Expression Of Recombinant Human Cardiotrophin-1 In E. Coli And Identification Of Its Biological Activity

Cardiotrophin-1 was not only considered as a cardioprotective cytokine,but also play a pleiotropic effects in other aspects. To indentify the activity that human cardiotrophin-1 could survive damaged cadiocyte and stimulate hepatoma carcinoma cell multiplicating; PQE-huCT expression vector was constructed and recombinant human cardiotrophin-1 (rhCT-1) was expressed in E. coli. RhCT-1 was refolded and reoxidated in a glutathione redox system (GSSG/GSH) and its biological activity was indentified by MTT test. PQE-huCT was efficiently expressed in E.coli M₁₅, but 95% of rhCT-1 was in the form of inclusion bodies; The purity of dissoluble rhCT-1 increased to 95% by Ni⁺-NTA affinity column after denaturation and renaturation; The cadiocyte cultures from neonatal rats which were cultivated in media with serum-free were subjected to lethal ischaemia and model of myocardial damage was established by adriamycin damaging cadiocyte. After these damaged cadiocyte cultures were treated with rhCT-1, it was found that rhCT-1 had a significant capability of surviving damaged cadiocyte and cause a dose -dependent increase in succinodehydrogenase ( SDH ) activity. To compare the activity of SDH and lactic dehydrogenase (LDH) of pre-treated group with treated group, it was found that the effect of pre-treatment was more significant; it implied that CT-1 might play a role in cardioprotection by inhibiting appoptosis in initial stage of myocardial damage. In the model of rat myocardial damage induced by isoprenaline at heavy dose.rhCT-1 could lighten obviously the reaction of denaturation , necrosis and leukocytes infiltration in rat myocardium. It aslo could obviously decrease the activity of aspartate aminotransferase(AST) , creatinkinase(CK) , lactate dehydrogenase-1 (LDH-1) in blood serum of rats compared with negative control. It was found that rhCT-1 also had the ability of stimulating Bel-7402 hepatoma carcinoma cell multiplicating. We had got dissoluble rh CT-1 with biological activity ;It underlay the base of application and research...