摘要
目的构建增强绿色荧光蛋白(EGFP)-人乳头瘤病毒16型变异株E7(HPV16-HBE7)重组质粒pEGFP-HBE7,研究HPV16-HBE7蛋白亚细胞定位,为进一步了解其生物学功能奠定基础。方法采用分子克隆技术,将HPV16-HBE7基因克隆在pEGFP-C1表达载体上,用脂质体法导入宫颈癌细胞中;West-ern印迹检测HBE7蛋白的表达;同时借助免疫荧光技术和EGFP-融合蛋白技术,采用激光共聚焦显微镜观察HBE7蛋白的亚细胞定位。结果重组质粒经PCR、酶切和测序鉴定,其目的片段大小、插入位点和核苷酸序列完全正确;结果表明,转染细胞HBE7蛋白的相对表达量其胞浆明显多于胞核;各个时间段HBE7蛋白均以胞浆分布为主,绿色荧光密集点状分布于细胞浆内,而野生株E7(WE7)蛋白分布在核内。结论人乳头瘤病毒16型变异株E7蛋白主要分布在细胞浆内,以胞浆为主的分布可能和HBE7基因发生突变后丢失核定位信号有关。
Objective To construct recombinant plasmid pEGFP-HBE7 and study the subcellular localization of HPV16-HBE7, as a basis for further research on its functions. Methods HPV16-HBE7 gene wase cloned into pEGFP-Cl by molecular cloning and transfected into cervical cancer HeLa cells by liposome. Western blot analysis were used to examine the expression of HBE7 gene. HBE7 subcellular localization was examined by laser confocal microscope by using immunofluores- cent staining and EGFP-protein fusion. Results PCR, enzyme digestion analysis and DNA sequencing showed that the target gene was cloned into expression vector. Western blot analysis revealed the fusion protein HBE7 could be mostly expressed in the cytoplasm of transfected cells. The results of transient transfection showed that HBE7 was primarily distributed in cytoplasm during its expression and green fluorescence was densely scattered in cytoplasm, while wild type E7 protein was concentrated in nucleai. Conclusion HBE7 protein was located in cytoplasm, which may be correlated with its loss of NLS after the mutations of HBE7 gene. The research of HBE7 gene and the function of its protein is still under way.
出处
《医学分子生物学杂志》
CAS
CSCD
2007年第1期1-5,共5页
Journal of Medical Molecular Biology
