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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。
This study screened cells stably expressing HPV18E5 protein and detected the effect of HPV18E5 on cell proliferation and cycle. The HPV18E5 gene was amplified by PCR, and the His-tagged pSecTag-HPV18E5 eukaryotic expression plasmid was constructed by digestion and ligation. The recombinant plasmids were transfected into Balb / c3T3 cells by liposome - mediated method, and the positive cell lines were screened with bleomycin - containing medium. The expression of HPV18E5 in the cells was identified by Western blot and immunoenzymatic assay. Using CCK-8 method, flow cytometry was used to detect the effect of HPV18E5 on cell proliferation and cell cycle. The results showed that the eukaryotic expression plasmid pSecTag-HPV18E5 was successfully constructed. The recombinant plasmid was transfected into Balb / c3T3 cells and screened with bleomycin 400μg / mL for 21 days to obtain a stable cell line expressing HPV18E5 protein. Compared with the control group, HPV18E5 was stable Cell proliferation of the cell lines significantly enhanced the cell cycle S phase ratio was significantly increased. Studies have shown that HPV18E5 affects cell proliferation and cell cycle. The results laid a foundation for further study on the biological characteristics and carcinogenesis of HPV18E5 protein.