Construction and Expression of Recombinant Adenovirus Encoding Tumor Suppressor Fhit and Its Biological Function in Colon Cancer Cells / 中国生物工程杂志

ABSTRACT

AIM:To construct a recombinant adenovirus carrying Fhit gene,a tumor suppressor in many types of cancer,and to observe its biological function on the proliferation of colon cancer cells.METHODS:Fhit gene was cloned from the fetal liver cDNA library using the PCR method.The PCR product was inserted into the T vector to construct the plasmid pMD18T-Fhit.The Fhit fragment from the pMD18T-Fhit was inserted into the vector ptrack-CMV to construct a shuttle plasmid ptrack-CMV-Fhit.After PmeI digested and linearized process,ptrack-CMV-Fhit was co-transformed into Escherichia coli strain BJ5183 together with the adenovirus backbone vector pAdEasy-1 to generate a recombinant adenovirus plasmid by homologous recombination.The adenovirus plasmid was identified by PacI digestion and transfected into 293A cells to package a recombinant adenovirus which expressed the Fhit protein.Furthermore,the adenovirus rAd-Fhit was infected into colon cancer cells,and the expression of the ectogenic protein was detected by Western blotting.Finally,the proliferation of colon cancer cells was observed in adenovirus-infected cells by the MTT assay.RESULTS:Constructed the recombinant adenovirus encoding Fhit gene and expressed it in colon cancer cells successfully.Detected that the proliferation of colon cancer cells was inhibited obviously in rAd-Fhit-infected cells with comparison to the control groups.CONCLUSION:Fhit may function as a tumor suppressor in colon cancer cells,and the adenovirus-mediated Fhit can be a novel strategy for the colon cancer therapeutics.