Infectious bursal virus and method for propagating bursal virus with chicken embryo cell line and bioreactor to prepare inactivated vaccine and combined vaccine

A bioreactor, bursal disease technology, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., can solve the problems of low culture cell density, increased concentration multiple, incomplete production process, etc. The effect of reducing production cost, increasing virus titer, and reducing labor intensity

Inactive Publication Date: 2011-11-30
POULTRY DISEASE RES INST OF HENAN AGRI UNIV
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Problems solved by technology

The spinner bottle primary cell (chicken embryo fibroblast) culture method is that the cultured cells adhere to the wall and grow on the inner wall of the glass culture flask. Although the operation technology requirements are low and the technology is mature and stable, it is not suitable for the current large-scale animal vaccine production. , mainly as follows: (1) the proliferation titer of infectious bursal disease virus on chicken embryo fibroblasts is not high, and it is necessary to increase the concentration multiple and increase the production cost, so the application effect is poor in practice; (2) the transfection Bottled chicken embryo fibroblast culture cannot adjust the culture conditions in a timely manner, and ca

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  • Infectious bursal virus and method for propagating bursal virus with chicken embryo cell line and bioreactor to prepare inactivated vaccine and combined vaccine

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Effect test

Embodiment 1

[0028] The isolation, cultivation and identification process of embodiment 1 HQ strain

[0029] In the early 1990s, the super-virulent infectious bursa virus that was prevalent in the world was introduced into China, causing a large number of morbidity and death in unimmunized chicks, with a mortality rate as high as 40-60%. In June 1992, the inventor collected the bursa of dead chickens from a chicken farm where infectious bursal disease broke out in a chicken farm in the suburb of Zhengzhou, Henan Province in June 1992 to isolate the virus and obtain a super-virulent strain. , chicken embryo kidney cells, chicken embryo fibroblasts after multiple passages and training, the pathogenicity of cytotoxicity is weakened, and the adaptability to cells is greatly enhanced. The toxic price on fibroblasts, TCID 50 ≤10 -6.0 / 0.1ml; toxicity on DF-1 cells, TCID 50 ≤10 -7.0 / 0.1ml, it is obviously HQ strain; after the cytotoxicity of HQ strain was cloned and purified and tested for ex...

Embodiment 2

[0030] Embodiment 2 The method for preparing inactivated vaccines and joint vaccines by using the torrent reactor Amprotein AP-20C to propagate infectious bursal virus comprises the following steps:

[0031] (1) Select bioreactor: torrent reactor Amprotein AP-20C, volume 7-10L;

[0032] (2) Select chicken embryo-derived passage cell line DF-1, which is a spontaneous immortal cell, a passage cell line that is very suitable for the growth of HQ strain, has no carcinogenicity, and was purchased from ACCT Company in the United States;

[0033] (3) Cultivation and selection of virus species for seedling production: the bursal cell-adapted virus HQ strain cultivated by the Poultry Disease Research Institute of Henan Agricultural University, and the preservation number is CGMCC NO.4935;

[0034] (4) Amplification of cell seed chains: use cell growth solution to cultivate cells for seedling production in square bottles or spinner bottles. Take the cells out of the liquid nitrogen tan...

Embodiment 3

[0048] Example 3 Determination of minimum immune dose of inactivated vaccine for infectious bursal disease in a rapid flow bioreactor cell line

[0049] According to the method and requirement described in above-mentioned embodiment 2, propagate infectious bursal virus liquid (TCID) on DF-1 cell with torrent bioreactor 50 for 10 -8.5 / 0.1ml) prepare three batches of infectious bursal disease inactivated vaccine, each batch is diluted into 1 / 2, 1 / 4, 1 / 8, 1 / 16 feather parts with the oil emulsion that does not contain the normal saline preparation of antigen, Respectively immunize 10 28-day-old SPF chickens with intramuscular injection of 0.5ml / piece, and set up 10 chickens as a control group without any immunization. Blood was collected 30 days after immunization, and the neutralizing antibody and agar antibody level of the bursa of each group were measured, and the virulent BC6-85 strain of chicken infectious bursal disease virus was used for oral inoculation, each 0.2ml (cont...

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Abstract

The invention relates to an infectious bursal disease virus (IBDV) HQ strain CGMCC NO.4935 and a preparation method of inactivated vaccines and combined vaccines for infectious bursal disease (IBD). The preparation method mainly comprises the following steps of: (1) carrying out chain amplification on cell seeds; (2) adding cell growth-promoting liquid into a sterilized bioreactor, and inoculating cells for preparing vaccines for suspension culture; (3) replacing maintenance liquid containing an IBDV strain when cells are of the maximum density, and continuing to culture; (4) collecting viruses in time, and measuring virus titer; and (5) inactivating virus liquid, and preparing inactivated vaccines and combined vaccines thereof for the IBD according to different proportions. The method provided by the invention increases the cell density, improves the virus titer, improves the vaccine titer, reduces the side reaction, reduces the labor intensity, lowers the production cost, improves the controllability of production processes, and ensures the uniformity and stability of product quality. The produced inactivated vaccines and combined vaccines thereof for the IBD have the advantagesof good safety and high immune efficiency and have a complete protective effect on the IBDV attack.

Description

technical field [0001] The invention relates to the technical field of veterinary biological products, in particular to an infectious bursal disease virus and a method for preparing an inactivated vaccine and a combined vaccine by propagating the bursal disease virus with chicken embryo cell lines and a bioreactor. Background technique [0002] Infectious Bursal Disease Virus (IBDV) belongs to the double-stranded RNA virus family and belongs to the double-stranded RNA virus genus. The pathogen mainly destroys the central immune organ of chickens - the bursa of Fabricius, causing the body's immunosuppression, leading to the death of infected chicks and the increase of infectivity to (other) pathogenic factors, and the decline of immune response to (other) vaccines. cause serious harm. At present, the disease is mainly prevented and controlled through vaccination. In addition to immunizing chicks with live vaccines, breeders are usually injected with inactivated vaccines to ...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/12A61K39/295A61P31/14C12R1/93
Inventor 王泽霖王川庆赵军陈陆王新卫杨霞常洪涛王雷周欣余彬辉宋羚羚高焕河
Owner POULTRY DISEASE RES INST OF HENAN AGRI UNIV
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