Stable expression of hepatitis C virus compound multi-epitope gene in the establishment of P815 cell clone.docVIP

 PAGE \* MERGEFORMAT 14 Stable expression of hepatitis C virus compound multi-epitope gene in the establishment of P815 cell clone Author: Wei 3-hua, Yin, Lei Ying-feng, HU Xing-bin, Jing Yang, Xin Lu, Sun Meng-ning, XU Zhi-kai [Keywords:] Hepatitis C virus Establishment of stably transfected P815 cell line expressing multiepitope gene of hepatitis C virus [Abstract] AIM: To construct a eukaryotic expression vector of HCV compound multiepitope gene and obtain a stably transfected P815 cell line. METHODS: The cDNA sequence Ct encoding truncated HCV core gene lacking parts of the carboxylterminal region was amplified by PCR and inserted into shuttle plasmid pDE22. HCV multiepitope gene Em from E2 and nonstructual proteins were synthesized and inserted into pDE22. CtEm was obtained by BamH Ⅰ / Hind Ⅲ and cloned into the eukaryotic expressing vector pcDNA3.1 (-) which was digested by BamH Ⅰ / Hind Ⅲ and the recombinant plasmid pcDNA3.1 (-) CtEm was obtained. After sequencing, pcDNA3.1 (-) CtEm was transfected to P815 with liposome and screened by G418 and obtained the stably transfeced cell line. RESULTS: The eukaryotic expression vector pcDNA3.1 ( -) CtEm was constructed, transfected to P815 and stably expressed HCV compound multiepitope gene, which was confirmed by RTPCR, IFA and Westernblot. CONCLUSION: A eukaryotic expression vector of HCV compound multiepitope gene has been constructed, stably transfected cell line obtained and the target cells established for analyzing CTL function in the study of HCV vaccine. [Keywords] hepatitis C virus; epitope; eukaryotic expression; transfection [Abstract] Objective: To construct hepatitis C virus (HCV) multi-epitope gene eukaryotic expression vector to obtain recombinant plasmid was stably transfected P815 cell clone. Methods: PCR amplified C-terminal part of the core area of the missing gene fragments Ct ; synthetic mimic epitope HCV E2 area, and NS3 ~ NS5 seven T cell epitope genes Em; were cloned into the s