BHK cell line stably expressing hamster TIGAR gene, and construction method and application thereof

A technology for stable expression and construction methods, applied in microorganism-based methods, genetically modified cells, and cells modified by the introduction of foreign genetic material, etc. The effect of increasing cell anti-apoptosis ability, increasing virus titer and prolonging survival time

Active Publication Date: 2019-08-06
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 construction method and application thereof
  • BHK cell line stably expressing hamster TIGAR gene, and construction method and application thereof
  • BHK cell line stably expressing hamster TIGAR gene, and construction method and application thereof

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Effect test

Embodiment 1

[0034] 1. Amplify hamster TIGAR gene and construct plasmid

[0035] 1. Extraction of RNA from BHK cells

[0036] (1) BHK cells were mixed according to 10 6 Cells were seeded in 6-well plates, and after confluence, the cells were lysed with 800 μL trizal and placed in centrifuge tubes.

[0037] (2) Add 200 μL of chloroform to the centrifuge tube, shake and mix thoroughly, and then centrifuge at 12000 rpm for 10 min at 4°C.

[0038] (3) Take the supernatant and put it into a new centrifuge tube, add an equal amount of isopropanol, mix well, put it at -20°C for precipitation for 30min, and centrifuge at 12000rpm for 10min at 4°C.

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

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

[0041] (6) Open the lid and dry it on a clean bench.

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

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

...

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Abstract

The invention relates to a BHK cell line stably expressing a hamster TIGAR gene, and a construction method and an application thereof. The cell line contains the TIGAR gene and has the preservation number of CCTCC NO:C201928. The hamster TIGAR gene is amplified firstly, and a plasmid is constructed; then the constructed plasmid is transferred into 293T cells with PAX and PMD2G plasmids at the sametime to form packaged lentivirus; after the BHK cells are infected by the packaged lentivirus, drug screening is performed, and then enlarged cultivation is performed to obtain the cell line. The cell line can increase the anti-apoptotic ability of cells, prolong the survival time of the cells, increase the viral titer of Newcastle disease in the cells, and successfully construct and screen high-yield cell lines suitable for the reproduction of Newcastle disease virus.

Description

technical field [0001] The invention relates to a BHK cell line stably expressing hamster TIGAR gene and its construction method and application. Background technique [0002] Newcastle disease (ND) is one of the serious diseases that endanger the poultry industry in my country, and a large number of vaccines are used every year to prevent the disease. At present, the vaccines used to prevent Newcastle disease virus in my country are all produced by using chicken embryos. The huge demand for chicken embryos has caused cost pressures on vaccine manufacturers. environmental pressure. The main bottleneck that Newcastle disease vaccine cannot be produced by cell culture is that the virus does not reproduce efficiently in conventional cell lines, and the yield is low. If the virus titer is to achieve the effect of the current chicken embryo vaccine, it needs a large amount of concentration. The economic benefit of producing Newcastle disease vaccine by cell culture method is not...

Claims

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Application Information

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Patent Type & Authority Applications(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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