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Method for preparing heat-resisting attenuated virus live vaccine for goatpox by using BHK21-C13 passage cell

A technology of BHK21-C13 and passaged cells, applied in the field of biomedicine, can solve the problems of unverified heat resistance effect and high cost, and achieve the effects of increasing venom titer, reducing batch differences, and reducing operation steps.

Inactive Publication Date: 2017-01-04
JINYUBAOLING BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing heat-resistant protective agent formula one (patent document CN104800842A) is: trehalose 50g / L, skimmed milk powder 20g / L, polyvinylpyrrolidone 20g / L, gelatin 10g / L, arginine 1.5g / L, vitamin C 2.5g / L; Formula 1 was used for goat pox sheep testicular primary cell live vaccine, but the cost was high, and its heat-resistant effect was not verified; Heat-resistant protective agent formula 2 (porcine pseudorabies live vaccine heat-resistant protective agent and frozen Drying Technology Research—Wang Ying: Gansu Agricultural University 2014 Master's Degree Thesis, Chapter 3 Screening of Porcine Pseudorabies Virus Heat-resistant Protective Agents, page 38) (W / V): sucrose 15.2%, enzymatic casein 7.6% , 4.0% gelatin, 1.0% trehalose; 2.0% bovine serum albumin, 2.0% arginine hydrochloride, 2% sodium glutamate, 2.0% sorbitol, and the rest are water for injection; formula two can be used for other viruses ( For example, attenuated live vaccines such as pseudorabies virus, etc., have not been used for goat pox attenuated live vaccines, and it is not yet known whether they are applicable

Method used

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  • Method for preparing heat-resisting attenuated virus live vaccine for goatpox by using BHK21-C13 passage cell
  • Method for preparing heat-resisting attenuated virus live vaccine for goatpox by using BHK21-C13 passage cell
  • Method for preparing heat-resisting attenuated virus live vaccine for goatpox by using BHK21-C13 passage cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Preparation of goat pox serum-free attenuated live vaccine virus liquid with BHK21 subcultured cells

[0055] The present invention provides a method for preparing goat pox serum-free attenuated live vaccine virus liquid by using BHK21 passaged cells by optimizing the culture conditions, that is, using BHK21 passaged cells to inoculate goat pox virus with serum-free culture, and cultivate the obtained goat pox virus liquid Higher TCID can be achieved 50 Titer (the virus content in every 0.1mL virus liquid ≥10 4.5 TCID 50 ).

[0056] The specific method for preparing goat pox serum-free attenuated live vaccine virus liquid with BHK21 passage cells of the present invention comprises the following steps:

[0057] 1. Cell selection and culture

[0058] In the past, goat pox live vaccines were prepared using conventional bovine or sheep testis primary cells and secondary cells as seed cells, but it was impossible to ensure that the bluetongue virus and other i...

Embodiment 2

[0073] Embodiment 2, preparation of heat-resistant goat pox attenuated live vaccine

[0074] The method for preparing the heat-resistant goat pox attenuated live vaccine of the present invention is to add a heat-resistant freeze-drying protective agent to the goat pox serum-free attenuated live vaccine virus liquid obtained in Example 1, and then undergo freeze-drying (freeze-drying) curves to obtain Heat-resistant goat pox attenuated live vaccine with good appearance and easy preservation. The appearance of the product obtained by the conventional freeze-drying curve is not up to standard, and the optimized freeze-drying curve of the present invention obtains a product with a good appearance. Adding heat-resistant protective agent does not have strict requirements on the cold chain: according to the new quality standards, ordinary attenuated live vaccines must be stored below -15°C, while heat-resistant goat pox attenuated live vaccines can be stored at 2-8°C.

[0075] The pre...

Embodiment 3

[0102] Embodiment 3, the detection of heat-resistant goat pox attenuated live vaccine

[0103] Select 3 batches of goat pox virus liquid obtained in Example 1 and culture it with BHK21 subcultured cells to F28 (choose 25-28 generations), and use the method in Example 2 to prepare 3 batches (batch numbers: S001, S002, S003) of heat-resistant type Goat pox attenuated live vaccine (both specifications are 100 doses / bottle), according to the method recorded in the "Regulations of the People's Republic of China for Veterinary Biological Products" inspection procedures for 3 batches of heat-resistant goat pox attenuated live vaccine finished products for routine testing , and increase the safety and heat resistance test of the purebred pregnant sheep of the vaccine. The specific test items and test results are shown in Table 2-Table 4.

[0104] The test results of routine testing items of the heat-resistant goat pox attenuated live vaccine are shown in Table 2. It can be seen that t...

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Abstract

The invention discloses a method for preparing a heat-resisting attenuated virus live vaccine for goatpox by using a BHK21-C13 passage cell. Based on the existing attenuated virus AV41 for goatpox, which has excellent immunogenicity, the method comprises the following steps: culturing 25-28th generations of virus solution with a heterologous BHK21 cell, and then adding an appropriate heat-resisting freeze-drying protective additive to obtain the heat-resisting attenuated virus live vaccine for goatpox. The passage cell vaccine used by the method is superior to the primary cell vaccine in the aspects of virus yield, homogeneity, purity and the like, not only guarantees the effective level of the vaccine and has a better heat-resisting protection effect, but also can ensure the safety of a purebred pregnant goat. As the existing BHK21 cell adopts a mature suspension culture process, the exploration of various parameters of adherent culture lays a solid foundation for the next promotion of suspension process.

Description

technical field [0001] The invention belongs to the preparation method of vaccines in the field of biomedicine, and in particular relates to a method for preparing goat pox serum-free attenuated live vaccine virus liquid with BHK21-C13 subcultured cells, and adding a heat-resistant protective agent to the product to obtain a heat-resistant type for veterinary use Goat pox attenuated live vaccine method. Background technique [0002] Goat pox is a highly contagious disease of goats caused by goat pox virus (GPV). Sick sheep manifested as elevated body temperature, papules or nodules, blisters, visceral pox lesions on the skin and mucous membranes of hairless or less hairy parts, abortion in pregnant ewes, and high mortality. Goat pox is a legally notifiable infectious disease stipulated by the World Organization for Animal Health (OIE), and it is listed as a first-class animal disease in my country. [0003] The existing vaccines for preventing goat pox are mainly attenuate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/275A61K9/19A61K47/26A61K47/42A61K47/18A61P31/20C12R1/93
CPCA61K39/12A61K47/183A61K47/26A61K47/42A61K9/19C12N7/00A61K2039/5254A61K2039/552C12N2710/24234C12N2710/24251
Inventor 韩四娥魏学峰刘国英范秀丽闫聪杜宇荣李超樊雅婷李雪峰张七斤张贵刚
Owner JINYUBAOLING BIO PHARMA CO LTD
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