Chromatographic Purification Of Coagulant EV71

Hand-foot-mouth disease is popular in children,which is caused by many enterovirus.EV71 is one main agents of HFMD in china these years.There has been a significant increase in EV 71 epidemic activity in the Asia-Pacific region since 1997,and has a propensity to cause severe neurological disease or death.There is no specific medicine and vaccine to therapy and prevention of the EV71.So it is important to research and develop one vaccine which can control the epidemic of EV71.The purpose of this study is the EV71 virus purification process for screening the best chromatographic parameters of preparation for the future vaccine research to provide reliable data.In this study, EV71 virus provided by the Division as a crude extract of materials,create a gel filtration and ion exchange chromatography purification process of combining, for the improvement of the purity of EV71 virus provides the best chromatographic combination of parameters.First,Sepharose 4 Fast Flow gel filtration chromatography parameters of screening,use of different elution flow rate, sample volume of different samples,different buffer solutions of different pH values and salt concentration on the EV71 intestinal virus for continuous crude extracts elution. The results show that the gel filtration elution flow rate, sample volume on the sample, buffer solution pH value and salt concentration on the EV71 virus can affect the EV71 virus in varying degrees the separation of crude extracts.Within the experimental range, select the elution flow rate was 60cm / h, the sample on the sample using 5% CV, pH 7.5 buffer solution and the salt concentration of 0.1mol / L,the EV71 virus on the separation of crude extracts ideal. Then use Q Sepharose XL anion chromatography media to gel as a crude extract of purified EV71 virus. Ion exchange chromatography of ionic strength factors, pH, temperature and flow rate, the paper mainly discusses the ionic strength and pH on the preparation of ion exchange EV71 virus. First linear gradient elution method, preparation of initial screening with EV71 virus equilibrium buffer and elution of sodium chloride concentration range of the concentration of sodium chloride buffer.Then at the same pH, alter the balance of sodium chloride in buffer were calculated under the buffer in each balance EV71 virus recovery and specific activity,choose the best balance between the content of sodium chloride buffer.Again in the same ionic strength conditions, changes in pH, by calculating the EV71 virus recovery and specific activity,choose the best pH value. Finally, the orthogonal method to verify the different ionic strength and pH value of the portfolio, which selected the best combination. This experiment can be seen that ionic strength and Ph value could affect the EV71 virus with different degrees of preparation.Within the experimental range, balanced buffer solution containing 200mmol/L sodium chloride and pH 7.5 as the best combination. Finally, after the first gel filtration chromato graphy,ion-exchange method of combining, the use of screening in front of the best chromatographic conditions for the experiment, EV71 virus has been the feasibility of high purity, with satisfactory results. The process is simple,low cost, high recovery and specific activity.