Seperation And Purification Of Equine Chorionic Gonadotropin

Equine chorionic gonadotropin(eCG),a kind of glycoprotein hormone which is also named as pregnant mare serum gonadotropin(PMSG),has been applied in breeding area widely on account of its significant role in stimulating follicle maturity and promoting spermiogenesis.Because of its low concentration in pregnant horse serum(60 IU/mL)and tendacy to deactivate,it is a big challenge to separate and purify eCG with higher specific activity,satisfied activity recovery,and moderate cost.In this paper,separation and pufication of eCG from pregnant mare serum was carried out by exploring proper separation method and optimized the conditions of ion exchange chromatography(IEC).Furthermore,feasibility and performance of purifying eCG with hydrophobic interaction chromatography(HIC)was investigated and a novle purification means to polish eCG with HIC was established.Finally,the relationship between the bioactivity and immunological activity of eCG and content of N-acetyl neuraminic acid(Neu5Ac)in eCG was studied.1)Screened methods for crude separation,optimized the crude separation factor:ascertained the best factor of metaphosphoric acid to subside impure protein.The specific activity of product disposed by crude separation was 80-150 IU/mg,and the activity recovery was above 90%.2)Applied the ion exchange chromatography(IEC)to purify further more.Screened the suitable media,loading condition and elution condition.By IEC,the specific activity of the final product was 1124.41 IU/mg,and the activity recovery was 53.68%.3)Investigated into separating and purifying eCG by hydrophobic interaction chromatography.Screened and compared the property of different hydrophobic absorbent.Explored the composition and ionic strength of loading buffer,determined the best composition of loading buffer.Optimized the elution condition.The specific activity of product was 1429.86 IU/mg.Activity recovery was 88.66%.Compared with IEC method,the yield of activity elevated.4)Explored the feasibility to represent bioactivity of eCG by Neu5Ac concentration.By pre-column derivatization,using HPLC to determine Neu5Ac concentration,optimized the derivatization time,derivatization temperature,OPD concentration.Investigated into the hydrolysis method and condition for Neu5Ac from eCG,optimized the hydrolysis time,hydrolysis temperature,TFA concentration.Established techniques to determinate Neu5 Ac concentration in eCG.Explored the relationship of Neu5Ac concentration and eCG activity.The research showed that the bioactivity and immunological activity was elevated with the rise of Neu5Ac concentration.