Establishment of Mammary Epithelial Cell Line Transfected with HSP72 and Its Expression Identification

doi:10.7668/hbnxb.2018.06.014

Abstract

Abstract: The objective of present study was to establish stably expressing HSP72 mammary epithelial cell (bMECs) line with expression vector of HSP72 slow virus constructed. The primers for PCR amplification purpose were designed and synthesized according to the template of bovine HSP72 gene sequence and was connected to plasmid virus. The bMECs were infected with the slow virus obtained after packaging. The cells with purpose gene or no-load virus were sorted out with puromycin and nuclear localization were determined by Hochest33342 method. The transfection efficiency was examined under an inverted fluorescence microscope. The positively expressed cell lines were verified by immunohistochemical technique and Western Blot method. The results showed that the titer of the lentivirus carrying HSP72 gene was about 1.0×10⁹ TU/mL, the optimum screening concentration of purinamycin was 2 μg/mL, the bovine HSP72 lentivirus infected cells expressed HSP72, and the infected cow mammary epithelial cells could see green fluorescence under the inverted fluorescence microscope, and the transfection efficiency could be over 90%. The results showed that normal cells and empty carriers infected cells, HSP72 showed low expression, transfected with HSP72 target gene carrier cells, HSP72 high expression and dark brown color, and Western Blot assay proved that the expression of HSP72 in milk gland epithelial cells transfected by lentivirus empty carrier was slightly lower than that of normal transfected cells. There was no statistical difference, but the expression of HSP72 transfected by lentivirus carrier with HSP72 gene was highly expressed, and the difference was very significant (P<0.01). Compared with the mammary epithelial cells transfected with the lentivirus empty carrier, the HSP72 of the cells transfected with the lentivirus carrier with HSP72 gene showed high expression, and the difference was very significant (P<0.01).In conclusion the bovine mammary epithelial cell line, which stably expressing HSP72 protein, was successfully established. It laid solid foundation for further studying HSP72 function in inflammatory response of bovine mammary epithelial cells.

Key words: HSP72, Stable transfection, Lentivirus vector, Mammary epithelial cells of dairy cows

CLC Number:

S823.03
Q78

YU Wenhui, ZHAN Xizhen, FENG Yanni, CAO Rongfeng, JIANG Zhongling, LI Huatao, TIAN Wenru. Establishment of Mammary Epithelial Cell Line Transfected with HSP72 and Its Expression Identification[J]. ACTA AGRICULTURAE BOREALI-SINICA, doi: 10.7668/hbnxb.2018.06.014.

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http://www.hbnxb.net/EN/Y2018/V33/I6/103

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