Construction And Expression Of Fusion Proteins With Both Fibrinolytic And Anticoagulant Avtivity And Studies On Their Functions;Studies On Sustained-release Dosage Form Of Pegylated Hirudin

To prolong the half-life of hirudin, polyethylene glycol was used to modify hirudin molecule. The pegylation degree of hirudin was optimized with the change in the ratio of PEG versus hirudin and pH in reaction system. Matrix-assisted laser desorption ionization time of flight mass spectrometry(MOLDI-TOF-MS),SDS-PAGE and HPLC determination indicated that the attachment of PEG produced a heterogeneous mixture of hirudin with different pegylation degree that differed by 5000 U. Purified products with gel filtration were obtained and the special activities were analyzed by chromogenic substrate TH. The activity of hirudin linked with 1 or 2 PEG chains kept intact but that of hirudin linked with 3 PEG decreased markedly. Activated partial thrombin time (APTT) of rabbit serum was analyzed to image the half-life of hirudin in vivo. The half-lives of hirudins with different pegylated degree were prolonged to different extent. So, under the optimization of modification conditions, two half-life-prolonged pegylated hirudins were obtained without activity loss.Despite successful lytic therapy of thromboembolic disorder, reocclusion of the damaged vessels or bleeding complication frequently reduces the therapeutic effect. To combine the benefits of thrombolytics and anticoagulants for prevention of vessel reocclusion and to alleviate their bleeding side effect, we designed three targeting bifunctional fusion protein, termed as (1) TFXH(tissue-type plasminogen activator linked with hirudin by FXa recognition peptide); (2) SFXH (staphylokinase linked with hirudin by FXa recognition peptide); (3) STH(staphylokinase linked with hirudin by thrombin recognition peptide). The aims of the design were to: (1) improve thrombolytic properties of tissue-type plasminogen activator or staphylokinase through the high affinity of hirudin's C-terminal for thrombin; (2) reduce bleeding side effect with the block of N-terminal of hirudin by tissue-type plasminogen activator or staphylokinase; (3) locally release the anticoagulant activity of hirudin by FXa or thrombin at the vicinity of thrombus.TFXH was constructed and expressed in Pichia pastoris. Tissue-typeplasminogen activator gine(tpa) and hirudin gene(hir) with FXa-recognition sequence codon at its 5'-terminal were obtained by RT-PCR and PCR respectively. The fusion protein geneftfah) was cloned into plasmid pPIC9K and electroporated into the genome of Pichia pastor is GS115. The expression of fusion protein was induced by methanol in shaking flask and secreted into the culture medium. Two forms of the fusion protein, single-chain and double-chain linked by a disulfide bond (due to the cleavage of t-PAat Arg275-Ile276), were obtained. The expression level of TFXH was not improved dramatically by fed-batch methanol induction under fermentation conditions. Affinity chromatography was used to isolate the fusion protein from fermentation supernatant. The intact fusion protein retained the fibrinolytic activity but lacked any anticoagulant activity. After cleaved by FXa, the fusion protein liberated intact free hirudin molecule to exert its anticoagulant activity.SFXH and STH were constructed and expressed in E.coli. The fermentation technology was optimized. It was shown that 12h-old seed guaranteed the fastest growth rate and the highest cell density. Fed-batch of 50% glucose and complicated medium led to higher expression level cf proteins. The fusion proteins were expressed in periplasm and could be extracted with freezing and thawing. Purification process of the fusion proteins were developed and optimized by gel filtration and ion exchange chromatographies. SFXH and STH with purity of above 96% were obtained. The fusion proteins retained plasminogen-activating and fibrinolytic activity from the domain of staphylokinase but no anticoagulant activity due to the extension of the N-terminus of hirudin. In vitro, SFXH showed effectively anticoagulant activity after cleavage with FXa. The results of immunohistology showed that both SFXH and STH could target to thrombus because of the high affinity of HV for thrombin.To investigate the function of SFXH and STH in vivo, two animal models were developed. In a mouse tail thrombosis model induced by ï¿¡ap/?a-carrageemn, at equimolar concentrations, an enhanced antithrombolytic activities of the SFXH and STH than staphylokinase alone were observed. In a venous stasis thrombosis model in the vena cava inferior of the rat, the wet weight of thrombus was analyzed to evaluate the thrombolytic and anticoagulation activities of SFXH and STH, and TI\ APTT> PT in plasma were analyzed to image the bleeding complication, respectively. The results showed that the wet weights of thrombus in SFXH and STH groups were smaller than that of staphylokinase and hirudin groups, indicating that the two fusion...