摘要
目的:建立稳定表达人巨细胞病毒UL23基因的HELF细胞系,研究病毒蛋白在宿主细胞内行为,为进一步研究人巨细胞病毒蛋白pUL23的功能提供依据。方法:通过PCR技术从人巨细胞病毒基因组中扩增出UL23基因,通过分子克隆技术构建重组逆转录病毒表达载体pLEGFP-N1-FLAG-UL23。将该载体导入Am-phoPackTM-293细胞,收获重组逆转录病毒,然后感染HELF细胞,HELF经持续G418抗性筛选后获得稳定表达UL23基因的细胞系。采用激光共聚焦显微镜观察病毒蛋白在细胞内的定位。结果:RT-PCR、Western blotting结果证实病毒基因UL23能够整合到宿主细胞基因组中,并能在宿主细胞中稳定表达病毒蛋白。共聚焦显微镜观察到病毒蛋白pUL23定位于细胞质,处于细胞核周边。结论:利用逆转录病毒载体介导的基因转移技术,成功构建了稳定表达UL23基因的转基因细胞系。该病毒蛋白在宿主细胞质中的定位,提示病毒蛋白发挥功能的空间位于细胞核周边,有利地推进了人巨细胞病毒蛋白pUL23功能研究的进程。
AIM：To establish HELF cell line with stable and effective expression of human cytomegalovirus （HCMV） UL23 gene as a powerful tool to study the situation of viral proteins in host cells in vitro. METHODS：UL23 gene was amplified by PCR from HCMV and then recombinant plasmid pLEGFP-N1 -FLAG -UL23 was constructed by molecular cloning techniques. Artificial retroviral viruses containing HCMV UL23 gene were obtained by transferring plasmid pLEGFP-N1-FLAG-UL23 into the AmphoPackTM-293 package cells. The HELF cells were infected by the artificial retroviral particles. The positive HELF cells which stably expressed the UL23 were obtained by G418 selection. The location of the viral proteins in the cells was observed by laser confocal microscope. RESULTS：UL23 gene was amplified by RT-PCR from the positive HELF cells,and the UL23 fusion protein was expressed correctly and confirmed by Western blotting in the positive cells,indicating that the UL23 gene was stably integrated into the chromosome of HELF cells. The viral UL23 protein expressed in HELF cells was located in the cytoplasm observed by means of laser confocal microscope. CONCLUSION：Transgene cell strain stably carrying UL23 gene is successfully constructed by retroviral gene transfer techniques. The location of HCMV pUL23 in the host cells implicates that this vial protein exerts its function in cytoplasm.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2010年第5期966-970,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30770106)
国家自然科学基金重大计划面上资助项目(No.90608024)
广东省自然科学基金资助项目(No.06025162)
作者简介
通讯作者周天鸿Tel：020-85228003；E-mail：tzth@jnH．edu．cn；
通讯作者 李弘剑Tel：020—85226360；E—mail：tlihj@jnu．edu．cn
