Construction And Identification Of Tumor Cells Specific Expression Vector Of Combination Gene Of HSV-TK And HIL-12 Driven By HTERT Promotor

Objective To do further investigation and explore the novel strategy of gene therapy on tumor, we construct the tumor cells specific expression vector of combination gene of HSV-TK and hIL-12 driven by hTERT promotor. Then try to identify the expression of the vector in cells to make a good preparation for the further research.Methods hTERTp, HSV-TK, IL-12p40 and IL-12p35 gene were amplified in the way of PCR from Hela cell gene bank, pNZTK2, pCMV6-XL4p40 and human spleen bank separately. By the way of SOE, we got IL-12p70 gene and did a point mutant on it. Then the promoter and two separated genes we successfully cloned were cloned on to the vector pShuttle to establish the combination gene expression vector as pShuttle-hTERTp-HSV-TK-linker-IL-12. Subsequently, the expression vector was identified by enzyme digestion and DNA sequencing. Then the expression vector was transfected into cell line 293T in the help of VigoFect and was identified of expression in the way of western-blot and immumofluorescence.Results The hTERTp, HSV-TK and IL-12p70 genes were successfully cloned to the expression vector and identified to be correct by enzyme digestion and sequencing, except the point mutant we successfully made. The constructed tumor cells specific expression vector was transfected to 293T cell. Then western-blot and immumofluorescence test was preformed. The contrast between the experimental group and blank control group demonstrated that the pShuttle-hTERTp-HSV-TK-linker-IL-12 vector was well expressed in 293T cell. Conclusion The pShuttle-hTERTp-HSV-TK-linker-IL-12 combination gene expression vector was successfully constructed and expressed as prediction. It may contribute to the further research of gene therapy on tumor.