Swine testicular clone cell line and production method of classical swine fever live vaccine

A live swine fever vaccine and porcine testis technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, animal cells, etc., can solve the problems of low virus titer, immune failure, vaccine contamination, etc., and achieve vaccine purity Good, simplifies the production process and improves the production efficiency

Active Publication Date: 2013-03-13
PU LIKE BIO ENG
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Problems solved by technology

However, in the vaccine production practice, it has been proved that the virus titer of the vaccine produced by the above-mentioned cells is not high, and the production process is unstable. In particular, the virus titer decreases rapidly with the increase of the cell passage number, resulting in a large difference between vaccine batches. Big
[0004] In addition, since bovine viral diarrhea virus and swine fever virus belong to the genus Pestivirus in the Flaviviridae family, the homology of the two viruses is very high, so bovine testicular cells or calf serum contaminated with BVDV will cause vaccine contamination, while my country The infection rate of viral diarrhea virus (BVDV) in the cattle herd is very high. Immunization of pigs with live swine fever vaccine produced by such animal cells often leads to immune failure and serious consequences

Method used

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  • Swine testicular clone cell line and production method of classical swine fever live vaccine
  • Swine testicular clone cell line and production method of classical swine fever live vaccine

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: the screening of ST clone cell and the method utilizing this cell production swine fever live vaccine

[0035] 1. Screening of mother ST cells

[0036] 1. Cell purity test: no mycoplasma contamination

[0037] 2. Exogenous virus detection Take bovine viral diarrhea virus (BVDV) as an example to illustrate the exogenous virus detection method.

[0038] Using PCR technology, bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV) detection was performed on ST cells. Results ST cells should not carry BVDV. Detection method: Digest monolayer ST cells grown in T25 cell flasks with trypsin, harvest the cells, centrifuge at 1000rpm for 10min, discard the supernatant, wash once with PBS, and collect the cell pellet by centrifugation. Refer to Invitrogen's TRIzol manual to extract total cellular RNA. The cDNA obtained after the reverse transcription of the extracted RNA was used as a PCR template, and BVDV-specific primers were used for PCR amplificatio...

Embodiment 2

[0046] Embodiment 2 ST clone cell culture and the method for producing classical swine fever virus

[0047] The obtained highly infectious ST clone cell strain ST-B2 (preservation number CCTCC C2011101) was digested with trypsin, then inoculated to produce seed batch virus synchronously, and cultivated in a 37°C incubator for 3-4d, according to 1:3 (volume ratio) At the same time, the supernatant was harvested as the venom for making seedlings for cryopreservation. The same method was used for at least 15 generations, and the virus supernatant of each generation was harvested as the venom for making seedlings.

[0048] 1. Determination of virus titer of CSFV on positive ST clone cell line ST-B2

[0049] The CSFV virus obtained by the method of passing the virus by carrying the virus was continuously multiplied and cultured on the highly infectious clone cell ST-B2, and the titer of the CSFV virus solution of each generation of the harvest was measured by the indirect immunoflu...

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Abstract

The invention provides a highly classical swine fever virus infective swine testicular clone cell strain ST-B2, which has a preservation number of CCTCC NO.C2011101, and a preparation method of the swine testicular clone cell. The method comprises the steps of: 1) subjecting a swine testicular cell to limited dilution, conducting cell cloning and subcell cloning, thus obtaining a subcellular clone strain; and 2) selecting a subclone strain of the swine testicular cell with a highest classical swine fever virus infection rate, i.e. the highly infective swine testicular clone cell of the classical swine fever virus. The invention also provides a method for preparation of a high titer classical swine fever virus solution and a classical swine fever live vaccine from the swine testicular clone cell. The swine testicular clone cell provided in the invention has a high classical swine fever virus infection rate, and the classical swine fever vaccine produced from the swine testicular cell line ST clone cell strain ST-B2 by a virus-carrying and virus transmission technique has high virus titer, hard exposure to BVDV (bovine viral diarrhea virus) pollution and good pureness. By the virus-carrying and virus transmission technique, a resurgent cell can undergo continuous passage to at least 15 generations. The cell resurrection and virus inoculation times can be reduced, the production process is simplified, and the production efficiency is improved.

Description

technical field [0001] The invention relates to a method and a product for producing a live vaccine of swine fever by animal cells, in particular to a method and a product for producing a live vaccine of swine fever by using a pig testis clone cell line. Background technique [0002] Classical swine fever virus (CSFV) is a highly contagious disease caused by classical swine fever virus (CSFV), which can infect pigs of different ages, sexes and breeds, with a mortality rate as high as 80-90%. Swine fever is classified as a Class A disease by the World Organization for Animal Health, and my country classifies it as a Class I disease. The attenuated swine fever rabbit vaccine successfully developed by Chinese scholars is currently recognized as a safe and effective vaccine in the world. With the help of intensive vaccination of C-strain vaccine and comprehensive prevention and control measures, relevant countries have effectively controlled swine fever and even eliminated swine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N7/02A61K39/187A61P31/14C12R1/91
Inventor 张许科孙进忠
Owner PU LIKE BIO ENG
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