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缺乏合适的ＨＣＶ感染细胞及小动物模型，是抗ＨＣＶ药物研究和开发的主要障碍之一。建立一种ＨＣＶ５＇ＮＣＲ调控荧光素酶基因的转基因细胞模型，可为以ＨＣＶ５＇ＮＣＲ及Ｃ基因５＇端为靶的反义寡核苷酸及特异性核酶等抗ＨＣＶ药物的评价及筛选创造条件。将ＨＣＶ５＇ＮＣＲ－Ｃ基因与荧光素酶基因的融合基因片段插入ｐＣＩ－ｎｅｏ表达载体，通过ＰＣＲ扩增、酶切反应、质粒大小检测及荧光素酶瞬间表达活性鉴定等试验，获得ＨＣＶ５＇ＮＣＲ调控荧光素酶的稳定表达质粒ｐＨＣＶ－ｎｅｏ４。将ｐＨＣＶ－ｎｅｏ４转染ＨｅｐＧ２细胞，经Ｇ４１８筛选，从１５０个克隆中获得一个荧光素酶活性表达为１４４３ＭＶ的细胞株（ＨｅｐＧ２．９７０６）。该株细胞内ＨＣＶ片段的ＤＮＡ及ｍＲＮＡ检测均呈阳性，接种培养板后，细胞荧光素酶活性表达持续１４４小时，无明显降低。该株细胞已传６０代，活性稳定表达已超过６个月
The lack of suitable HCV-infected cells and small animal models is one of the major obstacles to the research and development of anti-HCV drugs. To establish a transgenic cell model of HCV 5 ’NCR regulatory luciferase gene for evaluation and screening of anti-HCV drugs such as antisense oligonucleotides and specific ribozymes targeting on the 5’ end of HCV 5 ’NCR and C genes Create conditions. The fusion gene fragment of HCV 5 ’NCR-C gene and luciferase gene was inserted into pCI-neo expression vector, and HCV 5’ NCR was obtained through PCR amplification, restriction enzyme digestion, plasmid size detection and luciferase transient expression assay, Regulated luciferase stable expression plasmid pHCV-neo4. HepG2 cells were transfected with pHCV-neo4 and screened by G418 to obtain a cell strain (HepG2.9706) with a luciferase activity of 1443 MV from 150 clones. The DNA and mRNA of the HCV fragment in the strain were all positive. After inoculation of the plate, the luciferase activity of the strain persisted for 144 hours without any significant decrease. The strain has been passed 60 generations, the activity of stable expression has been more than 6 months