Establishment and Identification of Mammalian Stable Cell Lines Expressing HCMV Proteins pUL23 and pUL49
|Keywords||Human cytomegalovirus Retroviral Liposomal transfection pUL23 pUL49|
Objective: pUL23 and pUL49 of human cytomegalovirus (human cytomegalo virus HCMV) UL23, UL49 gene encoding the protein. UL23 is a viral growth of non-essential gene, its expression product pUL23 virus cortactin. UL49 is a virus gene required for its expression product pUL49 virus cortex protein, both viral protein function is unclear. Usually virus infected host cells after its own protein encoded by the host proteins or viral proteins protein interactions affect virus growth, reproduction, and the pathogenic process. Accordingly, most of the viral protein function places a host cell. Establish a stable expression of the the HCMV pUL23, and pUL49 mammalian cell lines, for the study pUL23 and pUL49, and the interaction of the host cell protein, and its role in the viral life cycle provides useful tools. Human foreskin fibroblast cells (human primary foreskin fibroblasts HFF) and human embryonic lung fibroblast cells (human embryonic lung fibroblasts HELF) is capable of HCMV-infected host cells in vitro, primary cytomegalovirus infection is better, therefore, generally using the original cultured cells as HCMV-infected model. The subject in primary cultured mammalian cells stably expressing exogenous protein cell lines were in-depth discussions. Methods: 1. HCMV pUL23 and pUL49 mammalian cell lines stably expressing establish 1) retroviral infection method ① build retroviral vectors pLEGFP-N1-UL23-FLAG pLEGFP-N-aimed at UL49-MYC; ② packaging containing pLEGFP-N1-UL23-FLAG the pLEGFP-Nl-aimed at UL49-MYC, pLEGFP-N1 retroviral and infected HFF, HELF cell; ③ respectively 100ug/ml, 700ug/ml concentration G418 selection stable expression pLEGFP-N1 -UL23-FLAG. pLEGFP-N1-aimed at UL49-MYC, pLEGFP-N1 HFF HELF cells. 2) the liposomal transfection Act ① construction of eukaryotic expression vector pCDNA3.1 ()-UL23-FLAG pCDNA3.1 ()-UL23-FLAG, pCDNA3.1 (); ② plasmid transfected into HELA cells; ③ 1200ug/ml concentration of G418 selection pCDNA3.1 ()-UL23-FLAG, pCDNA3.1 () HELA cells stably expressing. 2 .. stable expression of HCMV pUL23 and pUL49 mammalian cell lines identified 1) RT-PCR to identify viral proteins pUL23, pUL49 expression extraction of stable cell lines, RNA, done by RT-PCR, electrophoresis observations; 2) Western-blotting identification of viral proteins pUL23, pUL49 expression extraction of the protein of the stable cell lines, with specific antibodies; 3) intracellular immunofluorescence technique identification of viral proteins pUL23 pUL49 the expression in the stable cell lines intracellulare can be detected with an antibody specific pUL23, pUL49 expression. Three virus in HFF stable cell lines detected Growth kinetics 1) the Towne virus strains suggest infection stable expression pUL23, pUL49 HFF cell lines detected; 2) Towne strain of the virus in the stable expression of pUL23 multi pUL49 HFF cell lines step growth curve. Results: 1) by double digestion, PCR and sequencing proved retroviral the carrier pLEGFP-N-UL23-FL AG of pLEGFP-N1-aimed at UL49-MYC and eukaryotic expression vector pCDNA3.1 ()-UL23-FLAG build successful; 2) by observing the retroviral vector pLEGFP-N1 packaged retroviral infected HFF in HELF cells visible green fluorescence, indicating the success of the retroviral packaging; 3) stable cell lines by RT-PCR detected able to express the gene at the RNA level; 4) Western-blotting detection stable cell lines expressing the gene at the protein level; 5) immunofluorescence technique detected in the stable cell lines, the intracellular expression of pUL23 pUL49, and The pUL23 extracellular defaults located reported consistent. 6) the HFF stable cell lines the Towne growth dynamics and primary HFF cells did not differ significantly. Conclusion: In this paper, using the method of retroviral infection overcome HFF in HELF primary cultured cells transfected with the difficult situation, to build a stable expression HFF pUL23 and pUL49 The, HELF cell lines; RNA and protein levels detected pUL23 and pUL49 expression was observed in the stable cell lines pUL23 mainly localized in the nucleus, pUL23 reported positioning consistent positioning in the cytoplasm, pUL49, HFF stable Towne level of infection and replication in cell lines and primary HFF cells did not differ significantly . Therefore, in this study the stability of the expression the pUL23 and pUL49 HFF and HELF, cell lines can be used as a tool to study the viral proteins pUL23 and pUL49.