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Bhk cell line stably expressing hamster tigar gene and its construction method and application

A technology for stable expression and construction methods, which can be applied in the direction of microorganism-based methods, genetically modified cells, and cells modified by introducing foreign genetic materials, etc. problems, to achieve the effect of increasing virus titer, prolonging cell survival time, and increasing anti-apoptotic ability

Active Publication Date: 2021-08-03
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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Problems solved by technology

Studies have shown that when there are three mutants in the TIGAR gene, 10RHG 12 (Tigar-DRHG), 193PFK210 (Tigar-DPFK) and (H11A, E102A and H198A), they cannot resist the apoptosis of U2OS cells, and Tigar loses the ability to catalyze fructose -The ability of 2,6-bisphosphatase (Fru-2,6-P2), the deletion of the region 258-261 can still degrade Fru-2,6-P2 in Hela cells, but not with hexokinase (hexokinase, HK) interaction
[0006] Patent document CN103333916A discloses a method for establishing a BHK cell line adapted to chicken Newcastle disease virus proliferation and its application. It adopts traditional eukaryotic plasmid transfection and then antibiotic screening to construct a cell line. The stability of the cell line constructed by this method is Compared with the lentiviral packaging system, the difference is obvious

Method used

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  • Bhk cell line stably expressing hamster tigar gene and its construction method and application
  • Bhk cell line stably expressing hamster tigar gene and its construction method and application
  • Bhk cell line stably expressing hamster tigar gene and its construction method and application

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Effect test

Embodiment 1

[0034] First, amplify hamster TIGAR genes and build plasmids

[0035] 1. Extract the RNA of BHK cells

[0036] (1) BHK cells according to 10 6 The cells were seeded in a 6-well plate, and the cells were cleaved with 800 μl of TrizAl after longering and placed in the centrifuge tube.

[0037] (2) 200 μl of trichloromethane was added to the centrifuge tube, and after mixing, it was mixed with mixed mix, and was centrifuged at 4 ° C, 12000 rpm.

[0038] (3) Take the addition of the new centrifuge tube, and add isopropanol, and then pellets for 30 min, 4 ° C, 12000 rpm for 10 min after mixing.

[0039] (4) Abandon the supernatant, add 1 mL of 75% ethanol 4 ° C, 12000 rpm centrifuge for 10min, and discard the supernatant.

[0040] (5) Repeat step (4).

[0041] (6) Turn on the cover to dry.

[0042] (7) Add a DEPC 33.5 μL of water to dissolve RNA.

[0043] (8) Reverse transcription according to the following system

[0044]

[0045] After 42 ° C, it was putted at 75 ° C for 5 min.

...

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Abstract

The invention relates to a BHK cell line stably expressing hamster TIGAR gene and its construction method and application. The cell line contains the TIGAR gene, and the preservation number is CCTCC NO: C201928. First amplify the hamster TIGAR gene and construct a plasmid; then transfer the constructed plasmid, PAX and PMD2G plasmids into 293T cells at the same time to form a packaged lentivirus; after infecting BHK cells, carry out drug screening, and then expand the culture, that is obtained the cell line. The cell line can increase the anti-apoptotic ability of cells, thereby prolonging the survival time of cells and increasing the virus titer of Newcastle disease in cells; and successfully constructing and screening high-yielding cell lines suitable for the propagation of Newcastle disease virus.

Description

Technical field [0001] The present invention relates to a BHK cell line and its construction method and application of stabilizing hamster TiGAR genes. Background technique [0002] Newcastle Disease (ND) is one of the serious diseases of the poultry in my country, and a large number of vaccines should be used each year. At present, the vaccine used in my country for preventing new city disease viruses is the use of chicken embryos, which has caused cost pressures of vaccine production enterprises. At the same time, the chicken embryo is used as waste treatment, which has caused huge gains. Environmental pressure. The main bottleneck of the new city vaccine cannot be produced by cell culture is that the virus is not high in conventional cell lines, the output is low, and the viral titer is required to achieve the current chicken embryo vaccine. It takes a lot of concentration. Cell cultures have not produced new urban vaccines. Therefore, constructing a cell line supporting the h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867C12N7/00C12R1/91
CPCC12N5/0686C12N7/00C12N15/86C12N2510/00C12N2740/15043C12N2760/18151
Inventor 孟春春丁铲栗永华仇旭升孙英杰谭磊宋翠萍廖瑛刘炜炜
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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