【摘要】 目的应用基因芯片技术对pcDNA3.1(-)和pcDNA3.1(-)-p7分别转染的HepG2细胞的基因表达谱进行分析,筛选能被HCV p7蛋白反式调节的靶基因,研究p7蛋白的生物学功能。方法以含有HCV全基因组的pBRTM质粒作为模板,应用聚合酶链反应(PCR)扩增p7蛋白编码基因片段,以常规的分子生物学技术构建表达载体pcDNA3.1(-)-p7。以脂质体技术转染肝母细胞瘤细胞系HepG2,提取总mRNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1(-)的HepG2细胞进行DNA芯片分析并比较。结果构建的表达载体经过限制性内切酶分析和DNA序列测定,证实准确无误,提取高质量的总mRNA并逆转录成cDNA,进行DNA芯片技术分析。在1152个基因表达谱的筛选中,有1个基因表达水平显著上调,22个基因表达水平显著下调。结论 HCV p7蛋白是一种反式调节因子,p7基因的表达对于肝细胞基因表达谱有显著影响。更多还原

【Abstract】 Objective To investigate the biological functions of hepatitis C virus p7 protein and screen genes regulated by p7 protein by cDNA microarray technique.Methods The HCV p7 coding DNA fragment was amplified with polymerase chain reaction(PCR) with pBRTM plasmid containing the full length of HCV genome as the template.The expression vector of pcDNA3.1-p7 was constructed by routine molecular biological methods.The HepG2 cells were transfected by pcDNA3.1(-) and pcDNA3.1(-)-p7,respectively,using FuGENE6 transfection reagent.Total RNA was isolated and reverse transcribed.The cDNA were subjected for microarray screening with 1152 cDNA probes.Results The expression vector were constructed and confirmed by restriction enzyme digestion and DNA sequencing analysis.High quality mRNA and cDNA were prepared and successful microarray screening was conducted.From the scanning results,one gene was up-regulated and 22 genes were down-regulated by HCV p7 protein.Conclusions HCV p7 protein is a trans-regulator.The expression of p7 protein affect the expression spectrum of HCV infected hepatocytes.更多还原