运用重叠PCR技术构建HCV全长cDNA细胞培养适应性突变体

Construction of Full-Length Genome HCV cDNA with Cell Culture-Adaptive Mutations by the OE-PCR

目的:构建含有不同组合细胞培养适应性突变的丙型肝炎病毒(HCV)全长基因组cDNA。方法:根据HCV全长基因组cDNA序列及突变位点设计引物,运用重叠延伸PCR法对ns3和ns5a基因的E1202G、T1280I和S2197P位点进行细胞培养适应性突变,将突变后的片段分别克隆入pBluescriptIIKS(+)、pRSET-A载体,经测序正确后,分别连入含有HCV全长cDNA的质粒的H/FL相应位置,置换原未突变片段,并进行PCR及酶切鉴定。结果:经PCR、酶切和DNA序列测定证实,预期位点发生突变,而其他序列未发生随机突变。结论:构建了含有不同组合细胞培养适应性突变的HCV全长基因组cDNA突变体,为HCV在细胞内复制机制的研究和可稳定复制、表达的HCV体外培养体系的研究奠定了基础。

Objective： To construct full-length genome HCV cDNA clones which had several combinations of cell cuhureadaptive mutation. Methods： Designed the primers according to the full-length genome HCV eDNA sequence and the mutation sites. Cell culture-adaptive mutations were carried out by the method of overlapping extension PCR（OE-PCR） at the sites of E1202G, T1280I and S2197P in the genes of ns3 and nsSa. Then the fragments with mutation genes were individually cloned into pBluescriptIIKS（＋） and pRSET-A vectors. After validation by sequence test, they were transferred into the corresponding locations of H/FL in full-length genome HCV eDNA plasmid to replace the original fragments without mutations and identified by PCR and restriction enzyme digestion. Results： It can be proved that mutations were achieved at the expecting sites by PCR ,the restriction enzyme digestion and the sequence analysis, and there was no random mutation in other sequences. Conclusion： The full-length genome HCV eDNA which had a variety of combinations of cell culture adaption mutation was constructed. And this will provide a foundation for the research of the replication mechanism of HCV and for the establishment of a HCV culture system in vitro with stable replication and expression.