Secreted Eukaryotic Expression And Biological Activity Of Recombinent Fusion Protein Tumstatin-EGFP

objective Tumstatin,a latestly discovered angiogenesis inhibitor derives from precursor molecule known as colⅣ,shows a selective inhibition on endothelial cell proliferation as well as migration via specially binding toαvβ3 intergrin. As the other endogenesis inhibitors,tumstatin exhibits a significant anti-tumor effect with least toxicity , lowest risk of drug resistance and broad spectrum ,the research about it is to become one of the most focus therapy strategies on anti-tumor and maybe provide a optimum therapeutic candidate for future use.However, application of tumstatin is retricted by indirectly blocking tumor growth,long-term treatment cycle and so on,which must be improved to enhance its anti-tumor effect. Combined therapy with a variety of angiogenesis inhibitors or angiogenesis inhibitor together with other anti-tumor drugs may represent the trend of anti-tumor approaches of novel tumor therapy strategies proposed recently.In the set of our experiments,we construct a secreted eukaryon expressed vector for recombinant tumstatin-EGFP by the full-length gene of tumstatin and enhanced geen fluorescence protein gene under the indication of EGFP and its property of eliciting a classic T cell-mediated cytotoxic response in vivo. After obtatining stably cell line through transfecting fusion protein into Chinese hamster ovary cells(CHO),we analysed the fusion protein on its expression, and observed whether the fusion protein can retain both tumstatin for local angiogenesis inhibition,together with EGFP,which has green fluorescence expression ,and whether tumstatin could take EGFP along specially targeting vascular endothelial cell.Methods STL-EGFP , sig-EGFP and sig-tumstatin were separately amplified from pGEM-T/STL and PEGFP-C2 with their respective primers by overlap extension polymerase chain reaction (OSEing-PCR) and were inserted into eukaryotic expression plasmid pIRESneo3 to construct recombinant expression plasmid. After DNA sequence analysis and restriction enzyme digestion,CHO cells were transfected with the appropriate expression vectors of PIRESneo3/STL-EGFP ,PIRESneo3/ sig-EGFP , PIRESneo3/ sig-tumstatin and null vector by using lipofectamine 2000.We obtained stably cell line selected under the pressure of antibiotic G418(700mg/l) for consecutive three times. tumstatin-EGFP was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot. EGFP was detected by inverted fluorescence microscope.Experiments such as endothelial cell proliferation and inhibition assay, inducing apoptosis test were used to assess the biological activity of tumstatin-EGFP, in vitro matrigel tube formation assay contributed us to validate the fusion protein function of specially binding to endothelial cell.Results The recombinant eukaryotic expression plasmids were identified correct and the obtained cell line could stably express tumstatin-EGFP,which has intergrated into CHO cell genome by RT-PCR.Inverted fluorescence microscope showed that about 95% of the cells had positive fluorescent signals by counting in white or dark field.Western blot analysis with tumstatin polyclonal anlibody detected the expression of tumstatin-EGFP from the supernatant of CHO-STL-EGFP recombinant cell line culture medium ,which suggested STL-EGFP could secrete expression consistant with the predicted molecular weight about 55ku.The result demonstrated that we have obtained stably transfected recombinant cell line of CHO-K1/ pIRESneo3/STL-EGFP. Growth curve test shew that transfected cells grew lower than that of untransfected,it may think that exogenous gene could impact on acceptor cell growth.Endothelial proliferation and inhibition test together with inducing apoptosis assay indicated that tumstatin-EGFP not only suppressed endothelial cell proliferation but also displayed the property of induing endothelial cell apoptosis which has no significant deviation with tumstatin.In addition, in vitro matrigel tube formation assay had strongerly demonstrated that tumstatin-EGFP protein could inhibit vascular tube information. Furthermore,it had been provided unambiguous evidence that the protein of tumstatin-EGFP could specifically target to vascular endithlial cells in vitro test.Conclusion we have generated a bifunctional secrected fusion protein tumstatin-EGFP expressed by deferent genes fusion through recombinant DNA technology.We have successfully constructed a recombinant secrete eukaryotic expression plasmid of PIRESneo3-STL-EGFP and have established a CHO cell line stably expressing tumstatin-EGFP. Evidence existed that tumstatin and EGFP could keep their individual characteristics and immunogenicity with no interference with each other.Biologic activity tests such as endothelial proliferation and inhibition test ,inducing apoptosis test and in vitro matrigel tube formation assay have proved that the fusion protein remained tumstatin biological activity.Targetting test also identified tumstatin-EGFP could specially combine with endothelial cell in vitro. This work provided foundations for further study on the biologic activity ,targetting function and even a synergistic antitumor effect of the fusion protein tumstatin-EGFP,even would shed light on mechanism of action associated with tumstatin as well as its fusion protein.