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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。
Objective To investigate the correlation between the HBsAg chromatogram, the protein content and the HBsAg content of the purified recombinant Hansenula polymorpha (HP) by hydrophobic chromatography. Methods Eight batches of HP-HBsAg and five batches of validated test samples and five batches of HBsAg control samples expressed by Saccharomyces cerevisiae were purified by hydrophobic chromatography and detected by Lowry and Electrochemiluminescentimmunoassay (ECLIA) The protein content and HBsAg content in the purified sample were calculated and the recovery rate of HBsAg was calculated. The geometrical method was used to close the characteristic peak pattern of the ultraviolet monitoring spectrum, and the integral area method was used to calculate the graphical area of each closed characteristic peak using the digital sigmoid meter. A small amount of validation test 1 batch samples of the chromatographic peak and the secondary peak liquid collected electron microscopy. Results The average recoveries of HBsAg in 8 batches of HP-HBsAg and 5 batches of HP-HBsAg were 70% and 54%, respectively. HP-HBsAg hydrophobic chromatogram consists of two parts: the penetration peak and the target peak (HBsAg peak), the target peak can be seen two different peaks (main peak and secondary peak); control spectrum has the penetration peak and the target peak. Quantitative analysis of the target peak, the main peak, the peak and the target peak area ratio corresponding to their collection, protein content and HBsAg content ratio. Electron microscopy can be seen a small amount of verification test samples collected in the main peak HBsAg particle size uniformity, the second peak in the collection of HBsAg particle size heterogeneity. Conclusion Hydrophobically purified HBsAg expressed by recombinant HP has a good effect. The ratio of the peak shape to the collected volume, the amount of protein and the ratio of HBsAg can be obtained by the ratio of peak area, which provides a reference for the further purification of HBsAg.