摘要
目的构建热反应蛋白12(heat-responsive protein 12,HRSP12)慢病毒表达载体pWPI-HRSP12,包装慢病毒颗粒并感染宫颈癌细胞HeLa,分析过表达的HRSP12对HeLa细胞凋亡和增殖的影响。方法用反转录PCR扩增HRSP12全长编码顺序,构建慢病毒表达载体,利用人胚肾细胞HEK293T包装重组的慢病毒颗粒,感染HeLa细胞,用蛋白免疫印迹法检测剪切的半胱氨酸/天冬氨酸蛋白水解酶-3(caspase-3)并用MTS比色法检测HeLa细胞增殖的情况。结果编码全长HRSP12的cDNA片段为414bp,克隆至pWPI-linker载体成功构建了慢病毒表达载体pWPI-HRSP12,包装的慢病毒颗粒能够高效感染HeLa细胞,在细胞内表达Flag-HRSP12融合蛋白。HRSP12的过表达能够引起HeLa细胞caspase-3的剪切体增多,能够抑制HeLa细胞的增殖。结论成功构建了慢病毒表达载体pWPI-HRSP12,初步结果表明,HRSP12可能具有诱导HeLa细胞凋亡、抑制细胞增殖的活性,为进一步研究HRSP12的生物学功能奠定了基础。
Objective To construct a lentiviral vector pWPI-HRSP12 which was packaged into lentiviral particles and infected cervical carcinoma cell line HeLa and to analyze the effect of HRSP12 over-expressing on cell apoptosis and proliferation.Methods HRSP12 full length coding sequence was amplificated by reverse transcription PCR and lentiviral vector pWPI-HRSP12 was constructed.Next,recombinant virus particles were harvested from the culture medium of packaging cell HEK293T.Western blot was used to analyze the expression of Caspase3 and MTS was applied to analyze the cell proliferation.Results It was showed that the lentiviral particles infected HeLa cells with high efficiency and the expressed fusion protein flag-HRSP12 was also detected by Western blot.In addition,the over-expressed HRSP12 lead to the increase of cleaved-Caspase3 in HeLa cells.What＇s more,the over-expressed HRSP12 can inhibit the proliferation of HeLa cell.Conclusion A lentiviral expression vector pWPI-HRSP12 was successfully constructed,and the results showed that over-expressed HRSP12 may induces HeLa cells apoptosis and inhibits their proliferation,and these will lay a solid foundation for the study of biological functions of HRSP12 in the future.
出处
《医学研究杂志》
2014年第10期63-67,共5页
Journal of Medical Research
基金
国家自然科学基金资助项目(81272229)
