摘要
目的：构建丙型肝炎病毒（HCV）多表位基因的真核表达载体，获得重组质粒稳定转染的P815细胞克隆．方法：PCR方法扩增核心区羧基端部分缺失的基因片段Ct；合成HCVE2区模拟表位和NS3～NS5七个T细胞表位基因Em；分别克隆入穿梭质粒载体pDE22中．用BamHⅠ／HindⅢ双酶切pDE22得到复合多表位基因CtEm，再将CtEm亚克隆入真核表达载体pCDNA3，1（-），构建重组质粒pCDNA3．1（-）-CtEm，经酶切鉴定及测序正确后，通过脂质体转染至P815细胞，持续G418压力选择和有限稀释法克隆化获得稳定转染的细胞系，用RT—PCR，IFA，Western—blot证实该稳定转染细胞系可以表达多表位基因．结果：构建了真核表达载体pcDNA3．1（-）-CtEm，建立了稳定转染的P815细胞系．结论：构建HCV多表位基因的真核表达载体，稳定转染P815细胞，为进一步在疫苗研究中分析CTL功能建立了靶细胞．
AIM： To construct a eukaryotic expression vector of HCV compound muhi-epitope gene and obtain a stably transfected P815 cell line. METHODS： The cDNA sequence Ct encoding truncated HCV core gene lacking parts of the carboxyl-terminal region was amplified by PCR and inserted into shuttle plasmid pDE22. HCV multi-epitope gene Em from E2 and nonstructual proteins were synthesized and inserted into pDE22. CtEm was obtained by BarnH 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 muhi-epitope gene, which was confirmed by RT-PCR, IFA and Western-blot. CONCLUSION： A eukaryotic expression vector of HCV compound muhi-epitope gene has been constructed, stably transfected cell line obtained and the target cells established for analyzing CTL function in the study of HCV vaccine.
出处
《第四军医大学学报》
北大核心
2006年第1期38-41,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金(30371280)
关键词
丙型肝炎病毒
表位
真核表达
转染
hepatitis C virus
epitope
eukaryotic expression
transfection
