摘要： 为了构建HSP72慢病毒表达载体，建立稳定表达HSP72的牛乳腺上皮细胞系。以牛HSP72基因序列为模板，设计并合成引物，PCR扩增目的基因，连接到慢病毒表达质粒中，用包装获得的慢病毒感染牛乳腺上皮细胞，经一定浓度的嘌呤霉素筛选，Hochest33342法对细胞核定位，倒置荧光显微镜下观察转染效率，用免疫组化和Western Blot方法验证感染细胞是否稳定表达HSP72。结果显示，携带HSP72基因的慢病毒滴度约为1.0×10⁹ TU/mL；确定嘌呤霉素的最佳筛选浓度为2 μg/mL；牛HSP72慢病毒感染细胞表达HSP72，感染的奶牛乳腺上皮细胞在倒置荧光显微镜下可看到绿色荧光，转染效率可达90%以上；免疫组化结果表明，正常细胞和空载体感染细胞，HSP72呈现低表达，转染携带HSP72目的基因载体细胞，HSP72高表达，呈现颜色较深的棕色；Western Blot方法检测证实与正常转染细胞相比，慢病毒空载体转染的奶牛乳腺上皮细胞HSP72表达稍有降低，但无统计学差异，而携带HSP72基因的慢病毒载体转染的细胞HSP72呈现高表达，且差异极显著（P<0.01），与慢病毒空载体转染的奶牛乳腺上皮细胞相比，携带HSP72基因的慢病毒载体转染的细胞HSP72呈现高表达，且差异极显著（P<0.01）。成功建立稳定表达HSP72的牛乳腺上皮细胞系，可为研究奶牛乳腺炎分子调控机制以及抗性分子药物筛选提供新的理论依据和工作基础。

关键词: HSP72, 稳定转染, 慢病毒载体, 奶牛乳腺上皮细胞

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

中图分类号: 

• S823.03

• Q78