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Antigen of epidemic encephalitis live vaccine and preparation method and application thereof

A technology of Japanese encephalitis and Japanese encephalitis virus, applied in the field of antigens and vaccines containing it, can solve the problems of difficult control of exogenous viruses, many miscellaneous proteins, and large differences between batches, so as to prevent Japanese encephalitis Effects of virus infection, good immunogenicity, and small batch-to-batch variation

Inactive Publication Date: 2017-05-31
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The existing Japanese encephalitis vaccine antigen production methods for pigs include two methods, one is to inoculate mice with Japanese encephalitis virulence, collect the infected mouse brain tissue, and prepare the antigen through tissue homogenization. There are problems such as high biosafety risks, many foreign proteins, and unsuitability for large-scale production; the other is to use primary hamster kidney cells to culture attenuated vaccine strains, which has complex preparation processes for primary cells and control of exogenous viruses. Difficulty, large batch-to-batch differences, etc., especially the low toxicity of the cultured virus, which can only reach 5.5-7.0LgPFU / mL, which increases the production cost of the vaccine

Method used

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  • Antigen of epidemic encephalitis live vaccine and preparation method and application thereof
  • Antigen of epidemic encephalitis live vaccine and preparation method and application thereof
  • Antigen of epidemic encephalitis live vaccine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation and immunogenicity research of Japanese encephalitis virus attenuated strain adapted to Vero cells

[0025] (1) Adaptive passage of virus

[0026] Prepare Vero cell suspension with DMEM cell culture medium containing 8% calf serum, inoculate the culture flask according to the split ratio of 1:3, 37°C, 5% CO 2 Cultivate until the cells form a dense monolayer (about 48 hours in culture) for inoculation. Pour out the cell culture medium in the culture bottle before inoculation, wash it with PBS of pH 7.4 for 3 times, control the residual liquid after the last wash, and then inoculate attenuated Japanese encephalitis virus according to 5% of the culture medium volume strain (SA14-14-2). Adsorb at 37°C for 90 minutes after inoculation. After the adsorption is completed, DMEM maintenance solution containing 2% calf serum is added and incubated at 35°C. The cytopathic condition was observed daily, and the culture was terminated when the cytopathic rat...

Embodiment 2

[0039] Embodiment 2 adopts 3L rotating bottle to prepare Japanese encephalitis live vaccine virus liquid (antigen)

[0040] (1) Antigen preparation

[0041]Technical scheme 1: adopt 3L spinner bottle to cultivate Vero, culture medium is the DMEM cell culture medium that contains 8% calf serum, treat that Vero cell forms monolayer, discard cell culture medium, wash 3 times with the PBS of pH7.4, discard Remove the PBS washing solution; inoculate the F8 Japanese encephalitis virus attenuated strain (SA14-14-2 strain) adapted to Vero cells at 0.2MOI (PFU / cell number), the rotation speed of the bottle machine is 12 rpm, 37°C Adsorb for 1 hour; add 0.22ul membrane filter to sterilize pH7.6 DMEM solution (containing 20mL / L calf serum, 10g / L arginine hydrochloride, 5g / L PEG2000 and 5g / L 4-hydroxyethyl Piperazineethanesulfonic acid), cultured at a constant temperature at 35°C, and the rotation speed of the bottle spinner was 12 rpm; about 3 to 4 days, 75% of the cells had lesions, an...

Embodiment 3

[0051] Embodiment 3 adopts 15L rotating bottle to prepare swine Japanese encephalitis live vaccine

[0052] (1) Antigen preparation

[0053] The Vero cells were cultivated in a 15L spinner bottle, and the culture medium was DMEM cell culture medium containing 8% calf serum. After the Vero cells formed a monolayer, the cell culture medium was discarded, and the PBS with pH 7.4 was used to wash 3 times, and the PBS was discarded and washed. solution; attenuated Japanese encephalitis virus strain of the F8 generation (SA14-14-2 strain) inoculated with Vero cells at 0.02 MOI (PFU / cell number), the rotation speed of the spinner was 20 revolutions / hour, and adsorbed at 37°C for 1h; Virus culture maintenance solution DMEM (containing 20 mL / L calf serum, 10 g / L arginine hydrochloride, 5 g / L PEG2000 and 5 g / L 4-hydroxyethylpiperazineethanesulfonic acid) at pH 7.8, 35 Cultivate at a constant temperature under the condition of ℃, and the rotation speed of the spinner is 20 rpm; when 75%...

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Abstract

The invention relates to animal vaccines, and specifically discloses an antigen of an epidemic encephalitis live vaccine and a preparation method and application thereof. Vero cells are adopted to carry out roller bottle culture on epidemic encephalitis virus attenuated strains (SA14-14-2 strains); virus culture maintenance solution which comprises components such as 4-hydroxyethylpiperazine ethane sulfonic acid, arginine monohydrochloride and PEG2000 is used; the produced antigen has the advantages of being small in batch difference, good in immunogenicity and high in virus titer; and the epidemic encephalitis live vaccine prepared through the antigen is capable of effectively preventing the epidemic encephalitis virus infection of pigs.

Description

technical field [0001] The invention relates to an animal vaccine, in particular to an antigen of a Japanese encephalitis live vaccine and a vaccine containing it. Background technique [0002] The existing Japanese encephalitis vaccine antigen production methods for pigs include two methods, one is to inoculate mice with Japanese encephalitis virulence, collect the infected mouse brain tissue, and prepare the antigen through tissue homogenization. There are problems such as high biosafety risks, many foreign proteins, and unsuitability for large-scale production; the other is to use primary hamster kidney cells to culture attenuated vaccine strains, which has complex preparation processes for primary cells and control of exogenous viruses. Difficulty, large batch-to-batch differences, etc., especially the low toxicity of the cultured virus, which can only reach 5.5-7.0LgPFU / mL, which increases the production cost of the vaccine. Contents of the invention [0003] In orde...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02A61K39/12A61P31/14C12R1/93
CPCA61K39/12A61K2039/5254A61K2039/552C12N7/00C12N2770/24134C12N2770/24151
Inventor 马良周建民郝霖雨韩伟
Owner CHINA ANIMAL HUSBANDRY IND
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