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目的进一步研究丙型肝炎病毒５′ＮＣＲ对结构蛋白编码基因的表达调控。方法以拼接好的ＨＣＶ５′ＮＣＲ，Ｃ，Ｅ１和Ｅ２／ＮＳ１区基因共长２５４７个核苷酸片段克隆于逆转录病毒载体ＬＮＳＸ得重组体ｐＬＨＣ２５４７，用聚合酶链反应（ＰＣＲ）及核苷酸序列分析等方法对重组体进行鉴定。通过磷酸钙－ＤＮＡ共沉淀法把经鉴定的重组体转染入ＰＡ３１７细胞，用Ｇ４１８进行克隆筛选，以ＮＩＨ３Ｔ３细胞测其病毒滴度。提取转染细胞ＤＮＡ及培养上清ＲＮＡ分别用ＰＣＲ和逆转录ＰＣＲ进行鉴定。结果培养上清的病毒滴度为２×１０５ＣＦＵ／ｍｌ，ＰＣＲ及逆转录ＰＣＲ（ＲＴ－ＰＣＲ）分别可以从转染的细胞ＤＮＡ及培养上清中扩增出ＨＣＶ的基因片段。结论目的基因已整合到细胞的基因组中并得以表达
Objective To further study the regulation of the expression of structural protein coding genes by the 5 ’NCR of hepatitis C virus. Methods A total of 2547 nucleotide fragments of 5 ’NCR, C, E1 and E2 / NS1 genes were cloned into the recombinant retrovirus vector LNSX pLHC2547 and sequenced by polymerase chain reaction (PCR) and nucleotide Sequence analysis and other methods to identify the recombinant. The recombinant plasmids were transfected into PA317 cells by calcium phosphate-DNA co-precipitation method, cloned and screened by G418, and the virus titers were measured by NIH3T3 cells. The DNA of transfected cells and the supernatant RNA were extracted and identified by PCR and reverse transcription PCR. Results The titer of virus in culture supernatant was 2 × 105CFU / ml, and the gene fragment of HCV was amplified from the transfected cell DNA and culture supernatant by PCR and reverse transcription PCR (RT-PCR). Conclusion The target gene has been integrated into the cell genome and expressed