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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。
OBJECTIVE: To establish a HELF cell line stably expressing human cytomegalovirus UL23 gene and to study the behavior of the virus protein in host cells, so as to provide basis for further study on the function of human cytomegalovirus protein pUL23. Methods: The UL23 gene was amplified from human cytomegalovirus genome by PCR and the recombinant retroviral vector pLEGFP-N1-FLAG-UL23 was constructed by molecular cloning technique. The vector was introduced into Am-phoPackTM-293 cells, recombinant retrovirus was harvested and then infected with HELF cells. HELF cells were screened with G418 for stable expression of UL23 gene. Laser confocal microscopy was used to observe the localization of the viral protein in the cells. Results: The results of RT-PCR and Western blotting confirmed that the UL23 gene could be integrated into the host cell genome and could stably express the viral protein in the host cells. Confocal microscopy showed that the viral protein pUL23 was localized in the cytoplasm and located in the periphery of the nucleus. Conclusion: The transgenic cell line stably expressing UL23 gene was successfully constructed by retroviral vector-mediated gene transfer technology. The localization of the viral protein in the host cytoplasm suggests that the viral protein functions in the space surrounding the nucleus and favorably advances the functional study of human cytomegalovirus protein pUL23.