Method for preparing avian influenza virus and inactivated vaccine thereof with Vero passage cells

A technology of avian influenza virus and subcultured cells, which is applied in the field of preparation of avian influenza virus inactivated vaccines and inactivated vaccines using VERO subcultured cells. The effect of production costs

Active Publication Date: 2012-07-18
哈药集团生物疫苗有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the use of chicken embryos to produce influenza vaccines can only be incinerated to deal with the used chicken embryo residues, which not only increases costs, but al

Method used

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  • Method for preparing avian influenza virus and inactivated vaccine thereof with Vero passage cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 utilizes VERO passage cell to produce influenza virus inactivated vaccine

[0035] 1. Preparation of immobilized trypsin by chitosan nanoparticle embedding method

[0036] (1) Weigh chitosan with a degree of deacetylation of 85%, dissolve it with 1% acetic acid to make a chitosan solution with a concentration of 0.5-2 mg / mL, and pass it through a 0.22 μm filter. Weigh trypsin, dissolve it in deionized water to make a trypsin solution with a concentration of 500 μg / mL, and pass it through a 0.22 μm filter. Weigh sodium tripolyphosphate, dissolve it with deionized water to make a sodium tripolyphosphate solution with a concentration of 1.0-2.0 mg / mL, and pass it through a 0.22 μm filter.

[0037](2) Take 5 mL of chitosan solution, add 2.5-7.5 mL of 500 μg / mL trypsin solution dropwise, and stir magnetically for 3 minutes to obtain solution A;

[0038] (3) Solution A was magnetically stirred at 900-1300 r / min for 30 seconds at room temperature and under steri...

Embodiment 2

[0057] Embodiment 2 Utilize the vaccine produced by the method of the present invention to compare with the vaccine produced by chicken embryo

[0058] In order to verify the production performance of the new method, so while utilizing chicken embryos to prepare avian influenza inactivated vaccine (prepared according to the method described in Experimental Example 1.), VERO cells were also used to prepare 10 batches of inactivated vaccine (according to Example 1 Described method preparation.), now the comparison result of some of them indicators is summarized as follows, as described in table 1-6:

[0059] Table 1 Comparison of agglutination value of semi-finished erythrocytes by two production methods

[0060]

[0061] Table 2 Semi-finished product EID of two production methods 50 Compare (lg EID 50 )

[0062] batch 1 2 3 4 5 6 7 8 9 10 Embryo 7.4 7.5 7.7 8.0 8.2 8.4 8.0 6.8 6.9 8.1 cell seed...

experiment example 1

[0073] Experimental example 1 utilizes chicken embryo to produce influenza virus

[0074] 1. Preparation of H9 subtype avian influenza virus HY strain virus solution

[0075] (1) Inoculation: get the H9 subtype avian influenza virus HY strain as the seed virus for production, and properly dilute it with sterilized physiological saline (10 -3 ~10 -4 ), inoculate 0.1 mL into the allantoic cavity of each embryo, seal the pinhole after inoculation, and continue incubation at 36-37°C without turning the eggs.

[0076] (2) Incubation and observation of chicken embryos: After the chicken embryos were inoculated, the chicken embryos that died 48 hours ago were discarded. After that, the eggs were illuminated once every 6 hours, and the dead chicken embryos were taken out at any time until 96 hours, all the embryos were taken out, and placed in 2-8°C to cool for 4-24 hours.

[0077] (3) Harvesting of virus fluid: Take out the cooled chicken embryo, disinfect the air cell part with t...

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Abstract

The invention discloses a method for preparing an avian influenza virus and an inactivated vaccine thereof with Vero passage cells. According to the method disclosed by the invention, the Vero passage cells are adopted to replace chicken embryo to culture influenza virus, so that the problems of chicken embryo remnant and exogenous virus inflection are solved and the immunogenicity of the cultured virus is more stable. On the other hand, for avoiding the phenomenon that the cracking of HA is influenced because protease is acted by an inhibitor in a maintaining liquid to inactivate, pancreatinis coated with chitosan and then is added in a cell maintaining liquid, so that the pancreatin is slowly released in the maintaining liquid and the defect that the cracking of hemagglutinin is influenced because the pancreatin is inactivated in the reproduction process of viruses is overcome. In addition, the probability that the cells are polluted since the pancreatin is added for multiple timesis avoided. The virus cultured by the method disclosed by the invention is high in titer and favorable in stability and is suitable for large-scale vaccine production.

Description

technical field [0001] The invention relates to a method for preparing an inactivated vaccine, in particular to a method for preparing an inactivated vaccine of avian influenza virus by using VERO passage cells. The invention belongs to the technical field of animal husbandry and veterinary medicine. Background technique [0002] Avian influenza is an important disease that endangers the development of the poultry industry in the world, especially the outbreak of highly pathogenic avian influenza has brought a devastating disaster to the poultry industry. Since 2004, highly pathogenic avian influenza has broken out successively in the mainland of China, which not only severely hit the domestic poultry industry, but also had a serious impact on my country's economic and trade development. [0003] Since the successful cultivation of influenza virus in chicken embryos in 1933, chicken embryos have become the main source for people to obtain influenza viruses in large quantiti...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/145A61P31/16
Inventor 丁国杰张杨付丽杰李东伟焦利红吴金
Owner 哈药集团生物疫苗有限公司
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