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Method for preparing pseudorabies virus

A technology of pseudorabies virus and pseudorabies virus, applied in biochemical equipment and methods, viruses/bacteriophages, microorganisms, etc., can solve the problems of low virus yield, large batch-to-batch difference, low virus titer, etc., and increase vaccine production , Improved safety, and genetically stable effects

Active Publication Date: 2011-02-23
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention attempts to provide a large-scale production method of pseudorabies virus, which can solve the shortcomings of low virus yield, low virus titer and large batch-to-batch difference in the traditional spinner bottle culture mode, and can Eliminate the potential safety hazards of the virus produced in the existing tidal production method in the subsequent production of live vaccines, and achieve high-titer multiple harvests of virus liquid through optimized parameters, greatly improving the yield of the virus

Method used

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  • Method for preparing pseudorabies virus
  • Method for preparing pseudorabies virus
  • Method for preparing pseudorabies virus

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Embodiment 1 Utilizes Vero cells to continuously produce pseudorabies virus in a tidal bioreactor

[0057] Bioreactor: TideCell bioreactor, purchased from Saiyu Cell Technology Co., Ltd.

[0058] Carrier: BioNOC II carrier, purchased from Saiyu Cell Technology Co., Ltd.

[0059] CVD Nucleus Counting Kit: purchased from Saiyu Cell Technology Co., Ltd., Cat. No. AB0100, Lot No. 3007.

[0060] Glucose analyzer: purchased from Saiyu Cell Technology Co., Ltd.

[0061] Pseudorabies virus: Bartha-K61 strain (purchased from China Veterinary Drug Control Institute).

[0062] Medium: DMEM medium was purchased from GIBCO Company (product number: 12800-082, lot number: 1309922).

[0063]Trypsin: purchased from BD-Difco (product number: 215250, lot number: 5213753).

[0064] Fetal bovine serum: purchased from PAA company (product number: A 15-151, batch number: A64905-0972)

[0065] Glucose: purchased from SIGMA Company (article number: 7021, batch number: MFCD00063774).

[00...

Embodiment 2

[0073] Example 2 BHK-21 cells continuously produce pseudorabies virus in a tidal bioreactor

[0074] Cell culture medium: MEM medium was purchased from GIBCO Company (product number: 61100-087, batch number: 678277), and prepared into cell culture medium as described in the product manual.

[0075] Bovine serum: purchased from Hyclone Company (article number: SV30087.02, batch number: NSK0069)

[0076] Pseudorabies virus: Fa gene deletion strain (TK - / gE - / gI - ) (see Chinese patent CN101186902A for this strain, which is deposited in the China Center for Type Culture Collection (CCTCC) with the accession number V200002).

[0077] Other materials and reagents are the same as in Example 1.

[0078] The BHK-21 cells used in this example are deposited in the American Type Culture Collection (ATCC) with the accession number CCL-10. For details, see the ATCC web page http: / / www.atcc.org / ATCCAdvancedCatalogSearch / ProductDetails / tabid / 452 / Default. aspx? ATCCNum=CCL-10&Template...

Embodiment 3

[0080] Example 3 MDBK cells continuously produce pseudorabies virus in a tidal bioreactor

[0081]Cell culture medium: MEM medium was purchased from GIBCO Company (product number: 61100-087, batch number: 678277), and prepared into cell culture medium as described in the product manual.

[0082] Neonatal bovine serum (manufactured by GIBCO, product number 16010-14, batch number 693745)

[0083] Other material and reagent are with embodiment 1.

[0084] In this example, the MDBK cell (NBL-1) was deposited in the American Type Culture Collection (ATCC) with the accession number CCL-22. For details, please refer to the ATCC webpage. http: / / www.atcc.org / ATCCAdvancedCatalogSearch / ProductDetails / tabid / 452 / Default.aspx? ATCCNum=CCL-22&Template=cellBiology. In this embodiment, a tidal bioreactor with a carrier tank of 10 L is adopted. The steps, methods and parameters adopted are basically the same as those in Example 1, and the cell growth solution and cell maintenance solution us...

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Abstract

The invention relates to a method for producing a gene defect pseudorabies virus by culturing a passage cell in a tidal biological reactor. The method comprises the following steps of: (1) subcultring a recovery cell; (2) performing high-density cell culturing by using a specific biological reactor and a carrier system, attaching the cell on a vector, circulating a culture medium, filling oxygen and adjusting pH value and glucose; (3) performing virus infection by using the biological reactor, replacing the culture medium by using a virus culture medium, and inoculating the virus into the virus culture medium so as to ensure that the virus is fully adsorbed on the cell; (4) culturing and amplifying the virus under a specific condition; and (5) harvesting viruses so as to realize mass production.

Description

technical field [0001] The present invention relates to a production method of pseudorabies virus, more specifically, the present invention relates to a method for large-scale production of pseudorabies virus using mammalian cells in a carrier tank of a tidal bioreactor. Background technique [0002] Pseudorabies (PR) is an infectious disease common to a variety of animals, causing acute and fatal infectious diseases in livestock and wild animals, with fever, itching and encephalomyelitis as the main symptoms, and pigs are its natural susceptible hosts. It mainly causes abortion, stillbirth and mummified fetuses in pregnant sows. Piglet infection is mainly neurological symptoms, while adult pigs are mainly recessive infection. This disease has become the main cause of reproductive failure syndrome in breeding pigs. Vaccine immunization is the fundamental measure to prevent and control pseudorabies, and inactivated vaccines, attenuated vaccines and gene deletion vaccines are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02C12N7/00
Inventor 张许科孙进忠乔荣岑
Owner PU LIKE BIO ENG
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