分子尺寸排阻高效液相色谱法检测戊型肝炎类病毒颗粒  

Test for Hepatitis E Virus-like Particles by Size-exclusion High Performance Liquid Chromatography

作　　者：王宁[1] 王青波[1] 李静[1] 周思杭[1] 李天成 

机构地区：[1]北京生物制品研究所第四研究室,北京100024 [2]日本国立感染症研究所

出　　处：《中国生物制品学杂志》2011年第10期1220-1223,共4页Chinese Journal of Biologicals

摘　　要：目的采用分子尺寸排阻高效液相色谱(Size-exclusion HPLC,SE-HPLC)法检测戊型肝炎类病毒颗粒(Hepatitis Evirus-like particle,HE VLP)。方法分别采用超速离心法和Sephacryl S-200HR凝胶层析法制备HE VLP,对两种方法制备的HE VLP进行SDS-PAGE、Western blot、电镜观察、动态光散射及SE-HPLC分析,建立SE-HPLC定性、定量分析HE VLP的方法,并利用该方法对不同时间点培养上清HE VLP进行定量分析。结果 SDS-PAGE及Western blot分析显示,两种方法制备的HE VLP均可见相对分子质量约为50 000的目的蛋白条带,浓度和纯度基本一致,且具有相同的反应原性;电镜观察可见,超速离心法制备的HE VLP为直径约30 nm的均匀空心颗粒,层析纯化的HE VLP则为大量不规则的团块及少量空心颗粒;动态光散射分析显示,超速离心法制备的HE VLP只有1个峰,颗粒直径为27.2 nm,纯度大于98%,层析纯化的HE VLP颗粒半径2～120 nm不等;SE-HPLC分析显示,HE VLP含量在1～40μg范围内与峰面积呈线性相关,且精密性良好;利用该法能够对病毒细胞培养液中的HE VLP进行实时定量。结论 SE-HPLC可以对HE VLP进行定性、定量分析。Objective To test for hepatitis E virus-like particles（HE VLPs） by size-exclusion HPLC（SE-HPLC）.Methods HE VLPs were prepared by ultracentrifugation and Sephacryl S-200HR gel chromatography respectively,and analyzed by SDS-PAGE,Western blot,electron microscopy,dynamic light scattering and SE-HPLC,based on which a SE-HPLC method for qualitative and quantitative assay of HE VLPs were developed and used for quantitative assay of HE VLPs in culture supernatants at various time points.Results The HE VLPs prepared by the two methods showed target protein bands each with a relative molecular mass of about 50 000 on SDS-PAGE and Western blot profiles,of which the concentrations,purities and reactogenicities were basically in agreement.Electron microscopy showed that the HE VLPs prepared by ultracentrifugation were even hollowed particles at a mean diameter of about 30 nm,while those by chromatography consisted of mass irregular blocks and a small quantity of hollowed particles.Dynamic light scattering proved that the HE VLPs prepared by ultracentrifugation showed a single peak,at a mean diameter of 27.2 nm and a purity of more than 98%,while those by chromatography at radiuses ranged from 2 to 120 nm.SE-HPLC showed that the HE VLP content at a range of 1 ～ 40 μg was linearly related to the peak area.The developed method showed high precision and was suitable for the real-time quantitative assay of HE VLPs in cell culture of virus.Conclusion SE-HPLC may be used for qualitative and quantitative assay of HE VLPs.

关 键 词：分子尺寸排阻 高效液相色谱 戊型肝炎病毒 类病毒颗粒 

分 类 号：R373.2[医药卫生—病原生物学] R392-33[医药卫生—基础医学]