Recombinant Bovine Prion Protein Separation, Purification And Prostitution Raise Hopane Active Ingredient Extraction, Separation And Structural Identification

Purification of Recombinant Bovine Normal Prion Protein and Extraction , Separation and Structural Identification of Effective Components from EpimediiAbstractThe dissertation is composed two sections: l.The long chain and short chain fragments of recombinant bovine normal prion protein highly expressed by E.Coli were purified, repectively, by high performance hydrophobic interaction chromatography (HIC) in this dissertation. The separation conditions involved in this technological process were optimized. 2. The extraction of effective components from Chinese Herb Epimedii was also investigated. Five kinds of compounds in the extract were separated and prepared by semi-preparative high performance reversed phase chromatography (HPRPC), and their structures were investigated respectively.Section I :1. Purification of the long chain fragment of recombinant bovine normal Prion protein by HIC.The long chain fragment of recombinant bovine normal prion protein (rbPrPcL) highly expressed by E.Coli was purified by HPHIC . The extration of inclusion body and the effects of composition of mobile phase, stationary phase, pH of mobile phase, and gradient mode for HPHIC on the separation of rbPrP癓 were investigated. An optimum condition for the separation and purification of rbPrPcL by HPHIC was obtained. Both the purity and the mass recovery of rbPrP癓 are more than 90% only by one step with HPHIC. In addition, the refolding mechanism of unfolding rbPrP癓 by HPHIC is also discussed.2. fnrtflcatfea M Hie short chain frtgmefit of reeombinant bovine normal Prion protein by HIC.The short chain fragment of recombinant bovine normal prion protein (rbPrP S) highly expressed by E.Coli was purified by HPHIC. The effects of stationary phase, composition of mobile phase, pH of mobile phase, and gradient mode on the separation of rbPrP癝 were studied and compared. On the basis of which, an optimum condition for the separation and purification of rbPrP癝 by HPHIC was obtained.Under the optimum condition, the rbPrP L with purity protein was separated only by one step with HPHIC. The result of thin layer scanning of SDS-PAGE shows its purity to be 96%. The mass recovery of this protein is 87%.Section TJ:1. Extraction of effective components from Chinese Herb Epimedii.The extration efficiencies of icarrin from Chinese Herb Epimedii by aqueous solution of CHaOH, CHsC^OH, fatty acid were investigated and compared with each other. Fatty acid as an organic sovent was firstly investigated. The extraction efficiencies of icarrin by soaking at room temperature and fluxing were also compared. The result shows that the effective components of Chinese Herb Epimedii can be extracted by fatty acid aqueous solution with the most extraction efficiency. The effects of temperature, time and the ratio of liquid to solid on the extraction efficiency of icarrin were studied. An optimum extraction process of effective components from Chinese Herb Epimedii was obtained, and it is magnified to production grade.2. Preparation and structure determination of five compounds fromthe Chinese Herb Epimedii.Isolation and preparation of five main compounds from the extract of Chinese Herb Epimedii by semi-preparative RP桯PLC were studied. The effects of the type of organic solvents in mobile phase on the separation of the extract was studied and compared. Other conditions, such as sample injection and the concentration of organic solvent in mobile phase were also studied. Structures of the five compounds wereidentified by UV, IR, MS, 1HNMR and 13CNMR. A new compound exandraside A was isolated from the genus for the first time.