HCV p7蛋白反式调节新基因3及其剪切体克隆化和生物信息学分析

目的:筛选HCV p7病毒蛋白反式调节的新靶基因p7TP3,克隆p7TP3基因及其不同剪切体基因序列,并进行功能分析,探讨丙型肝炎病毒(HCV)p7病毒蛋白在HCV致病过程中的作用．方法:依据我们构建的真核表达载体pcDNA 3．1(-)-p7转染肝母细胞瘤细胞系HepG2,提取总RNA,逆转录为cDNA后进行基因表达谱芯片分析,利用生物信息学技术获得新基因 p7TP3及其可变剪切体的编码序列,设计特异性引物,并对其进行克隆化研究. 结果:经测序鉴定获得新基因p7TP3的编码序列,并发现了p7TP3的不同剪切体,对 D7TP3基因组进行分析,获得剪切体的编码序列,并进行了克隆化研究及生物信息学分析．p7TP3(416 bp)编码蛋白质序列比 p7TP3(477 bp)少TQNTDVYPGLAVFFQNAL IFFSTLIY 26个氨基酸残基,其余序列相同．结论:HCV p7蛋白是一种典型的病毒基因组编码的具有反式调节作用的蛋白．利用分子生物学技术与生物信息学分析,发现并鉴定了HCV p7TP3反式调节作用的新的靶基因 p7TP3及其剪切体,为阐明HCV p7蛋白的反式调节作用及其机制开辟了新的研究方向．

Cloning and bioinformatic analysis of new gene p7TP3 and its spliced variant transregulated by hepatitis C virus p7 protein

AIM: To identify and clone a new gene p7TP3 and its spliced variant transregulated by hepatitis C virus p7 protein, and to explore the role of HCV p7 protein in the pathogenesis of HCV infection. METHODS: Based on the construction of expressive vector pcDNA3.1(-)-p7, hepatoblastoma cells HepG2 were transfected with plasmid DNA of pcDNA3.1(-)-p7, and then the total RNA was extracted from the cells. Reverse transcribed cDNA was subjected for microarray assay. The coding sequence of the new gene and its spliced variant were obtained by bioinformatic methods. Polymerase chain reaction (PCR) was conducted to amplify p7TP3 gene and its spliced variant using specific primer. The structure was detected and the function was predicted by bioinformat-ics methods. RESULTS: Sequence analysis confirmed that the coding sequence of p7TP3 gene and its spliced variant were cloned successfully and bioinfor-matically analyzed. p7TP3 (416 bp) with Mr 14 165.4 is identical to p7TP3 (477 bp) with Mx 17 935.7 except a deletion of 26 amino acid residue (TQNTDVYPGLAVFFQNALIFFSTLIY). CONCLUSION: HCV p7 protein is a potential transregulator. A novel gene and its spliced variant have been identified as the new target trans-regulated by HCV p7 protein.