新基因NS5ATP4表达产物下调细胞周期素B2基因表达的研究

Down-regulation effect of NS5ATP4 on transcription activity of cyclin B2 gene promoter

目的探讨新基因NS5ATP4表达产物对细胞周期素B2(cyclinB2)基因启动子转录的调节作用。方法根据文献确定细胞周期素B2的启动子区域,聚合酶链反应(PCR)扩增细胞周期素基因因启动子(B2p),克隆至真核报告基因表达载体pCAT3Basic中,构建pCAT3cyclinB2p报告载体;以该质粒转染肝癌细胞系HepG2细胞系,用酶联免疫吸附法(ELISA)检测报告基因氯霉素乙酰转移酶(CAT)的表达活性并与pcDNA3.1()NS5ATP4共转染HepG2细胞系,用ELISA法检测CAT的表达活性。结果成功获得细胞周期素B2启动子的正确克隆。pCAT3cyclinB2p和pcDNA3.1()NS5ATP4共转染的HepG2细胞的CAT表达活性是pCAT3Basic空载体的10倍,pCAT3cyclinB2p的0.14倍。结论本文克隆的细胞周期素B2启动子有顺式调节下游基因表达的活性,NS5ATP4对细胞周期素B2基因的转录具有下调作用。

Objective To investigate the transregulating effect of NSSATP4 on cyclin B2 gene promoter. Methods Polymerase chain reaction （PCR） technique was employed to amplify the DNA sequence of cyclin B2 promoter using HepG2 cell genomic DNA as template, and the product was subcloned into pCAT3-Basic at Kpn I and Bgl II sites, the resulted vector was named pCAT3-cyclin B2p. pCAT3-cyclin B2p was transfected into the hepatoblastoma cell line HepG2, then cotransfected with pcDNA3.1 （-）-NSSATP4 by FuGENE 6 transfection reagents. The HepG2 cells transfected with pCAT3-Basic was used as negative control. The activity of CAT in HepG2 cells transfected was detected by an enzyme-linked immunosorbent assay （ELISA） kit after 48 hour, which reflected the transregulating function of pcDNA3. 1 （-） ： NSSATP4 on cyclin B2p gene promoter. Results The report vector pCAT3-cyclin B2p has been constructed and had been confirmed by restriction enzyme digestion and DNA sequencing. The expression of CAT in HepG2 cells co-transfected with pCAT3- cyclin B2p and pcDNA3.1（-）-NSSATP4 was 10 times as higher as that of pCAT3-Basic, and 0.14 as higher as that of pCAT3-cyclin B2p. Conclusion NSSATP4 can clown-regulate the expression of cyclin B2 gene.