新基因TAHCCP1编码蛋白对NS3TP6基因启动子转录活性的调节

Transcriptional regulation of new gene TAHCCP1-coding protein on NS3TP6 promoter

目的探讨丙型肝炎病毒(HCV)核心蛋白反式激活基因TAHCCP1编码蛋白对NS3TP6启动子转录活性的调节。方法以我室前期克隆的TAHCCP1基因表达谱芯片结果为基础,利用生物信息学方法确定NS3TP6基因的启动子区域(NS3TP6-p),聚合酶链反应(PCR)扩增并克隆至真核报告载体pCAT3-basic中,构建重组报告质粒pCAT3-NS3TP6-p;以该质粒单独或与pcDNA3.1(-)-TAHCCP1共转染肝癌细胞系HepG2细胞,用酶联免疫吸附法(ELISA)检测氯霉素乙酰转移酶(CAT)的表达活性;观察细胞中表达的TAHCCP1蛋白对NS3TP6启动子转录活性的调节。结果构建的重组表达载体pCAT3-NS3TP6-p在HepG2细胞中能够启动报告基因CAT表达,表明克隆的NS3TP6启动子有指导下游基因转录表达的活性;共转染实验中pCAT3-NS3TP6-p与pcDNA3.1(-)-TAHCCP1组CAT的表达活性是对照组的3.1倍,说明TAHCCP1蛋白能够在转录水平反式激活NS3TP6基因启动子活性。结论本实验验证了基因芯片的实验结果准确性,进一步完善了新基因TAHCCP1的生物学功能,为深入理解HCV核心蛋白的反式激活调节机制提供了新的依据。

Objective To investigate the regulatory effects of a new gene TAHCCP1-coding protein transactivated by hepatitis C virus core protein on NS3TP6 gene promoter. Methods Based on the expressing profiles data of TAHCCP1 gene as cloned previously, the sequence of NS3TP6 gene promoter was identified in GenBank by bioinformatics, amplified from HepG2 genome by polymerase chain reaction （PCR）, and then cloned into pCAT3-basic reporter vector. The HepG2 cell line was transfected by pCAT3-NS3TP6-p alone, or co-tranfected by pCAT3-NS3TP6-p and pcDNA3.1 （-）-TAHCCP1. The chloramphenicol acetyltransferase （CAT） activity was detected by an enzyme-linked immunosorbent assay （ELISA） kit and the regulatory effect of TAHCCP1 protein on the transcriptional activity of NS3TP6 promoter was investigated. Results In the transfection experiment of HepG2, pCAT3-NS3TP6-p had higher activity of reporter CAT expression than that of pCAT3-basic, which suggested NS3TP6 promoter has the transcription activity. The expression level of CAT in co-transfection of pCAT3-NS3TP6-p and pcDNA3.1 （-）-TAHCCP1 was 3.1 times as high as that of pCAT3-NS3TP6-p plasmid alone, which indicated that TAHCCP1 protein could transactivate the expression of NS3TP6 gene. Conclusions The results from microarray of TAHCCP1 has been proved accurate, and the biochemical function of novel gene TAHCCP1 has been improved, which bring some new clues for studying the transactivation of HCV core protein.