Construction Of Deletion Mutants Of Bifunctional Chimeric Proteins Possessing Anti-tumor And Thrombolytic

According to clinical studies, thromboembolic diseases such as deep veinthrombosis (DVT), pulmonary embolism (PE), disseminated intravascularcoagulation (DIC), migratory superficial thrombophlebitis (MST), portal veinthrombosis (PVT) and arterial thromboembolism (AT) often appeared in those cancerpatients who got renal cells tumors, pancreatic tumors, ovarian tumors, liver tumorsand other tumors. Clinical tumor-associated thrombosis was called Trousseausyndrome. Thrombosis plays an important role in tumor metastasis and angiogenesisof patients. At the same time, thrombosis can lead to high morbidity and mortality.Above all, tumor and thrombosis have a very close relationship.Staphylococcal enterotoxin C2(SEC2) is one of bacterial superantigens producedby Staphylococcus aureus. It can be attributed its superantigenic activity to cross-linkmajor histocompatibility complex class II molecules and the variable region of the βchain (Vβ) of the T cell receptors (TCR) and activate a large number of T cells in verylow concentration, resulting in the release of massive cytokines, which will produce avery strong immune response and a significant tumor inhibition. As tumorimmunotherapy agent in clinic, SEC2is used to cure malignant tumor including lungcancer, colorectal cancer, liver cancer, ovarian cancer et al. Staphylokinase (Sak) is atypical drug of the third generation thrombolytic drugs. Compared with the traditionalthrombolytic agents, Sak has many merits and characters including strongerthrombolytic activity, lower immunogenicity, higher specificity and fewer allergicreactions. So Sak is a safe and reliable therapy medicine against cardiovasculardisease at present.Based on broad-spectrum anti-tumor effect of SEC2and effectively thrombolyticactivity of Sak, chimeric proteins SEC2-linker-Sak and Sak-linker-SEC2wereobtained in our lab, not only have the same thrombolysis effectiveness as Sak, butalso have the superantigen activity as SEC2which effectively cure malignant tumor.But as a kind of potential gastrointestinal toxins, enterotoxin C2can cause foodpoisoning with vomiting, abdominal cramp and severe diarrhea. And it was not easy to be injected and absorbed. Because of its large molecular weight (approximately27.6kDa, with239amino acids), it can cause allergic reactions and side effects invivo. Thus the clinical application was limited. Respectively truncated of the17amino acids and the132amino acids from SEC2N-terminal and C-terminal, theresultant small peptide had no side effects but kept super-antigen activity. Besides，deleting the10amino acids from Sak N-terminal through modern molecular biologytechniques, the staphylokinase mutant still have the same thrombolytic activity aswild type Sak.Based on pre-research, fusion proteins with low weight were obtained. Proteinswere purified through Ni2+-NTA His Bind Agarose column. The thrombolyticfunction and antiitumer activities of chimeric proteins were analysized by fiber-plateprocess and MTT methods, respectively. Results were as follow:1) Construction of expression vector. Vector pET28a-Δsak-linker-Δsec2wasconstructed，and then the linker between Δsak and Δsec2was removed by PCR.After blunting, kinating and ligating, the vector was transformed into E.coliBL21(DE3) and confirmed by restriction enzyme digesting and sequencing. AndpET28a-Δsec2-linker-Δsak was constructed by the above method.2) Preparation of chimeric protein. The recombinant E.coli BL21(DE3) inoculatedin LB mediums containing40μg/ml kanamycin was cultured until OD600=0.6.1mM isopropy-β-D-thiogalactoside (IPTG) was added at30℃for4to5h so as toinduce expression of chimeric proteins. Cells were collected and lysised byultrasonic, and proteins were purified through Ni2+-NTA His Bind Agarosecolumn. After removed histidine tag of N-terminal by enterokinase cutting andpurified through Ni2+-NTA His Bind Agarose column, chimeric proteinΔSak-ΔSEC2andΔSEC2-ΔSak was obtained.3) Western blot identify of chimeric proteins. Rabbits were immunized by purifiedSEC2as antigen to get polyclonal anti-SEC2specifically. Antibody titer wasdetermined by indirect elisa, and the titer was102,400. Chimeric proteinsΔSEC2-ΔSak and ΔSak-ΔSEC2were determined through western blot. Itdemonstrated that both chimeric proteins can bind with SEC2specificly.4) Bioactivities assay of chimeric proteins. Thrombolytic activity and lymphocyte proliferation activity and antitumor activity in vitro of chimeric proteins wereanalyzed through fiber-plate process and MTT methods respectively. Resultsshowed that chimeric proteins not only possessed the same thrombolyticactivities with Sak, but also stimulated lymphocyte proliferation and inhibited thegrowth of823cells in vitro. To sum up, chimeric proteins obtained in this studyhave the superantigen activity as SEC2which effectively cure malignant tumor.The toxic negative effects was avoided，and molecular weight was reducedbecause of removing the amino acids related to emetic activity and unrelated tosuperantigen activity. Besides, the chimeric proteins have the same thrombolyticactivity as Sak. This study will laid a foundation for the development of noveldrug of anti-tumor and thrombolysis in clinic.