Expression Vector Construction,expression And Biological Activity Identification Of Eh-l-Fc

Cardiovascular disease had become the number one killer threatening the human health and life in recent years.Thrombosis is the main inducement of cardiovascular disease.Once formed,thrombus often formed irreversible consequences.Anticoagulant is an important drug in the prevention of thrombosis.Hirudin is a new drug and has strong inhibitory effects on thrombin,which leads to a sharp increase in coagulation parameters.Neroudin has better targeting and effects on reducing non-specific bleeding side than that of hirudin.The serum half-life of neorudin is about 1-2h caused by the smallmolecular weight of 7.3 KD which was easily eliminated by kidney filtration.To prolong the half-life of recombinant Neorudin,a new form of recombinant Neorudin(EH-L-Fc)was constructed by fusion of the Neorudin gene and the coding sequence for fragment of human IgG1 with linkers peptide,and the function of EH-L-Fc was analyzed.The expression of E.coli has the advantages of easy-operating,short expression cycle.The eukaryotic expression system has the advantage of protein expression modification.In this study,the prokaryotic and eukaryotic expression vectors were constructed to obtain fusion protein.Methods: The fusion gene was obtained by SOE-PCR and was constructed into the expression vector pET-24 a and pcDNA3.1.The recombinant expression vector pET-24a-pelB-eh-L-Fc was transfered into E.coli BL21,the recombinant protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The recombinant expression vector pcDNA3.1-Eh-L-Fc was transfected into Chinese Hamster Ovary cells(CHO)by liposomes.Stable gene expression cell lines were selected by Geneticin(G418)resistance screening.The expression of EH-L-Fc protein in the cell culture supernatant was measured by Western blotting.The monoclone was selected from the resistance screening polyclone by limiting dilution analysis.The fusion protein was purified by Protein A affinity chromatography,and then the purity of the EH-L-Fc protein was analyzed by SDS-PAGE and reversed phase high performance liquid chromatography(RP-HPLC).The fusion protein was cleaved by coagulation factor Xa and the anticoagulant activity was determined in vitro by fibrin clot method.Results: The results showed that the recombinant expression vector pET-24a-pel B-eh-L-Fc and pcDNA3.1-Eh-L-Fc was successfully constructed.Recombinant EH-L-Fc was detected by SDS-PAGE in E.coli.The stable cell line CHO-pcDNA3.1-Eh-L-Fc was obtained.The expression product is a dimer with a molecular weight of 72168 Da.The purity of the recombinant protein was 93.9% with a 96.9 ATU/mg anticoagulant specific activity after cleaved by FXa.The complete recombinant EH-L-Fc has no anticoagulant activity.Conclusion: The SDS-PAGE results showed that recombinant EH-L-Fc existed in the form of inclusion body in E.coli.The stable CHO cell line harboring the recombinant Eh-L-Fc was successfully obtained.The EH-L-Fc fusion protein has a high purity and bioactivity after cleavage by FXa which provides an important foundation for the further research of EH-L-Fc with a prolonged half-life.