Establishment And Identification Of Mammalian Stable Cell Lines Expressing Hcmv Proteins Pul23 And Pul49

Aim:pUL23 and pUL49 are encoded by HCMV UL23 and UL49 gene. UL23 is a non-essential gene for viral's growth, but UL23 is a essential gene for its growth.They are all tegument proteins,but their fuction are unclear. Usually,viral's protein interact with itself or host protein after infecting, affecting virus growth, reproduction and pathogenesis. Therefore, the function venue of the viral protein is in host cell. Construct expression of pUL23 and pUL49 mammalian cell lines Stably, providing an enabling tool for research pUL23 and pUL49 interact with host cell proteins, and its role in the virus life cycle. HCMV can replicate in Human foreskin fibroblast cells and human embryonic lung fibroblast cells. In this article,we discussion a method in depth about to establish expression of foreign protein stably in primer cell lines.Methods:1.Establishment of mammalian stable cell lines expressing HCMV proteins pUL23 and pUL491) Retrovirus infection methodâ‘ Construction of retroviral vector pLEGFP-N1-UL23-FLAG, pLEGFP-N1-UL49-MYC;â‘¡Package contains pLEGFP-N1-UL23-FLAG, pLEGFP-N1-UL49-MYC, pLEGFP-N1 retrovirus and infection of HFF, HELF cells;â‘¢Stable expression pLEGFP-N1-UL23-FLAG, pLEGFP-N1-UL49-MYC, pLEGFP-N1 of HFF and HELF cells were selected by 100ug/ml and 700ug/ml G418.2)Lipofection methodâ‘ Construction of eukaryotic expression vector pCDNA3.1 (+)-UL23-FLAG;â‘¡lipofection pCDNA3.1 (+)-UL23-FLAG, pCDNA3.1 (+) into HELA cells;â‘¢Stable expression pCDNA3.1 (+)-UL23-FLAG, pCDNA3.1 (+) of HELA cells were selected by 1200ug/ml G418;2. Identification of mammalian stable cell lines expressing HCMV proteins pUL23 and pUL491) RT-PCR identification of viral proteins pUL23, pUL49 expression;2) Western-blotting identification of viral proteins pUL23, pUL49 expression;3) Immunofluorescence Identification of intracellular viral proteins pUL23, pUL49 expression in stable cells. 3.Detect viral growth kinetics in HFF stable cell lines1) Detect the infection capability of Towne with HFF cell line which expression pUL23, pUL49 stably.2) Using multi-step growth curve assay to detect the level of virus propagation in stable cell lines.Results:1) The restriction enzyme digestion, PCR and sequencing proved that retro viral vector pLEGFP-N1-UL23-FLAG, pLEGFP-N1-UL49-MYC and eukaryotic expression vector pCDNA3.1 (+)-UL23-FLAG was successfully constructed;2)By observed pLEGFP-N1 retroviral vector packaging the retro virus, infection of HFFand HELF cells showed green fluorescence, indicated successful retrovirus packaging;3) Stable cells were detected the target gene expression at RNA level by RT-PCR;4) were found the target gene expression at the protein level by Western-blotting;5) Immunofluorescence assay showed that pUL23, pUL49 can be expressed within stable cell lines and pUL23 location match with the literature;6) The growth kinetics of Towne were not significantly difference between HFF stable cell lines and primary HFF cells.Conclusion:In this paper, constructed a stable expression of pUL23 and pUL49 of HFF,HELF cells successfully,the methods overcome the problem that primary cells were difficult to tansfection. We detected the expression of pUL23 and pUL49 in both RNA and protein levels,and observed pUL23 was localized in the cytoplasm while pUL49 was localized in the nucleus,.The pUL23 location were consistent with the reported.The replication of Towne between HFF stable cell lines and primary HFF cells with no significant difference. Therefore, HFF and HELF cells which can expression of pUL23 or pUL49 stably can serve as tool to research the function of pUL23 and pUL49.