pEGFP-IFI16表达载体构建及其表达对Hep-2细胞增殖的影响  

Construction of pEGFP-IFI16 Expression Vector and the Influence of Its Expression on Hep-2 Cell Proliferation

作　　者：虞游[1] 张艳欧[1] 赵文铖[1] 白坚[1] 陈建明[1] 

机构地区：[1]杭州师范大学生命与环境科学学院,浙江杭州310036

出　　处：《杭州师范大学学报（自然科学版）》2013年第1期35-39,共5页Journal of Hangzhou Normal University(Natural Science Edition)

基　　金：浙江省钱江人才计划项目(2010R10062)

摘　　要：RT-PCR扩增的IFI16基因连接到pUCm-T载体并测序鉴定,经测序正确的IFI16基因再连接到pEGFP-C1载体构建pEGFP-IFI16重组质粒,重组质粒pEGFP-IFI16转染Hep-2细胞后用荧光显微镜及半定量RT-PCR分析其表达情况,并用流式细胞仪测定细胞生长曲线分析其表达对Hep-2细胞增殖的影响.结果显示pEGFP-IFI16重组质粒构建正确,转染Hep-2细胞后荧光显微镜下观察到绿色荧光信号,半定量RT-PCR结果显示转染后Hep-2细胞IFI16基因条带亮度明显升高,细胞生长曲线测定结果显示转染后Hep-2细胞从第二天起其增殖速度变慢、至第三天时其增殖速度明显慢于对照细胞的增殖速度.说明成功构建了能在Hep-2细胞中表达EGFP-IFI16融合蛋白的pEGFP-IFI16重组质粒,pEGFP-IFI16重组质粒体表达能抑制Hep-2细胞的增殖.IFI16 gene amplified by RT-PCR was ligated into the pUCm-T vector and confirmed by sequencing, and then ligated into the pEGFP-C1 vector to construct the pEGFP-IFI16 recombinant plasmid. The recombinant plasmid pEGFP- IFI16 transfected into Hep-2 cells and its expression was analyzed with fluorescent microscope and semi-quantitative RT- PCR, and the influences of its expression on Hep-2 cell proliferation was further analyzed through assaying cell growth curve using flow cytometry. The results showed that the pEGFP-IFI16 recombinant plasmid was correctively constructed, and after transfeeting Hep-2 cells, the green fluorescent signals was observed with fluorescent microscope, and the semi- quantitative RT-PCR result showed that the band brightness of IFH6 gene was obviously increased after transfection in Hep-2 cells, meanwhile it was found from cell growth curve assay that after transfection Hep-2 cell proliferation was slowed down from the second day on and to the third day the cell proliferation rate was obviously slower than that of the control cells. All results illustrated that the pEGFP-IFI16 recombinant plasmid expressing EGFP-IFI16 fusion protein in Hep-2 cells was successfully constructed and its expression could inhibit Hep-2 cell proliferation.

关 键 词：IFII6基因 EGFP-IFI16融合蛋白 HEP-2细胞 增殖 

分 类 号：Q253[生物学—细胞生物学] R363.1[医药卫生—病理学]