Preparation method and product of avian influenza vaccine

A technology of bird flu and bird flu virus, which is applied in the field of preparation of live bird flu vaccine, can solve the problems of wasting the immune potential of the body, and achieve the effects of improving equipment utilization, good safety, and saving equipment space

Pending Publication Date: 2019-04-26
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

[0004] It has been reported that fully suspended MDCK cells are used to cultivate avian influenza virus, but the cells are heterologo

Method used

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  • Preparation method and product of avian influenza vaccine
  • Preparation method and product of avian influenza vaccine
  • Preparation method and product of avian influenza vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Acclimatization of DF-1 cells adapted to the serum-free medium and growing in a suspension state The DF-1 cells were acclimatized by gradually lowering the serum to adapt to the culture at low concentration of serum. When DF-1 cells growing stably in MEM medium containing 10% newborn bovine serum grow to the mid-logarithmic growth phase, replace them with MEM medium containing 5% newborn bovine serum until the cells grow to 80%-90% At confluence, trypsinize the cells to a cell density of 2.0 x 10 5 The cells / ml were subcultured in MEM medium containing 5% newborn bovine serum. After several generations, the survival rate of DF-1 cells in the MEM medium containing 5% newborn bovine serum was maintained at more than 90%, and maintained a relatively fast growth rate, which was used to further reduce serum acclimation culture. In the same way, DF-1 cells were gradually adapted to the culture condition of MEM containing 1% newborn bovine serum. The DF-1 cells ada...

Embodiment 2

[0032] The preparation of embodiment 2 bird flu vaccines

[0033] 1. The DF-1-XF cells obtained in Example 1 were domesticated and cultured according to the ratio of 0.75×10 6 cells / ml is the initial inoculation density of the cells, inoculated into shaker flasks, and carried out shaking culture in a shaker. After 48 hours of culture, let the cells settle. Discard the supernatant after the cells have completely settled, and add the same volume of virus growth solution; press The avian influenza virus was inoculated with a dose of MOI of 0.01, and TPCK-pancreatin at a final concentration of 4 μg / ml was added at the same time, and the virus liquid was harvested after culturing for 72 hours. Other suspension culture conditions: pH control is 7.2, dissolved oxygen (DO) is 50%, temperature is 36°C, stirring speed is 100r / min;

[0034] 2. Inactivation

[0035] Add the virus liquid that has passed the sterility test into the formaldehyde solution, so that the final concentration of...

Embodiment 3

[0049] The preparation of embodiment 3 bird flu vaccines

[0050] 1. The DF-1-XF cells obtained in Example 1 were domesticated and cultured according to the ratio of 0.75×10 6 cells / ml is the initial inoculation density of the cells, inoculate them into shaker flasks, and carry out shaking culture in a shaker. After 36 hours of culture, let the cells settle. Discard the supernatant after the cells have completely settled, and add the same volume of virus growth solution; press Avian influenza virus was inoculated at an MOI of 0.0001, and TPCK-pancreatin at a final concentration of 8 μg / ml was added at the same time, and the virus liquid was harvested after culturing for 72 hours. Other suspension culture conditions: pH control is 7.2, dissolved oxygen (DO) is 50%, temperature is 36°C, stirring speed is 100r / min;

[0051] 2. Inactivation

[0052] Add the virus liquid that has passed the sterility test into the formaldehyde solution, so that the final concentration of the form...

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Abstract

The invention discloses a preparation method of an avian influenza vaccine. The preparation method comprises the following steps: (1) carrying out acclimation culture on CEF (Chicken Embryo Fibroblast), thus obtaining sub-culture CEF which can adapt to a serum-free culture medium and is in a single-cell suspended growth state; (2) inoculating the sub-culture CEF obtained through acclimation in step (1) to a shake flask for carrying out shaking culture; (3) inoculating an avian influenza virus to the sub-culture CEF after the shaking culture, and carrying out propagation culture of the virus; (4) harvesting a virus solution; (5) preparing the avian influenza vaccine after deactivating the virus solution. According to the preparation method disclosed by the invention, the pollution risk andthe production cost are remarkably reduced, the production time of the avian influenza vaccine is effectively shortened, and the production scale can be conveniently amplified; the prepared avian influenza vaccine is good in safety and high in immunoprotection effect.

Description

technical field [0001] The invention relates to a preparation method of an avian influenza vaccine and a product thereof, in particular to a method for preparing an avian influenza vaccine by adopting a serum-free suspension culture technique and the obtained avian influenza vaccine product, and belongs to the field of preparation of a live avian influenza vaccine. Background technique [0002] Most of the traditional production methods of avian influenza vaccines use the chicken embryo culture system, which is expensive to produce, and the residue may cause allergic reactions; and the chicken embryos used for vaccine production are planned supply, which cannot cope with large-scale outbreaks of avian influenza. Therefore, it is difficult to control the quality and quantity of the avian influenza vaccine produced by the chicken embryo culture system, and it is urgent to develop a continuous, stable, large-scale and economical and effective animal cell culture system to improv...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N7/00
CPCC12N5/0656C12N7/00C12N2500/90C12N2760/16134C12N2760/16151C12N2760/16163Y02A50/30
Inventor 刘鑫莹孙心李建华宋扬闫妍李应鹤李鑫武啸王宁蒋迪
Owner 哈药集团生物疫苗有限公司
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