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Efficient proliferation method and application of H5 subtype avian influenza virus

An avian influenza virus, high-efficiency propagation technology, applied in the biological field, can solve the problems of high mortality of chicken embryos, low chicken embryo culture titer, etc., and achieve the effects of increasing the number of live embryos, reducing the risk of contamination, and increasing the virus content

Active Publication Date: 2020-12-15
广东永顺生物制药股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Based on the low culture titer of chicken embryos of existing H5 subtype avian influenza virus and the high mortality rate of chicken embryos after inoculation, the present invention aims to provide a method for efficiently multiplying H5 subtype avian influenza virus, said method It can increase the virus content in allantoic fluid and the production of allantoic fluid during chicken embryo culture, and at the same time reduce the mortality rate of chicken embryos, which has positive significance for the improvement of production efficiency

Method used

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  • Efficient proliferation method and application of H5 subtype avian influenza virus
  • Efficient proliferation method and application of H5 subtype avian influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Seed virus dilution is on the impact of chicken embryo production recombinant avian influenza virus H5N1 subtype Re-11 strain

[0025] The company found in research and development that aspartic acid, ZnCl 2 and epidermal growth factor have a certain influence on the growth of chicken embryos and the proliferation of viruses in chicken embryos. In order to obtain the most suitable seed virus dilution and improve the effect of virus proliferation in non-free chicken embryos, the company optimized aspartic acid acid, ZnCl 2 and the amount of addition of epidermal growth factor, through early research and development, it is found that the most suitable concentration of aspartic acid is 1.5 mg / mL, therefore, in the present invention, further ZnCl 2 and epidermal growth factor and its dosage have been further optimized test, the specific test design is as follows:

[0026] The culture method of H5 subtype avian influenza virus, after the H5 subtype avian infl...

Embodiment 2

[0036] Example 2: Efficient propagation method of recombinant avian influenza virus H5N1 subtype Re-11 strain

[0037] Step 1, get the H5 subtype avian influenza virus seed and do 10-fold serial dilution with the seed virus dilution liquid, get 10 -4 Inoculate 10-11-day-old non-immune chicken embryos into the allantoic cavity with diluted virus liquid, 0.1ml per embryo, and continue to incubate at 37°C after inoculation, without turning the eggs;

[0038] Step 2: After inoculation of the chicken embryos, put eggs twice a day, discard the chicken embryos that died before 24 hours, take out the dead chicken embryos at any time after 24 hours, and take out all the live embryos at 72 hours, and the air chamber is upright , placed in a refrigerator at 2-8°C for 12-24 hours.

[0039] Step 3: Take out the chicken embryos that have been cooled for 12 to 24 hours, and after surface disinfection, push them into the harvesting operation room, use a fully automatic harvester to absorb th...

Embodiment 3

[0048] Embodiment 3: the method for H5N1 subtype avian influenza virus inactivated vaccine

[0049] Step 1, get recombinant avian influenza virus H5N1 subtype Re-11 strain virus seed and do 10 times serial dilution with seed virus dilution, get 10 -4 Inoculate 10-11-day-old non-immune chicken embryos into the allantoic cavity with diluted virus liquid, 0.1ml per embryo, and continue to incubate at 37°C after inoculation, without turning the eggs;

[0050] Step 2: After inoculation of the chicken embryos, put eggs twice a day, discard the chicken embryos that died before 24 hours, take out the dead chicken embryos at any time after 24 hours, and take out all the live embryos at 72 hours, and the air chamber is upright , placed in a refrigerator at 2-8°C for 12 hours.

[0051] Step 3: Take out the chicken embryos that have been cooled for 12 hours, and after the surface is sterilized, push them into the harvesting operation room, use a fully automatic harvester to absorb the ch...

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Abstract

The invention relates to an efficient proliferation method and application of an H5 subtype avian influenza virus, and mainly relates to improvement of a virus seed diluent. According to the efficientproliferation method and application of the H5 subtype avian influenza virus, a phosphate buffer solution is used as a basis, and aspartic acid, ZnCl2 and an epidermal growth factor are added, so that efficient proliferation of the H5 subtype avian influenza virus in SPF chicken embryos and non-immune chicken embryos is realized; in a vaccine preparation process, an antigen meeting a production standard can be obtained only by centrifuging and filtering treatment, and the virus content and HA titer which are the same as those of an antigen solution concentrated by a traditional production process can also be achieved; and the level of an immunized HI antibody has no significant difference from that of an HI antibody obtained by the traditional process. Compared with the traditional process, the efficient proliferation method has the advantages that concentration treatment is not needed, so that the production cost, particularly the cost of concentration equipment and a concentration process, is reduced, the production efficiency is improved, and the pollution risk in product production can be reduced.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a high-efficiency propagation method and application of an H5 subtype avian influenza virus. Background technique: [0002] Avian Influenza (Avian Influenza) is a severe infectious disease of poultry caused by the Orthomyxoviridae Influenza A virus, which is classified as a Class I infectious disease by the OIE. Death, can also infect mammals and humans. According to the difference in the pathogenicity of avian influenza virus, avian influenza can be divided into low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI), in which the H5 subtype is one of the HPAI, and the bird is the subtype of the H5N1 virus. As a natural host, studies have shown that the H5N1 avian influenza virus can break the species barrier, making mammals more likely to cause disease and causing a pandemic. Different countries in the world adopt culling or vaccine immuniz...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/145A61P31/16C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/552A61P31/16C12N7/00C12N2760/16134C12N2760/16151
Inventor 林德锐陈坚李永红韩翡巫月生涂玉蓉李静肖永珍余玲娜陈汉林
Owner 广东永顺生物制药股份有限公司
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