Survivin反义RNA/HSP70双基因表达载体的构建及鉴定

Construction and Indentification of Survivin Antisense RNA/HSP70 Double-Gene Expression Vector

目的:构建Survivin反义RNA/HSP70双基因表达载体,为后期转染肿瘤细胞,抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡以及激发机体有效的特异性抗肿瘤免疫应答提供重要的实验材料。方法:应用RT-PCR从Jurkat细胞中获得Survivin cDNA片段,反向插入pIRES2-DsRed2质粒载体中,经酶切和测序鉴定所构建的Survivin反义RNA表达载体是否成功;应用RT-PCR从HepG2细胞中获得的HSP70 cDNA片段,定向插入到pIRES2-DsRed2质粒载体中;经菌落PCR、酶切和测序鉴定所构建的HSP70表达载体是否正确。结果:经酶切和测序鉴定证明Survivin反义RNA表达载体已成功构建;经菌落PCR、限制性酶切和测序鉴定证明HSP70表达载体已成功构建。结论:本实验已成功构建了Survivin反义RNA/HSP70双基因表达载体,为进一步研究提供了实验基础。

Objective：An Survivin antisence RNA/HSP70 double-gene expression vector was constructed to provide important experimental material for the further research,which was used to transfect the tumor cells ,inhibit proliferation of tumor cells ,induce apoptosis of tumor cells and stimulate the specific anti-tumor immune response effectively. Methods ：A cDNA fragment of survivin obtained by RT-PCR was inserted into a plasmid vector named pIRES2-DsRed2 in the reverse direction. Survivin antisense RNA was confirmed using restriction enzyme digesion and DNA sequencing. A HSP70 cDNA fragment which was obtained from the HepG2 cell using RT-PCR was inserted into pPIRES-DeRed2. Results ：It was verified that the Survivin antisense RNA expression vector had been successfully constructed by restriction enzyme digestion and DNA sequencing. By colony PCR, reserietion enzyme digestion and DNA sequencing,HSP70 expression vector had been proven to be successful constructed. Conclusions ： It indicate that an survivin antisense RNA/ HSP70 double-gene vector is successfully estabilished,which laid an experimental foundation for further study.