前列腺特异性双基因表达载体pIRES-PSMAe/p-TK-Cx43的构建及鉴定

目的 构建前列腺特异性双基因表达载体pIRES-PSMAe/p-TK-Cx43,为前列腺癌基因治疗实验研究奠定基础. 方法获取Cx43基因并克隆至pMD19-T Simple载体;合成HSV-TK基因并克隆到pIRES载体的MCS A中,得pIRES-TK;获取PSMAe/p并克隆至pIRES-TK中替换CMV启动子,得到pIRES-PSMAe/p-TK;将Cx43基因克隆到pIRES-PSMAe/p-TK的MCS B中,得到pIRES-PSMAe/p-TK-Cx43,对此质粒双酶切鉴定并测序.pIRES-PSMAe/p-TK-Cx43转染人前列腺癌细胞株LNcap,RT-PCR观察TK及Cx43基因表达. 结果合成的各质粒经双酶切、琼脂糖凝胶电泳可见目的 基因条带;pIRES-PSMAe/p-TK-Cx43经测序与预期设计相符.pIRES-PSMAe/p-TK-Cx43转染LNCaP细胞,RT-PCR显示成功表达TK及Cx43 mRNA. 结论成功构建了含HSv-TK及Cx43的双基因表达载体pIRES-PSMAe/p-TK-Cx43.

Construction and identification of prostate-specific double gene expression vector pIRES-PSMAe/p-TKCx43

Objective To construct the prostate-specific double gene expression vector pIRESPSMAe/p-TK-Cx43 and establish the foundation for experimental prostate cancer gene therapy research. Methods Cx43 gene was amplified and cloned into pMD19-T Simple vector. HSV-TK gene was then synthesized and cloned into multiple clone site (MCS) A of the eukaryotie vector plRES. The new plasmid was named plRES-TK: PSMAe/p was obtained and cloned into plRES-TK by replacing CMV promoter. The new plasmid was named plRES-PSMAe/p-TK; Fourth, Cx43 gene was cloned into the MCS B of pIRES-PSMAe/p-TK and the new plasmid was named pIRES-PSMAe/p-TK-Cx43.This plasmid was identified by double digestion with Sal Ⅰ/Not Ⅰ and sequenced; Finally, LNCaP cells were transfected by the plasmid plRES-PSMAe/p-TK-Cx43 and the mRNAs expression of HSV-TK gene and Cx43 gene was tested by RT-PCR. Results The plasmids synthesized in this experiment were double digested respectively and the specific bands of the inserted genes were confirmed by RTPCR. plRES-PSMAe/p-TK-Cx43 was in line with the expected design by DNA sequencing. The mRNAs of TK gene and Cx43 gene were expressed and successfully confirmed by RT-PCR after LNCaP cells transfected with pIRES-PSMAe/p-TK-Cx43. Conclusion Double gene expression vector pIRES-PSMAe/p-TK-Cx43 containing HSV-TK gene and Cx43 gene is constructed successfully.