Evaluation Of Killing Effect Of Aif-T18 Fusion Protein On TEM1 Positive Cells In Vitro

Background and Objective:The morbidity of malignant tumor is gradually increased in recent years,which seriously threaten people's lives and health.Some treatments,e.g.surgery,radiotherapy,chemotherapy,and targeted therapy,were used clinically,while their drawbacks limited their further applications.Therefore,to find a new target for specifically killing tumor cells has important clinical significance.Tumor endothelial cell marker molecule 1(TEM1)can be abundantly expressed in virtually all solid tumors,particularly in ovarian cancer cells,but not or hardly expressed in normal cells.Thus,TEM1 can be used as tumor angiogenesis therapy markers,which laid the foundation for developing antibodies targeting the tumor microenvironment cells.In our previous studies,we had screened a high affinity specific single chain antibody(sc Fv T18)using yeast antibody library screening which could combine human and murine TEM1 proteins.In the present study,we prepared a fusion protein which combined the sc Fv T18 and apoptosis-inducing factor(AIF)for targeting and killing tumor vascular endothelial cells and our results provided evidence that the fusion protein might be developed a potential anti-tumor drug.Methods:The p IRES-TEM1-GFP recombinant plasmid was constructed by genetic engineering techniques.The plasmid was transfected to MS1 cells and the TEM1 positive cell lines(MS1-TEM1)were selected using G418 screening.Then the p ET302-Aif-T18 and p ET302-Aif recombinant plasmids were constructed,transformed into BL21 strain and the fusion proteins were expressed by IPTG induction.Finally,flow cytometry and MTT assay were used to evaluate the affinity and killing effects of the fusion proteins on MS1 and MS1-TEM1 cells.Results:We successfully constructed the MS1-TEM1 stable cell line and Aif-T18 fusion protein expressing system.The flow cytometry results indicated that Aif-T18 fusion protein possessed a strong binding capacity to MS1-TEM1 cells,and the Aif-T18 protein could effectively kill the MS1-TEM1 cells which were treated with the protein for 72 hours.Conclusion:We demonstrated that Aif-T18 fusion protein specifically targeted and killed the MS1-TEM1 cells,suggesting that the fusion protein may be developed as a new anti-tumor drug in future.