HCV5′NCR和结构蛋白编码序列的克隆及对PA317细胞的转染

Cloning of HCV 5′NCR,C,E1,E2/NS1 sequences and its transfection into the PA317 cell line

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。

Objective The study was performed as basic research preparing for further investigation on HCV gene regulation. Methods The target gene was a 2547bp HCV cDNA (HCV2547) ligated by PCR and inserted into the sma Ⅰ site of pGEM 3Zf(+)previonsly,containing the whole 5′NCR,C,E1,E2/NS1.Using Pst Ⅰ and EcoRI ,the target gene was cut and cloned into the hind Ⅲ site of a retroviral vector LNSX and a recombinant pLHC2574 was yielded thereafter,which was further analysed by polymerase chain reaction (PCR) and sequenced.By means of co precipitation with calcium phosphate,the recombinant pLHC2547 was transfected into PA317 cells.The lines were screened with G418 and the virus titer in supernatant of the colones was determined by NIH3T3 cells.DNA was extracted from transfected cells and tested by PCR with HCV specific primers.RNA was also extracted from the supernatant of cell culture and was tested by RT PCR.Results The virus titer was 2×10 5 CFU/ml.Both the result of PCR,RT PCR showed positive. Conclusion HCV2547 sequence had been integrated into the genomic groups of cells and the target gene had been expressed.