稳定表达HCV 1a NS5A和NS5A-ΔISDR细胞株的构建  

Construction of HeLa Cell Lines Stably Expressing HCV 1a NS5A and NS5A-ΔISDR

作　　者：廖继佩[1] 薛小平[1] 徐志凯[1] 尹文[1] 雷迎峰[1] 吕欣[1] 杨敬[1] 

机构地区：[1]第四军医大学微生物学教研室,陕西西安710032

出　　处：《生物技术通讯》2005年第1期1-4,共4页Letters in Biotechnology

基　　金：国家自然科学基金项目(30371280)

摘　　要：以pBRTM/HCV-3011模板,通过PCR法扩增ns5a、LISDR(左端序列)和RISDR(右端序列),分别经XhoⅠ/EcoRⅠ、XhoⅠ/HindⅢ和EcoRⅠ/HindⅢ双酶切,连入穿梭质粒pEGFP-N3中,构建重组质粒pEGFP-N3-ns5a和pEGFP-N3-ns5a-ΔISDR。对重组质粒进行酶切分析和序列测定。将这2种重组质粒通过电穿孔法转染HeLa细胞,然后在G418选择压力下进行有限稀释法筛选,用RT-PCR和荧光显微镜鉴定。经酶切鉴定和基因测序证实,重组穿梭质粒已插入目的片段ns5a、LISDR和RISDR。RT-PCR和荧光显微镜检测到目的基因的表达。以上结果说明成功构建了真核表达载体pEGFP-N3-ns5a和pEGFP-N3-ns5a-ΔISDR,目的基因在HeLa细胞中得到表达,为研究HCVNS5A中是否存在抗干扰素治疗的功能蛋白提供了实验材料。The gene fragments of ns5a, LISDR and RISDR were amplified from pBRTM/HCV-3011. The ns5a was cloned into XhoI and EcoRI sites of pEGFP-N3 to construct pEGFP-N3-ns5a expressing vector, and LISDR and RISDR were sequentially cloned into XhoI/HindIII and HindIII/EcoRI sites of pEGFP-N3 to construct pEGFP-N3-ΔISDR(ISDR deletion). Then the recombinant eukaryotic expression vectors were identified by enzyme digestion analysis and sequencing. HeLa cells were transfected by electroporation with the two recombinant vectors. Cells were grown for 36 h in the standard media used to propagated each cell lines, after which G418 was added to 400 μg/mL to select for plasmid-bearing cells. HeLa clones expressing target genes were derived by limiting dilution at a concentration of 0.3 cells per well. The expression of NS5A and NS5A-ΔISDR in transfected HeLa cells were verified by RT-PCR and fluorescent microscope. Enzyme digestion analysis and DNA sequencing results showed that target genes have been cloned into the eukaryotic expression vector pEGFP-N3. The expression of NS5A and NS5A-ΔISDR have been demonstrated by RT-PCR and fluorescent microscope. The recombinant eukaryotic expression vectors have been constructed and expressed successfully in the transfected HeLa cells. It is possible to use HeLa cell lines stably expressing HCV NS5A and NS5A-ΔISDR to detect whether the resistance to interferon therapy of some HCV genotypes is related to ns5a.

关 键 词：丙型肝炎病毒 干扰素敏感决定区 HELA细胞系 增强型绿色荧光蛋白 丙型肝炎 真核表达载体 

分 类 号：R512.63[医药卫生—内科学] R373.21[医药卫生—临床医学]