Expression,purification And Functional Evaluation Of Neorudin-human IgG-Fc Fusion Protein

Cardiovascular diseases have become the main reason of death among Chinese citizens in recent years,and the formation of thrombosis is associated with cardiovascular diseases.Therefore,the intervention of thrombosis by anticoagulants may reduce the risk of cardiovascular diseases.Although hirudin has strong thrombin inhibiting effect and anticoagulant activity,the serious bleeding side effects limited the population in patients.Neorudin,a derivative of hirudin,was developed as a novel recombinant anticoagulant with a feature of low bleeding risk.Considering the short half-life,EH is need repeated medication to maintain the therapeutic effect.To prolong the half-life of EH,the fusion proteins of the Fc segment of human IgG and EH were prepared.We hoped to obtain fusion protein that not only obviously prolong the half-life of EH,but also not affect the anticoagulant activity.According to the gene sequences of EH,human IgG4-Fc fragment and GS linker peptide,we synthesized the recombinant gene sequence suitable for translating and expression in Chinese hamster ovary cells(CHO).The Fc-EH and Fc-L-EH gene were amplified by PCR and cloned to the pcDNAHC vector.The recombinant expression vector pcDNAHC-Fc-EH and pcDNAHC-Fc-L-EH were transfected into CHO cells by liposomes.The highly expressed cells were screened by MSX.The expression of fusion protein was detected by ELISA assay and Western Blot.The highly expressed cells were expanded with bioreactor.The target protein was purified by Protein A affinity column.And the fusion protein concentration was measured by Lowry method.The purity of the fusion protein was calculated by SDS-PAGE.The relative molecular weight and C-terminal amino acid sequence of the two fusion proteins were determined by mass spectrometry.The anticoagulant activity of fusion proteins in vitro was detected by clot methods,and the antithrombotic effect was studied by a jugular vein thrombosis rat model.The half-life of the fusion protein was tested in rat by chemiluminescence immunoassay(CLIA).In this study,the recombinant expression vector of pcDNAHC-Fc-EH and pcDNAHC-Fc-L-EH were obtained and the cell lines with high expression of Fc-EH or Fc-L-EH fusion proteins were screened after the vectors were transfected into CHO cells.The molecular weight of two targeting proteins is 68476 Da and 70542 Da respectively.Both C-terminal amino acid sequence of Fc-EH and Fc-L-EH fusion proteins are correct.The purity of two fusion proteins is 98.9%and 97.8%respectively.The anticoagulant activity of the fusion protein Fc-EH is 256 ATU/mg while Fc-L-EH is 64 ATU/mg.The result of jugular vein thrombosis rat model showed that Fc-EH dose dependent inhibited the formation of thrombus and the antithrombus activity of Fc-EH was comparable with EH.However,the bleeding side effect of Fc-EH together with EH was significantly lower than low molecular weight heparin.The result of pharmacokinetic study in vivo showed that the Fc-EH had an elimination half-life of 33.9～53.9h.In summary,the fusion protein Fc-EH with a similar anticoagulant activity to EH was obtained,moreover,the half-life of Fc-EH extends about 20-fold that of EH.The development of Fc-EH provided a different strategy for the study of anticoagulant.