| Cancer is one of diseases which is threating the human being health. Though thetradition treatment methods can temporary control the development of the cancer，those methods can not solve this problems of reappearing, metastasis and transferfundamentally. Because of the development of the various biologic subjects, there aresome new treatment methods such as gene therapy, immunization therapy, targetedtherapy, induced apoptosis therapy and inhibited new vessel formation therapy areaccepted by public gradually in recent years. Those biologic treatment methods tosome degree solve those disadvantage of the weak targetting character and strong sideeffect. Those new methods can remit the suffering of the cancer patient on clinic,extend the survival time of the cancer patient. Therefore biologic treatment hasbecome a tide which the scientists cure the cancer.According to cancer biologic treatment advance tide of multi-target, multi-signalpathway and multi-approach, this paper chooses30amino acids from the IL-2aminoterminal which can enhance the immunity of the body,60amino acids from theVasostatin120~180amino acid sites which can inhibit the new vessel formation and167amino acids from the TRAIL114~281amino acid sites which can induce cellsapoptosis as experimental material. We use genetic engineering method to recombineIL-2(1-30), Vasostatin(120-180)and TRAIL(114-281)gene, beside, we add a RGD targetpeptide at the linker to create a multifunctional gene which can express a IVT fusionprotein. Hope to produce a multifunctional cancer treatment drug.The international and domestic recombinant protein expression usually use E. colior pichia pastorios system. E. coli bacteria is a prokaryote expression system, itsexpression proteins have many defects such as easy to produce inclusion body,protein biologic activity and protein expression efficiency low. But pichia pastorios isa eukaryotic expression system. its expression proteins can overcome these problems.Therefore, we choose pichia pastorios GS115to express this fusion protein.In the chapter2, through the reasonable design and optimization of primers Weconstruct pPIC9-IVT eukaryotic expression vector， and then transform the pPIC9-IVT into GS115by electricity transform. After the initial expression, wescreen the high expression Mut+IVT engineering bacterium. In Chapter3, we focuson the expression conditions and purification conditions optimization of the IVTengineering bacterium. After purification conditions optimization, the proteinpurification can already be electropHoresis pure, molecular weight of33KD. In theChapter4,we focus on the effect concentration research of IVT protein to HUVECIVT cells, NCI-446cells, SMMC cells. We discovered that the IVT concentrationfrom10μ g/ml began to suppress HUVEC cells proliferation. When the IVTconcentration get to20μ g/ml. The inhibition ratio exist significant differences. TheIVT concentration from20μ g/ml began to significantly inhibit NCI-446cells andSMMC cells proliferation. The cell proliferation inhibition rate get to24.4%±3.8%,20.8%±3.5%, and show a dose-effect. In order to further study biological activity ofIVT, we process the HUVEC cells with20μ g/ml，10μ g/ml IVT and found IVTsignificantly suppress vessel formation, process the NCI-446cells and SMMC cellswith20μ g/ml IVT and find that both NCI-446cells and SMMC cells are inducedapoptosis pHenomenon after dyeing by Hoechst. The induction apoptosis effects toSMMC cells are obvious. But, the induction apoptosis effects to NCI-446cells is notvery good. Which factor lead to this differences between different cell lines is notclear. The induction apoptosis mechanism remains to be studied further.The preliminary research results show that the new IVT fusion protein hasmulti-function biologic activities which can control the vessel formation and inducethe cancer cells apoptosis. This results provide theory basis for the development ofnew type multifunction targeted drugs. IVT fusion protein has wide applicationprospects. |
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