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

Abstract

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. Key words: Prostatic neoplasms Genetic vectors; HSV-TK/GCV; Prostate specific membrane antigen promoter/enhancer(PSMAe/p); Connexin 43