稳定表达人巨细胞病毒pUL49蛋白人胚肺成纤维细胞系的建立  

Construction of HCMV UL49 recombinant retroviral vector and establishment of stable cell line

作　　者：宫君原[1] 肖小平[1] 员月明[1] 李月琴[1] 张欣[1] 邹弈[1] 李弘剑[1] 周天鸿[1] 

机构地区：[1]暨南大学生命科学技术学院,广东广州510632

出　　处：《中国现代医学杂志》2010年第23期3521-3525,3529,共6页China Journal of Modern Medicine

基　　金：国家自然科学基金重大计划面上项目(No:90608024);国家自然科学基金面上项目(No:30770106)

摘　　要：目的用逆转录病毒载体介导的基因转移方法,建立稳定表达人巨细胞病毒(HCMV)UL49基因的HELF细胞系。方法首先应用RT-PCR从感染HCMV的HELF细胞RNA中扩增UL49基因,克隆到逆转录病毒载体pLEGFP-N1上,以此构建重组逆转录病毒载体pLEGFP-N1-FLAG-UL49,并转染AmphoPack-TM-293细胞,收集逆转录病毒,感染HELF细胞,经G418抗性筛选出阳性细胞。结果经IFA、RT-PCR及Western blot检测到UL49基因在稳定细胞系中表达。结论 HELF-PLEGFP-N1-FLAG-UL49稳定细胞系构建成功。此细胞系为进一步研究HCMV pUL49蛋白的功能提供了一个有力工具。【Objective】 To establish a human embryonic lung fibroblast（HELF） cell line which stably expresses human cytomegalovirus（HCMV） UL49 gene.【Methods】 HCMV UL49 gene was amplified by RT-PCR from HCMV infected HELF cells and then cloned into pLEGFP-N1 vector using standard procedures to develop recombinant plasmid pLEGFP-N1-FLAG-UL49.The packaging cell line,AmphoPackTM-293 cells were transiently transfected by pLEGFP-N1-FLAG-UL49 to generate retrovirus carrying UL49 DNA,which can be used to infect HELF cells.Cells integrated the retrovirus were selected by antibiotic G418.【Results】 Protein encoded by HCMV UL49 gene was detected in the retrovirus-infected HELF cells by IFA,Western blot and RT-PCR assay.【Conclusion】 HELF cell line stably expressing UL49 has been established,which may provide a powerful tool for functional research of UL49.

关 键 词：人巨细胞病毒 逆转录病毒 稳定细胞系 

分 类 号：R34[医药卫生—基础医学] Q737[生物学—分子生物学]