Establishment of stably transfected P815 cell line expressing multiª²epitope gene of hepatitis C virus

    WEI Sanª²Hua, YIN Wen, LEI Yingª²Feng, HU Xingª²Bin, YANG Jing, L¦a Xin, SUN Mengª²Ning, XU Zhiª²Kai

    Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct a eukaryotic expression vector of HCV compound multiª²epitope gene and obtain a stably transfected P815 cell line. METHODS: The cDNA sequence Ct encoding truncated HCV core gene lacking parts of the carboxylª²terminal region was amplified by PCR and inserted into shuttle plasmid pDE22. HCV multiª²epitope gene Em from E2 and nonstructual proteins were synthesized and inserted into pDE22. CtEm was obtained by BamH¢ñ/Hind ¢ó and cloned into the eukaryotic expressing vector pcDNA3.1(-) which was digested by BamH¢ñ/Hind ¢ó and the recombinant plasmid pcDNA3.1(-)ª²CtEm was obtained. After sequencing, pcDNA3.1(-)ª²CtEm was transfected to P815 with liposome and screened by G418 and obtained the stably transfeced cell line. RESULTS: The eukaryotic expression vector pcDNA3.1(-)ª²CtEm was constructed, transfected to P815 and stably expressed HCV compound multiª²epitope gene, which was confirmed by RTª²PCR, IFA and Westernª²blot. CONCLUSION: A eukaryotic expression vector of HCV compound multiª²epitope gene has been constructed, stably transfected cell line obtained and the target cells established for analyzing CTL function in the study of HCV vaccine. ......