Method and special primer for rapidly breeding ST (swine testis) sensitive cell lines of hog cholera virus lapinized virus strain (C strain)

A technology for sensitive cells and swine fever rabbits, applied in the field of bioengineering, can solve problems such as unsatisfactory immune effect, and achieve the effect of overcoming low culture titers

Active Publication Date: 2016-08-24
JINYUBAOLING BIO PHARMA CO LTD
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0005] The object of the present invention is to provide a kind of method of the sensitive cell line of ST (pig testis cell) of rapid breeding of classical swine fever latheization strain (C str...

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  • Method and special primer for rapidly breeding ST (swine testis) sensitive cell lines of hog cholera virus lapinized virus strain (C strain)
  • Method and special primer for rapidly breeding ST (swine testis) sensitive cell lines of hog cholera virus lapinized virus strain (C strain)
  • Method and special primer for rapidly breeding ST (swine testis) sensitive cell lines of hog cholera virus lapinized virus strain (C strain)

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Embodiment

[0049] Embodiment, rapid selection of classical swine fever rabbitization attenuated strain (C strain) ST sensitive cell line

[0050] The present invention uses RT-PCR technology and real-time fluorescent quantitative RT-PCR technology to rapidly select the method for the sensitive cell line of classical swine fever rabbitization strain (C strain) ST (pig testicular cell), comprising the following steps:

[0051] 1) Obtain ST monoclonal cells

[0052] ST cells (purchased from the American Culture Collection (ATCC)) without exogenous virus contamination were incubated at 37°C, 5% CO 2 Cultured in the incubator for 48h, digested with 0.2% (mass / volume, W / V, unit g / 100mL) trypsin into single scattered cells, then counted the cells, and then used 15% (volume / volume, V / V, Unit mL / 100mL) Serum is diluted in DMEM medium to a cell concentration of 10-100 cells (cells) / mL (generally, trypsin digestion solution and serum addition amount only need to mark the ratio), and pipette Add s...

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Abstract

The invention discloses a method for rapidly breeding ST (swine testis) sensitive cell lines of a hog cholera lapinized virus strain (C strain) by virtue of a reverse transcription PCR (RT-PCR) (reverse transcription-polymerase chain reaction) technology and a real-time fluorescent quantitative RT-PCR technology. The method comprises the following steps: 1) acquiring ST monoclonal cells; 2) inoculating the hog cholera lapinized virus strain (C strain); 3) extracting RNAs of various batches of hog cholera lapinized virus liquid (virus liquid) which are continuously harvested, and determining that ST monoclonal cell lines can continuously infect the hog cholera lapinized virus strain (C strain) by virtue of the RT-PCR technology; and 4) determining that the hog cholera lapinized virus strain of which the number of copies is 10<6> copies/mL or above are the ST sensitive cell lines of the hog cholera lapinized virus strain (C strain) by virtue of the real-time fluorescent quantitative RT-PCR technology. With the application of the method disclosed by the invention, the sensitive cell lines can be rapidly provided for the production of hog cholera lapinized virus vaccines, so that a vaccine production cycle is shortened and vaccine cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method for rapidly selecting and breeding ST-sensitive cell lines of the attenuated classical swine fever rabbit strain (strain C) by using RT-PCR technology (i.e., reverse transcription PCR) and real-time fluorescent quantitative RT-PCR technology method. Background technique [0002] Classical swine fever virus (CSFV) is an acute, febrile, highly contagious and fatal severe infectious disease caused by classical swine fever virus (CSFV). This disease is the swine infectious disease with the most occurrence, the greatest harm and the widest prevalence. In recent years, the number of dead pigs due to disease in China accounts for 8%-10% of the total number of pigs raised, and one-third of them are caused by swine fever. The World Organization for Animal Health (OIE) lists swine fever as one of the 16 statutory infectious diseases in category A, and my country also defi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/686C12Q1/701
Inventor 商晓桂张贵刚郝鹏魏学峰刘国英范秀丽韩志玲宋志刚孔彩萍王艳杰
Owner JINYUBAOLING BIO PHARMA CO LTD
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