含热休克蛋白70和绿色荧光蛋白双顺反子慢病毒载体的构建与鉴定

目的构建并鉴定人热休克蛋白70(Hsp70)和增强型绿色荧光蛋白(EGFP)基因双顺反子慢病毒表达载体,以探索Hsp70对细胞保护作用的影响。方法通过基因重组技术将人Hsp70连接到含EGFP的慢病毒载体中,包装并收集病毒,感染人胰腺癌细胞系Aspc-1细胞后,通过荧光显微镜观察EGFP的表达,同时利用反转录-聚合酶链反应(RT-PCR)和蛋白质印迹(Westernblot)技术检测Hsp70基因的表达情况。结果酶切和测序鉴定Hsp70-IRES-EGFP双顺反子慢病毒载体构建正确,包装病毒感染细胞后荧光显微镜观察到细胞发绿色荧光,RT-PCR和western blot检测均表明感染病毒后细胞Hsp70的表达量明显增加。结论成功构建了Hsp70-IRES-EGFP双顺反子慢病毒载体,为后续研究Hsp70的功能和作用机制奠定了一定的基础。

Construction and identification of bicistronic lentivirus expression vector for Hsp70 and EGFP gene coexpression

Objective To construct a bicistronic lentivirus expression vector carrying Hsp70 gene and EGFP, and detect its expression in human cancer cells after infection. Methods Hsp70-IRES-EGFP bicistronic lentivirus expression vector was construeted by linking human Hsp70 cDNA with IRES-EGFP lentivirus vectors. Virus were packed by co-transfecting pMDL,pVSVG, prey plasmid with Hsp70-IRES-EGFP vector followed by infecting Aspc-1 cells. The infected cells were observed under fluorescence microscope for detecting EGFP expression, and measured by RT-PCR and western blot for detecting Hsp70 gene expression. Results The bicistronic lentivirus expression vector eoexpressing Hsp70 and EGFP genes was constructed and verified by DNA se- quencing. The EGFP and Hsp70 gene expressions were detected in the infected cells. Conclusions The Hsp70-IRES-EGFP bicistronic lentivirus expression vector has been constructed suecessfully,wilich has＇laid some foundation for studying the function of Hso70 and its mechanism.