HCV 1a/1b型嵌合体能在HepG2细胞内复制与表达

Replication and Expression of HCV 1a/1b Chimeric Genome in Transfected HepG2 Cells

采用HCV 1a/1b嵌合体cDNA构建表达质粒转染HepG2细胞,以免疫组化和Western blotting检测HCV蛋白表达,RT-PCR检测HCV正、负链RNA,研究丙型肝炎病毒(HCV)1a和1b型嵌合体全长cDNA在HepG2细胞中的复制和表达。结果证明,转染细胞中检测到分子量约70kDa的HCVNS3蛋白,转染细胞连续传20代,仍能检测到HCV正、负链RNA。表明该HCV嵌合体可以在细胞中复制和表达,HCV 1b型的RNA依赖的RNA聚合酶(RdRp)可以起始含1a型非编码区的病毒复制。HCV 5′端非翻译区第11、12、13、34和35位核苷酸改变可不影响其与核糖体结合。3′非翻译区9400,9403和9407位核苷酸改变,9435位缺失“A”,9409,9410位及9495,9496,9497位分别插入“TT”和“AAT”可不影响RORp的生物活性。本研究对阐明HCV复制和翻译机制有重要意义。

To investigate the replication and expression of Hepatitis C virus(HCV) 1a and 1b chimeric cDNA in HepG2 cells, HCV 1a/1b chimeric cDNA was used to construct an expression plasmid for the transfection of HepG2 cells. HCV protein and HCV genomic RNA and anti-genomic RNA in the transfected HepG2 cells and the culture supernatants were detected by immunocytochemical staining, Western blotting and RT-PCR respectively. The results showed that HCV NS3 protein with about 70kDa was detectable in the transfected HepG2 cells, HCV genomic RNA and anti-genomic RNA were found positive both in the HepG2 cells and the culture supernatants for more than 20 generations. HCV 1a/1b chimera can replicate and express in HepG2 cells, suggesting that the RNA-dependent RNA polymerase (RdRp)of HCV 1b can initiate the replication of HCV containing genotype 1a untranslated region(UTR) The alterations of 5' UTR of HCV 1b at nucleotides 11, 12, 13, 34 and 35 do not influence its binding to ribosome. The 3' UTR at nucleotides 9400, 9403 and 9407, the deletion 'A' at nucleotide 9439 and insertions 'TT' and 'AAT' at nucleotides 9409, 9410 and 9495, 9496, 9497, do not significantly influence the RdRp binding and activities of HCV 1b. This HCV chimeric cDNA can be of value in the studies of HCV replication and expression.