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

Objective: To construct the prostate-specific double gene expression vector pIRES-PSMAe/p-TK-Cx43 and Lay the foundation for experimental research of gene therapy for prostate cancer. Methods: First, Cx43 gene was amplificated and cloned into pMD19-T Simple vector； Second, HSV-TK gene was synthesized and cloned into multiple clone site(MCS) A of the eukaryotic vector pIRES. The new plamid was named pIRES-TK； Third, PSMAe/p was obtained and cloned into pIRES-TK by replacing CMV promoter. The new plamid was named pIRES-PSMAe/p-TK；Fourth, Cx43 gene was cloned into the MCS B of pIRES-PSMAe/p-TK and the new plamid was named pIRES-PSMAe/p-TK-Cx43. This plasmid was identified by double digestion with SalⅠ/NotⅠand sequenced； Finally, LNcap cells were transfected by the plasmid pIRES-PSMAe/p-TK-Cx43 and the mRNAs expression of HSV-TK gene and Cx43 gene was observed by RT-PCR assay. Results: The plasmids synthesized in this experiment were double digested respectively and the specific bands of the inserted genes were observed by Agarose gel electrophoresis. pIRES-PSMAe/p-TK-Cx43 was in line with the expected design by DNA sequencing. The mRNAs of TK gene and Cx43 gene were successfully expressed by RT-PCR assay after the transfection in LNCaP cells by pIRES-PSMAe/p-TK-Cx43. Conclusion: Double gene expression vector pIRES-PSMAe/p-TK-Cx43 containing HSV-TK gene and Cx43 gene was constructed successfully.