Antitumor effects of recombinant vectors carrying CDglyTK suicide gene on nasopharyngeal carcinoma cell in vitro / 中国病理生理杂志

ABSTRACT

AIM: To construct the recombinant adenovirus carrying fusion suicide gene CDglyTK with the C promoter(Cp),one of the key factors in controlling Epstein-Barr virus latent gene expression,and to investigate if the Cp mediates the expression of CDglyTK in CNE1 cells and kills the cancer cells specifically.METHODS: The tk,cd,Cp sequences were amplified by PCR and subcloned into corresponding sites of pDC316 vector with directional cloning method to construct the pDC316-CP-CDglyTK.The plasmid was analyzed by DNA sequencing and enzyme digestive method.The recombinant adenovirus of Ad-Cp-CDglyTK was packaged,amplified and purified in 293 cells,and the virus titre was determined by TCID50 method.The CDglyTK gene expression in CNE1 and NP69 were examined by reverse transcription-polymerase chain reaction(RT-PCR) after in vitro transfection in CNE1 and NP69 cells.The killing effect of Ad-Cp-CDglyTK/GCV+5-FC on CNE1 cells was detected by MTT method.RESULTS: The results of restriction enzyme digestion and DNA sequencing showed that the tk,cd,and Cp gene were inserted into the pDC316 plasmid in correct orientations.The titer of the recombinant adenovirus was 5.6?1012 TCID 50/L.The Cp fragment was amplified from the total RNA of the transfected CNE1 cells by RT-PCR.The mRNA of CDglyTK gene expression was not detected in NP69 cells.The MTT results showed that after administration of GCV and 5-FC,the killing effects of fusion gene were much better than those of single gene therapy.CONCLUSION: The C promoter specifically mediates the expression of CDglyTK in CNE1 cells.The Ad-Cp-CDglyTK/GCV+5-FC has much better killing effects on CNE1 cells than single gene.