To Construct An TAT-Apoptin Prokaryotic Vector、Soluble Expression And In Vitro Activity Research

Object: Tumor is a major threat to human life. There is no specific drug by now.The traditional treatment options such as operation, chemotherapy and other meanspresent is poor effective. Them have many defect.for example, you use them todestroy tumor cells, them also destroy normal cells of the body and tumor recurrencerate is very high and so on.Apoptin as a specific anticacer drugs is isolated from CAV.The Apoptin whichonly apoptosis tumor cells has no toxic side effects for the normal body. The object ofthis study is to development a tumor therapy which use the Apoptin protein.Protinwhich only destroy tumor cell is suit for tumor therapy,because the half life period ofprotein is not too long and dosage can be chosed.Apoptin has good function to destroy tumor cells. The biological activity is safeand harmless to normal cells. But it’s effective targets must be in cells. Proteinmolecule must enter the interior of the cell in order to play it’s biological activity.Apoptin protein does not have the ability to penetrate the cell membrane. So wedecided to combine the Apoptin gene with the TAT genes which can carry theexogenous molecules into the placenta to build the TAT-Apoptin fusion gene, it’sexpression product has an ability to enter the placenta by itself.To optimize the condition of expression in order to get the most native protein.Method:PCR was Used to isolate apoptin gene from the chicken anemia virusDNA.To construct the Apoptin prokaryotic expression plasmid. Then the recombinantwas transfered into E.coli BL21(DE3) to express the Apoptin protein. The result ofthe expression of apoptin protein was detected.Synthetic TAT sequences was used to construct recombinant expression plasmidof pET30a-TAT-EGFP. The expressive fusion protein was purified and incubated withtumor cells. The reporter gene EGFP was detected to know the penetrating activity ofTAT sequences which is synthesized.We combined the TAT genes and Apoptin gene by means of biologicalengineering to construct the fusion genes of prokaryotic expression vector, using the prokaryotic expression system to express the fusion protein. we optimized theexpression conditions to get the most native protein. The biological activity ofpurified fusion protein was detected by the MTT assay.Result: Through experiment, we successfully isolated the Apoptin gene from CAVDNA and onstructed a prokaryotic expression system of Apoptin gene and prove thatthe Apoptin gene can be successfully expressed in E.coli BL21(DE3).By synthetic methods, we successfully obtained the TAT sequence. theprokaryotic expression vector of TAT-EGFP fusion gene was constructed. AfterTAT-EGFP fusion protein was incubated with Hela for1h, the strong fluorescentsignal can be detect. We proved the penetrating activity of our synthetic TATsequences is satisfied.We successfully constructed the pET-30a-TAT-Apoptin plasmid and thepGEX-6p-1-TAT-Apoptin plasmid. The fusion protein was successfully expressed inE.coli BL21(DE3). We optimized the expression conditions. The best expressioncondition of pET-30a-TAT-Apoptin plasmid is20℃,0.5mmol/LIPTG, overnight. thepGEX-6p-1-TAT-Apoptin plasmid is20℃,0.1mmol/LIPTG, overnight. The rate ofnative protein was raised to82%from23%under our optimization condition. Wepurified inclusion bodies by His tag and purified native soluble protin by GST tag.The purified protein was used to test the biological activity of fusion protein.The result of MTT assay is that the survival rate is only10.86%, p<0.001.