论文部分内容阅读
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 detect Hepatitis virus-like particle (HE VLP) by size-exclusion HPLC (SE-HPLC). Methods HE VLPs were prepared by ultracentrifugation and Sephacryl S-200HR gel chromatography. The HE VLPs prepared by the two methods were analyzed by SDS-PAGE, Western blot, electron microscopy, dynamic light scattering and SE-HPLC. -HPLC qualitative and quantitative analysis of HE VLP method, and the use of the method of culture supernatant HE VLP at different time points for quantitative analysis. Results SDS-PAGE and Western blot analysis showed that the HE VLP prepared by the two methods showed a molecular weight of about 50,000 of the target protein band, the concentration and purity of the same, and has the same reactogenicity; electron microscopy can be seen, HE VLPs prepared by ultracentrifugation were uniform hollow particles with a diameter of about 30 nm. The chromatographic purification of HE VLPs showed a large number of irregular aggregates and a few hollow particles. Dynamic light scattering analysis showed that HE VLPs prepared by ultracentrifugation only had 1 peak, particle diameter of 27.2 nm, purity greater than 98%, chromatographic purification HE VLP particles ranging from 2 to 120 nm in radius; SE-HPLC analysis showed that HE VLP content in the range of 1 ~ 40μg linear with the peak area Related, and good precision; the use of this method can be real-time quantitative HE VLP in the virus cell culture medium. Conclusion SE-HPLC can qualitatively and quantitatively analyze HE VLPs.