Construction And Identification Of Tissue Specific Eukaryotic Expression Vector For Suicide Gene Combined With Cvtokine Gene

Objective To construct five kinds of double promoter eukaryotic expression vectors, each contains HSV-TK gene droved by UPII promoter and combine with IL-2、TNF-α、TRAIL、IL-2-TNF-α、IL-2-TRAIL respectively.Methods UPII cDNA、TK cDNA、IL-2cDNA、TNF-p cDNA、TRAIL cDNA were amplified by polymerase chain reaction(PCR). Two fusion genes IL-2-TNF-a (IT)、IL-2-TRAIL(ITR) were respectively constructed by overlap-PCR. All of the gene fragments were subcloned into pMD18-T vector.The recombined plasmids were transformed into competent cell TOP10F’. Identify the recombinants by PCR, digestion and DNA sequencing.Correct sequencing will be subcloned in pBudCE4.1in which two strong promoters CMV and EF-la were employed. EF-la promoter was replaced with UPII promoter, and then a new vector named pBud-UPII/CMV was constructed. The restriction enzymes were employed for digestion to verify the validity of pBud-UPII/CMV. TK was subcloned in the backward position of UPII promoter and the new recombined plasmid was named pBud-UPII-TK/CMV. IL-2, TNF-α、TRAIL、IT、ITR were subcloned in the backward position of CMV promoter respectively. The five new eukaryotic expression vectors pBud-UPII-TK/CMV-IL-2、 pBud-UPII-TK/CMV-TNF-α、pBud-UPII-TK/CMV-TRAIL pBud-UPII-TK/CMV-IT、pBud-UPII-TK/CMV-ITR were constructed.These recombined vectors were transformed into competent cell TOP10F’respectively, and then were identificated by PCR and digestion.Results PCR、digestion and DNA sequencing showed that the amplification of UPII promoter、TK、IL-2、TNF-α、TRAIL was successful. The results of PCR and digestion showed that the EF-1α promoter of pBudCE4.1was replaced with UPII promoter, so the construction of pBud-UPII/CMV was successful. The identification of pBud-UPII/CMV、pBud-UPII-TK/CMV-IL-2、pBud-UPII-TK/CMV-TNF-α、 pBud-UPII-TK/CMV-TRAIL、pBud-UPII-TK/CMV-IT was positive result.Conclusion Five kinds of double promoter eukaryotic co-expression vectors (pBud-UPII/CMV、pBud-UPII-TK/CMV-IL-2、pBud-UPII-TK/CMV-TNF-α pBud-UPII-TK/CMV-TRAIL、pBud-UPII-TK/CMV-IT) were successfully constructed. This research achievement has prepared the experimental ground for the further study, which could detect the expression of vectors in bladder cancer cells and these vectors’effect on the treatment of bladder cancer.