重组汉逊酵母表达的乙型肝炎病毒表面抗原疏水层析图谱的峰形规律分析

目的探讨疏水层析纯化重组汉逊酵母(Hansenula polymorpha,HP)表达的乙型肝炎表面抗原(HBsAg)层析图谱与蛋白含量及HBsAg含量的相关性。方法采用疏水层析纯化8批HP-HBsAg小量试验样品和5批小量验证试验样品及5批酿酒酵母表达的HBsAg对照试验样品,采用Lowry法和电化学发光法(Electrochemiluminescentimmunoassay,ECLIA)分别检测纯化样品中蛋白含量和HBsAg含量,并计算HBsAg的回收率;通过几何法封闭紫外监测图谱特征峰图形,用数字求积仪采用积分法求出各封闭特征峰的图形面积,并进行分析;取小量验证试验1批样品的层析主峰和副峰收液进行电镜观察。结果 8批HP-HBsAg小量试验和5批HP-HBsAg小量验证试验HBsAg的平均回收率分别为70%和54%。HP-HBsAg疏水层析图谱由两部分构成:穿透峰和目标峰(HBsAg峰),目标峰可见高低不同的两个峰(主峰与副峰);对照图谱中有穿透峰和目标峰。对目标峰进行量化分析,主峰、副峰与目标峰的面积比值分别与其所对应的收集量、蛋白含量和HBsAg含量比值相关。电镜观察可见小量验证试验样品主峰收液中HBsAg颗粒大小均一,副峰收液中HBsAg颗粒大小不均一。结论疏水层析纯化重组HP表达的HBsAg效果良好,通过峰形之间面积比值,可获得峰形所对应的收集量、蛋白量和HBsAg量比值,为HBsAg的进一步纯化提供了参考。

Graph pattern of Hansenula polymorpha-derived hepatitis B surface antigen purified by hydrophobic interaction chromatography

Objective To investigate the relationship of graph pattern of Hansenula polymorpha(HP)-derived hepatitis B surface antigen(HBsAg) purified by hydrophobic interaction chromatography(HIC) to HBsAg and protein contents.Methods Eight batches of the HP-HBsAg samples for small-scale preparation,five batches of samples for small-scale verification test and five batches of Saccharomyces cerevisae(SC)-derived HBsAg as control were purified by hydropobic chromatography,and determined for protein and HBsAg contents by Lowry method and electrochemiluminescent immunoassay(ECLIA),based on which the recovery rate of HBsAg was calculated.The UV-monitored graph was geometrically divided into several sections,of which the area was integrated by digital planimeter.The main and side peaks of one batch of samples for small-scale verification test were collected for electron microscopy.Results The mean recovery rate of eight batches of HP-HBsAg samples for small-scale preparation was 70%,while that of five batches for small-scale verification test was 54%.The HIC pattern consisted of breakthrough peak and target(HBsAg) peak,while the target peak appeared as a main peak and a side peak.The pattern of control samples appeared as breakthrough peak and target peak.Further quantitative analysis of the target peak showed that the area ratios of main,side and target peaks were related to those of collected liquid,protein and HBsAg contents.Electron microscopy showed that the sizes of HBsAg particles were even in the collected main peak,while were uneven in the side peak.Conclusion HIC was effective for purification of HP-derived HBsAg.By comparison of peak area graphed by UV-monitor,the contents of collected liquid,protein and HBsAg were calculated.It provided a reference for further purification of HBsAg.