The Construction Of Pegfp-n1-linamarase Fusion Gene In Eukaryotic Expression Vector And Its Expression In HepG2 Cells

Gene therapy is one of the hot issues of modern medical research work.Hepatocyte-targeted gene therapy is attached more and more important.In recent years,hepatocyte-targeted gene therapy made a rapid progress.Hepatocyte is an ideal target site of directional gene transduction in that liver is one of organs that communicate the blood directly.The treatment of primary liver carcinoma gives priority to operation ombined with post operation irradiation and chemistry therapy,but the curative effect is still dissatisfactory.Therefore,it makes sense to explore new therapy.Recently,the gene therapy of malignant tumor is developed rapidly and there are many methods about the gene therapy that include:suicide gene therapy,immunologic gene therapy,drug resistance gene therapy,angiostatin gene therapy and soon.The sucide gene therapy is one of the most potential approach of antitumor.Suicide gene therapy which is also named enzyme-prodrug therapy,encode enzyme that convert nontoxic prodrugs into highly toxic metablites.Cells transfected with suicide genes are targeted for specific negative selection,which can be induced by administration of the corresponding produg.Among the enzyme/produg combinations,two of the best characterized system are herpes simplex virus thymidine kinase(TK) / ganciclovir (GCV)and Escherichia Foli cytosine deaminase (CD)/ 5-flouroeytosine (5-FC).In this study,We construeted eukaryotic expression plasmids pEGFP-N1-lis,in order to find more effective suicide gene therapy strategy. OBJECTIVE: The aim of this study was to construet eukaryotic expression vector containing lis/lin sucide gene. The pEGFP-N1-lis were transferred into HepG2 cells through electroporation,in order to find a new way to cure the tumor.METHODS: The primer of linamarase were designed and composed at the base of linamarase′s DNA sequence. Linamarase gene which abstracted from casava were amplified by Rt-PCR and sequenced. The gene extended productions were separately inserted into clone vector pEGFP-N1, then the recombination plasmids pEGFP-N1-lis were obtained And were also identified by enzyme digestion. The pEGFP-N1-lis were transferred into HepG2 cells through electroporation,then stable virus-producing cell HepG2-lis were established after genetiein(G418) selection.Rt- PCR was resorted to demonstrate the successful transduction and transcription of linamarase gene. Expressions of related protein products were detected by Western-Blot.RESULTS:We sueeessfully cloned the lis gene and analyzed the lis gene sequence,which basically accorded with the genebank.Later,we inserted the cloned lis gene into clone vector pEGFP-N1. We successfully constructed the recombination expression plasmids pEGFP-N1-lis,which were identified by PCR and enzyme digestion.The pEGFP-N1-lis were transferred into HepG2 cells through electroporation, stable cell HepG2-lis were established after genetiein(G418) selection. Expression of EGFP protein was found by fluorescence microscopy,about1600bP Positive band were seen through RT-PCR,and expression of lis Protein was found by Western-Blot in transient transfected HepG2 cells,which showed that suicide gene could be transeribed and expressed in gene-transferred HepG2-lis cells.CONCLUSION:The construction eukaryotic expression vector pEGFP-N1-lis will lay a basis for carrying out further studies on the function of lis gene. These results suggest that lis/lin system is an ffective gene therapy and will lay some foundation for clinieal treatment of the hepatocellular Carcinoma.