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Large-scale production method of fowl Marek's disease virus

A technology for chicken Marek's disease and Marek's disease, which is applied in the field of large-scale production of chicken Marek's disease, can solve the problems of poor yield and quality of chicken Marek's disease vaccine, and achieves low cost, controllable conditions and strong immunity. Effect

Active Publication Date: 2013-07-31
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of this, the main purpose of the present invention is to provide a large-scale production method of chicken Marek's disease virus, to solve the problem of poor yield and quality of chicken Marek's disease vaccine

Method used

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  • Large-scale production method of fowl Marek's disease virus
  • Large-scale production method of fowl Marek's disease virus
  • Large-scale production method of fowl Marek's disease virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Large scale culture of chicken Marek's virus

[0045] Using the above-mentioned tidal reactor, the preparation steps are as follows:

[0046] (1) Prepare chicken embryo fibroblast (CEF) seed liquid, so that the total number of cells reaches 2.0 × 10 9 indivual.

[0047] (2) In the above-mentioned tidal bioreactor, add the CEF cell seed solution described in step (1) to the tank containing the carrier, and add 500L of M199 cell growth containing 4% (v / v) bovine serum to the medium bag solution, and connect the carrier tank and the medium bag at the same time.

[0048] (3) At 37°C, pH 7.2, 3.5% CO 2 To culture cells in the environment, set the culture parameters as follows, cell adsorption procedure:

[0049] The liquid level rise rate (up rate) is 2600ml / min, the liquid level top residence time (hold time) is 15s, the liquid level decline rate (down rate) is 2600ml / min, the liquid surface bottom residence time (hold time) is 10s; the adsorption procedure ...

Embodiment 2

[0056] Embodiment 2 The correlation comparison of different culture system propagation chicken Marek's virus

[0057] The above-mentioned tidal bioreactor was compared with a 3000mL spinner bottle culture for chicken Marek's virus. The conditions and methods used in the spinner culture were: 37°C constant temperature greenhouse, 8r / h culture in a spinner machine.

[0058] 1) To produce chicken Marek's virus in a 3000ml spinner bottle, prepare chicken embryo fibroblasts (CEF) at a final concentration of 1.5×10 6 Inoculate cells / ml into 3000ml spinner bottle, add cell growth medium to 300ml. Put it in a 37°C greenhouse and culture it on a spinner bottle machine for 5r / h. Cultivate the cells in this way until 24h to inoculate chicken Marek's virus. Before inoculation, pour out the cell growth medium in the 3000ml spinner bottle, and inoculate according to the multiplicity of infection 0.02. , supplemented with cell maintenance solution to 300ml, placed in a 37°C greenhouse and c...

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Abstract

The invention discloses a large-scale production method of fowl Marek's disease virus, which uses a bioreactor to produce fowl Marek's disease virus in a cell microcarrier suspension culture system. The method comprises the following steps: inoculating chick embryo fibroblast for preparing virus into a carrier tank containing culture fluid and microcarrier, and evenly mixing the fibroblast and microcarrier, so that the fibroblast is attached to the microcarrier; in a proper culture environment, providing sufficient nutrients and a proper gas environment for the fibroblast, so that the fibroblast can grow to 5-10 times of the inoculation concentration on the microcarrier; changing for a cell maintenance liquid, preparing the fowl Marek's disease virus into a virus suspension, and enabling the virus suspension onto the fibroblast; culturing the virus in a proper culture environment; after continuously culturing for 3-4 days, harvesting the fibroblast to obtain a cell suspension; and filling the cell suspension into an ampoule bottle, sealing the ampoule bottle, putting in a programmed cooling instrument, and storing in liquid nitrogen. The method has the advantages of large production scale, high single-batch yield and lower production cost.

Description

technical field [0001] The invention relates to a large-scale production method of chicken Marek's virus, in particular to a method for large-scale production of chicken Marek's virus by a tidal bioreactor. Background technique [0002] Marek's disease (Marek's disease, MD) is a contagious neoplastic disease of chickens caused by Marek's disease virus (MDV) of the herpesviridae family, characterized by lymphoid tissue hyperplasia and tumor formation, in the peripheral nerve , gonads, iris various organs, muscle and skin mononuclear cell infiltration. While MD causes tumors and death clinically, more importantly, it damages the immune organs of infected chickens, resulting in immunosuppression, resulting in decreased body resistance and reduced or no immune response to vaccination, causing complications of other diseases And secondary infection, causing huge economic losses. [0003] Vaccination is still one of the effective ways to control Marek's disease. A key point in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12R1/93
Inventor 张许科孙进忠白朝勇
Owner PU LIKE BIO ENG
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