HPV18 E5蛋白稳定表达株的筛选及对细胞增殖和周期的影响

Screening of the Stable Expressing HPV18 E5 Protein Cell Line and its Influence on Cell Proliferation and the Cell Cycle

本研究筛选能稳定表达HPV18E5蛋白的细胞株,并检测HPV18E5蛋白对细胞增殖及周期的影响。通过PCR方法扩增得到HPV18E5基因,经酶切和连接构建含His标签的pSecTag-HPV18E5真核表达质粒。运用脂质体介导的方法将重组质粒转染Balb/c3T3细胞,并用含有博来霉素的培养基筛选阳性细胞株。利用Western blot和免疫酶法鉴定HPV18E5在细胞中的表达。运用CCK-8法、流式细胞术检测HPV18E5对细胞增殖及细胞周期的影响。结果显示pSecTag-HPV18E5真核表达质粒构建成功;重组质粒转染Balb/c3T3细胞后,经400μg/mL博来霉素筛选21d,得到稳定表达HPV18E5蛋白的细胞株;与对照组相比,HPV18E5稳定表达细胞株的细胞增殖能力明显增强,细胞周期中S期的比值明显增加。研究表明HPV18E5对细胞增殖及细胞周期有影响。本结果为进一步深入研究HPV18E5蛋白的生物学特性及致癌作用奠定了基础。

We wished to screen the cell line that stably expresses the HPV18E5 protein, and to ascertain the influence of HPV18E5 protein on cell proliferation and the cell cycle. The HPV18E5 gene was amplified by the polymerase chain reaction. Then, the His-tag pSecTag-HPV18E5 eukaryotic expression vector was constructed by digestion ligation and connection. The recombinant plasmid was transfected into Balb/c3T3 cells with lipofectamine, and positive cell lines were screened by a culture medium containing bleomycin. HPV18E5 expression in cells was confirmed by western blotting and immuno-enzymatic methods. The in- fluence of HPV18E5 on cell proliferation and the cell cycle were detected by Cell Counting Kit 8 and flow cytometry, respectively. The pSecTag-HPV18E5 eukaryotic expression vector was constructed. After 21- day selection in a culture medium containing 400 μg/mL bleomycin, stably expressing HPV18E5 protein cells were harvested. Compared with control groups, cell proliferation in HPV18E5 stably expressed cells was obviously increased, as was the S phase in the cell cycle. Our results suggested that HPV18E5 influ ences cell proliferation and the cell cycle. Our study has laid the foundation of the biologic properties of HPV18E5 protein, which will aid further studies on the mechanism of action of carcinogenesis.