Method for large-scale production of chicken Marek's 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, high quality and good virus quality. Effect

Active Publication Date: 2011-11-30
PU LIKE BIO ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of this, the main purpose of the present invention is to provide a large-scale production method o

Method used

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  • Method for large-scale production of chicken Marek's virus
  • Method for large-scale production of chicken Marek's virus
  • Method for large-scale production of chicken Marek's virus

Examples

Experimental program
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Example Embodiment

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

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

[0046] (1) Prepare chicken embryo fibroblast (CEF) seed solution to make the total number of cells reach 2.0×10 9 One.

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

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

[0049] Liquid surface rise rate (up rate) 2600ml / min, liquid surface top residence time (hold time) 15s, liquid surface fall rate (down rate) 2600ml / min, liquid surface bottom residence time (hold time) 10s; adsorption process lasts 4h ; Switching cell culture program: liquid surface rise rate (up rate) 2...

Example Embodiment

[0056] Example 2 Correlation comparison of different culture systems for breeding chicken Marek's disease virus

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

[0058] 1) To produce chicken Marek’s virus in a 3000ml spinner flask, first prepare chicken embryo fibroblasts (CEF) at a final concentration of 1.5×10 6 Inoculate cells / ml to 3000ml spinner flask, add cell growth solution to 300ml. Place the cells in a greenhouse at 37°C for 5r / h culture on a spinner flask. In this way, the cells will be inoculated with chicken Marek's virus for 24h. Before inoculation, pour out the cell growth solution in the 3000ml spinner flask and inoculate the cells according to the multiplicity of infection 0.02 , Add cell maintenance solution to 300ml, place it in a greenhouse at 37°C and inc...

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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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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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