易发生聚集的重组HBcAg病毒样颗粒的纯化*

目的:探索针对易发生聚集的重组HBcAg病毒样颗粒(VLP)的有效纯化方案。方法:培养的大肠杆菌经IPTG诱导重组HBcAg蛋白的表达,菌体超声破碎后的离心沉淀用含有不同浓度尿素的PBS缓冲液重悬溶解,经密度梯度离心并结合电镜观察对VLPs行为进行分析鉴定。以Sepharose 4 FF凝胶过滤层析在选定的尿素条件下纯化沉淀溶解液,纯化获得的目的蛋白进一步在含30%山梨醇的PBS中脱盐去除尿素。整个过程以SDS-PAGE及电镜进行各步骤样品中目的蛋白的分析。结果:含有1mol/L尿素的PBS缓冲溶液重悬超声沉淀,可有效溶解聚集的VLPs,在蔗糖密度梯度离心中显示典型HBcAg VLPs的行为,且电镜观察颗粒形态结构完整。经1mol/L尿素下凝胶过滤,VLPs进一步获得纯化。在脱尿素过程中流动相采用含30%山梨醇的PBS,有效避免了VLPs在尿素去除后重新聚集。结论:尿素与山梨醇的联合应用,为具有聚集现象的VLPs纯化制备提供了一种有效解决方案。

The Purification Procedure for the Recombinant HBcAg Virus-like Particle Easy to Generate Aggregation

Objective: To explore an effective purification procedure of recombinant HBcAg virus like particles (VLPs) that are prone to aggregation. Methods: The expression of recombinant HBcAg protein was induced by IPTG in cultured E. coli. The centrifugation precipitates of bacteria after ultrasonic fragmentation were resuspended and dissolved in PBS buffer with different concentrations of urea. The VLPs behavior was analyzed and identified by density gradient centrifugation and electron microscopy. The precipitate solution was purified by Sepharose 4 FF gel filtration chromatography under the selected urea conditions, and the purified target protein was further desalinate to remove urea with PBS containing 30% sorbitol. The entire process was analyzed by SDS-PAGE and electron microscopy. Results: The ultrasonic precipitates resuspensed with PBS buffer containing 1mol/L urea was effective dissolve the aggregated VLPs, which showed the behavior of typical HBcAg VLPs in sucrose density gradient centrifugation, and morphology and structure of the particales were complete by electron microscope. VLPs were further purified after 1mol/L urea gel filtration. In the process of urea removal, PBS containing 30% sorbitol was used as the mobile phase, which effectively avoided the reaggregation of VLPs after urea removal. Conclusion: The combined application of urea and sorbitol provides an effective solution for the purification and preparation of VLPs with aggregation phenomenon.