

[0056] 1) Virus inoculation and cultivation: After properly diluting the H9 subtype avian influenza virus with sterilized physiological saline, an automatic inoculation machine was used to inoculate chicken embryos. Each embryo was inoculated with 0.1 mL, and placed at 37°C with a humidity of 70% of the incubator incubation for 72h;

[0057] 2) Harvesting of virus liquid: through inspection, discard dead embryos and weak embryos, take out all live embryos, cool at 5°C for 15 hours, and then disinfect with 75% alcohol, harvest chick embryo allantoic fluid with a fully automatic harvester, The virus fluid must be harvested, and the hemagglutination value is ≥1:2 7 ;

[0058] 3) Coarse filtration of the virus liquid: Centrifuge the harvested virus liquid at a speed of 11,000 rpm for 35 minutes to remove large particles of impurities in the ant...

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Abstract

The invention relates to a method for concentrating and purifying an avian influenza antigen and an avian influenza antigen, belonging to the field of antigen concentrating and purifying. The method for concentrating and purifying the avian influenza antigen of the present invention comprises the following steps: 1) Concentrating the avian influenza virus liquid through a 1000KD membrane bag to 1 / 10-1 / 8 of the volume of the virus liquid to obtain reflux liquid A; Add sterilized PBS to the reflux liquid A to make it the same volume as the avian influenza virus liquid in step 1), and then concentrate it to 1 / 10-1 / 8 of the volume of the virus liquid through a 1000KD membrane bag to obtain the reflux liquid B ; 3) Repeat the operation in step 2) for reflux liquid B for 1-5 times to obtain reflux liquid C; 4) add NaCl and PEG6000 to the reflux liquid C obtained in step 3), mix well, let stand, centrifuge, and collect the precipitate , that is. With the concentration and purification method of the invention, the removal rate of miscellaneous proteins can reach more than 95%, and the content of endotoxin is obviously reduced.

Description

technical field [0001] The invention relates to a method for concentrating and purifying an avian influenza antigen and an avian influenza antigen, belonging to the field of antigen concentrating and purifying. Background technique [0002] Avian influenza (Avian Influenza, AI) is caused by Orthomyxoviridae type A influenza virus, which is a very harmful infectious disease to humans and poultry. Various forms such as mild respiratory disease caused by low-pathogenic virus strains, egg production decline to acute pathogenic diseases caused by highly pathogenic strains. Since 1988, H9N2 subtype AI has continued to occur in most parts of my country. In recent years, in the investigation of avian influenza in some commercial laying hen farms and chicken professional villages in my country, it was found that H9 subtype AIV positive chickens accounted for 93.67% of the total AIV antibody positive chickens, indicating that H9 subtypes exist in most areas of my country AIV is still...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/02C12N7/00
CPCC12N7/00C12N2760/16121C12N2760/16151
Inventor 徐进骆爱芳丁少成郭晓庆谢梅梅李厚伟樊慧丽
Owner 河南后羿生物工程股份有限公司
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