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Proliferation of H5 subtype avian influenza virus

An avian influenza virus, virus technology, applied in the direction of antiviral agents, viruses/phages, biochemical equipment and methods, etc., can solve problems such as increasing pollution and increasing product costs

Pending Publication Date: 2017-05-31
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the breeding method of avian influenza virus mainly relies on chicken embryos, and the increase of virus content is mainly achieved through the method of concentration, but concentration also significantly increases the cost of the product and increases the chance of contamination

Method used

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  • Proliferation of H5 subtype avian influenza virus
  • Proliferation of H5 subtype avian influenza virus
  • Proliferation of H5 subtype avian influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The avian influenza virus propagation method of the present embodiment comprises the following steps:

[0025] 1. Preparation of virus diluent: add 3g of amino acid combination and 5g of vitamin combination to every 1000ml of phosphate buffer solution, filter and sterilize with 0.22um filter, set aside.

[0026] Phosphate buffer formula of pH7.0 is: NaH 2 PO 4 2H 2 O 1.56g, Na 2 HPO 4 12H 2 O 3.58g, Nacl9g, add water to 1000ml.

[0027] The amino acid combination formula is: 40% aspartic acid, 30% proline and 30% glutamine.

[0028] The vitamin combination formula is: vitamin B1 30%, vitamin B3 40% and vitamin B7 30%.

[0029] 2. The propagation of the virus, the method is as follows:

[0030] 1) Take Re-6, Re-7, and Re-8 strains, respectively dilute them 10,000 times with the above diluent, inoculate 10-day-old SPF chicken embryos, 0.1ml / piece, culture at 36°C, and harvest undead embryonic urine for 72 hours Cystic fluid, collect allantoic fluid, measure HA to...

Embodiment 2

[0038] Vaccines were prepared from the above-mentioned harvested virus fluids for comparison of immune efficacy

[0039] (1) Antigen solution preparation

[0040] 1. Each 100ml of the virus liquid before the propagation and purification in Example 1.

[0041] 2. Each 300ml of the virus liquid before propagation and purification in Example 1 was concentrated to 100ml.

[0042] 3. Each 100ml of the virus liquid after propagation and purification in Example 1.

[0043] (2) Determination of the virus content of the antigen solution The HA titer and EID of the (1) antigen solution were determined respectively 50 . It can be seen from the test results that even if the unpurified virus liquid is concentrated 3 times, its virus content is difficult to reach the virus content of the purified virus.

[0044]

[0045] (3) Inactivation of the antigen solution The above-mentioned virus solutions were inactivated with 0.05% formaldehyde solution for 16 hours, and stored at 2-8°C. Af...

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Abstract

The invention provides a method for proliferation of an H5 subtype avian influenza virus. The H5 subtype avian influenza virus is diluted by a virus diluent and is inoculated into 10-day-old SPF chicken embryo and then the chicken embryo is cultured at 36 DEG C. The allantoic fluid of the chicken embryo not dead within 72 hours is collected and stored at 40 DEG C; the collected allantoic fluid is then diluted with the virus diluent in a ratio of 10 times. The virus liquid with a dilution degree of 10<6> -10<9> is selected to inoculate into a 10-day-old SPF chicken embryo and then the chicken embryo is cultured at 35.5 DEG C. The allantoic fluid of the chicken embryos not dead within 72 hours is collected and then mixed with the allantoic fluid of a high dilution degree and highest HA titer to obtain a seed liquid. The collected seed liquid is Continuously passed down and then is mixed with the allantoic fluid with a high dilution degree and the HA titer of 10log2, 9log2, and 10log2 to obtain the virus solution for the preparation of vaccine. The method of the invention can save the cost of preparing the vaccine and improve the production efficiency, so as to overcome the technical defects of low virus content in the existing strains for preparing vaccine and that the univalent vaccine can only be achieved by the concentration of the multivalent vaccine.

Description

technical field [0001] The invention belongs to the technical field of research and development of poultry biological products, and in particular relates to a method for multiplying H5 subtype avian influenza virus. Background technique [0002] Since 2003, highly pathogenic avian influenza virus has been widely prevalent in our country and many countries in the world, which not only caused huge losses to the poultry industry, but also constituted a huge threat to human health. In order to better prevent the spread of bird flu virus, the Ministry of Agriculture approved 8 enterprises to produce H5 subtype bird flu vaccine. At present, the breeding method of avian influenza virus mainly relies on chicken embryos, and the increase of virus content is mainly realized through the method of concentration, but the concentration also obviously increases the cost of the product and increases the probability of contamination. Therefore, only by increasing the virus content of the st...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/145A61P31/16
Inventor 宫晓孙健徐保娟李陆梅郭伟伟朱艳梅胡潇
Owner YEBIO BIOENG OF QINGDAO
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