Construction Of A Bifunctional Fusion Protein And Evaluation Of Its Function As Anti-tumor And Thrombolytic And Tumor-targeted

With the deepening understanding of cancer and other major diseases,human beings are facing more and more clinical challenge.It was found in long-term clinical care,that cancer patients often have an underlying disease which indicated the venous thromboembolism(VTE)complications.In a survey,including 5,451 patients with ultrasound-confirmed deep vein thrombosis,32%of all patients are finally diagnosed with cancer.The formation of thrombosis in cancer patients has a complex mechanism,while the drugs for thrombosis or tumor therapy are all single targted.Therefore,it weigh a lot to construct a fusion protein which has dual functions of antitumor and thrombolytic in the treatment of cancer complication with thrombosis.Octreotide could specific combine with somatostatin receptors which will be expressed in tumor cell by specific binding.According to the relationship between octreotide and somatostatin receptors,octreotide carried fusion protein of dual functions and put them into chimeric protein.It will be an important implications for the treatment of cancer cases complicated with thrombosis.The natural SEC2 had been truncated 17 amino acids in the N terminal and 132 amino acids in the C terminal and in this way the mutant protein ASEC2 retained superantigen activity,non-toxic side effects obtained.At the same time,the mutant protein ASAK was also obtained by truncating staphylokinase 10 amino acids in the N terminal,but it had no effect on thrombolytic activity by genetic engineering technology.Based on the former research,fusion proteins which could target to tumor were constructed by using gene engineering techniques.Ni-NTA was adopted on the purification of fusion protein.The fibrinolysis activity and antitumor activity were respectively using of the fibrin plate method and MTT assay.The specificity and tumor targeting function were detected by Western bolt and Co-Immunoprecipitation.The results are as follows.1)Construction of recombinant plasmid:Single-stranded DNA oct-? sec2-?sak,oct-? sak-? sec2 were obtained by the PCR method with the pET-2 8a-? sec2-?sak and pET-28a-? sak-? sec2 as templates and the forward primer inserted octreotide gene.The oct-? sec2-? sak,oct-? sak-?sec2 gene were ligated into the pET-28a(+)experssion vector and the oct-?sec2-?sak,oct-?sak-?sec2 genes were partially digested with EcoR I-Xho I.The plasmids of pET-28a-?sec2-?sak and pET-28a-?sak-?sec2 were trandsformed into Escherichia coli BL21(DE3).Correct clones were identified by restricted length analysis and verified by sequencing from Sangon,Shanghai.2)Purification and expression of fusion proteins:Escherichia coli with pET-28a-oct-?sec2-?sak and pET-28a-oct-? sak-?sec2 grown on LB culture medium contain kanamycin.When reached the logarithmic phase,the Escherichia coli were treated 3.5-4 h by 0.5 mM IPTG at 26?.Microorganism were collected and disrupted by ultrasonic cell disruptor.Keep the supernatant after the disrupted mixture centrifuged 8000 r/min3 10min.Use the Ni-NTA resin to purify the expressed proteins.The fusion proteins with 6×His tags were freeze-drying and then the tags were cut off by thrombin.OCT-?SAK-ASEC2 and OCT-?SEC2-?SAK were obtained after mixture of fusion protein and 6xHis tags processed the Ni-NTA again.Purificated effect of fusion proteins was analysed by SDS-PAGE.3)Activation of fusion proteins:The anti-tumor,thrombolysis and targeting effects of recombinant proteins were verified by MTT experiments,fibrin plate method,Western blot and Co-Immunoprecipitation respectively.The results showed that new fusion proteins have been successfully expressed and purified in E.coli,and the good effects of fusion proteins shows potential applicable to in targeting therapy for tumor.By all accounts,it showed that recombinant proteins can significantly stimulate proliferation of T cells,and inhibit the growth of gastric cancer cell line BGC-823.The targeting of recombinant protein was verified in vitro by Western bolt.New recombinant proteins could be new antitumor biological preparation which can target to tumor,inhibit the growth of tumor and promote thrombolysis.