The Role Of Fusion Protein TTF-EG3287 In The Targeted Embolization Of Human Colon Cancer Xenografts In Nude Mice

Research backgroundCurrently,there are some problems in the clinical application of anti-tumor embolization,such as narrow indications,small dosage,high cost and difficulty in intervetion.We plan to design a new peptide that uses the drug targeting of a certain monoclonal antibody to load procoagulant factors to induce tumor vascular thrombosis with a targeted and efficient manner.Based on this research idea,we successfully constructed the recombinant gene of fusion protein tTF-EG3287.EG327 is a bicyclic polypeptide encoded by exons 7 and 8,and tTF is a truncated tissue factor that activates exogenous coagulation reaction.The fusion protein tTF-EG3287 plays an anti-tumor effect and must firstly be accurately localized to the tumor vascular endothelial cells by the carrier molecule EG3287,and effector molecule tTF induces tumor vascular thrombosis,resulting in tumor cell ischemic necrosis,thereby achieving therapeutic goals.MethodsThe construction and transformation steps of the recombinant plasmid tTFEG3287 had been completed on the basis of previous experiments.LB plates were selected for high expression strains and cultured overnight,and then expanded at a ratio of 1:100.IPTG was added to the logarithmic phase to add inducer,and culture was continued for 6-8 hours;bacteria were collected uniformly and electrophoresed on a 12% SDS-PAGE gel.Coomassie brilliant blue staining was used to verify and compare the expression of different proteins,and the induction concentration and time of IPTG were continuously optimized.After ultrasonication,the inclusion body complexes were washed repeatedly,centrifuged,and repeatedly stirred to dissolve and purified by nickel column.Protein renaturation was performed in a PBS system with gradient urea.Immunofluorescence confocal microscopy and thrombelastography were used to detect protein activity.The colorectal cancer xenograft models were constructed and randomly divided into 5 groups: Saline group(PBS group),tTF-EG3287 treatment group and tTF treatment group.The protein was administered intermittently by tail vein injection of tumor-bearing nude mice.Tumor volume before dosing.ResultsIn this experiment,the concentration of fusion protein tTF-EG3287 reached 2.1mg/mL,purity >85%,and the in vitro procoagulant activity and cell-targeting binding effect was obvious.In the experiment of tumor-bearing nude mice,continuous treatment for 27 days,the average volume of transplanted tumor in nude mice of tTFEG3287 and tTF treatment group were smaller than that of Saline control group.Compared with the control group,there was no significant difference between the tTF treatment group and the tTF-EG3287 treatment group(n=5,P<0.01),TGI(inhibitory rate)of tumor-bearing nude mice bearing tTF-EG3287 was 79.2%(n=5,P<0.01),and TGI of tTF was 48.5%(n=5,P>0.05).In vivo imaging experiments tTF-EG3287 could be enriched in tumor tissues in tumor-bearing nude mice,and was rarely enriched in other normal tissues except lungs,but tTF was not specifically targeted.After 24 hours,organ imaging showed tTF-EG3287 could stably and persistently accumulate in tumor tissue,but tTF was almost not enriched in various organs and tumors.Pathological section HE staining showed mixed thrombus formation in tumor vessels of tTFEG3287 treatment group,and tTF treatment group no obvious ischemic necrosis was found in all organs,and the performance was basically normal.ConclusionsThis experiment had optimized the purification and activity identification of the protein of the inclusion body.The fusion protein tTF-EG3287 had a stable biological action and could be accurately distributed in cancer tissue in a transplanted tumor of nude mice with colorectal cancer,and was targeted to embolize the tumor blood vessel,inhibiting the growth of tumor cells.