摘要目的:在毕赤酵母细胞中稳定表达经连接肽连接的HBsAg和水痘-带状疱疹病毒糖蛋白(glycoprotein，g)E的重组蛋白，并进行初步纯化及颗粒性验证。方法:构建HBsAg-gE的四拷贝重组表达质粒，经酶切线性化后电转化至毕赤酵母KM71细胞中，甲醇诱导表达。采用免疫印迹法和ELISA检测表达的重组蛋白，经蔗糖密度梯度离心和凝胶排阻层析初步纯化后进行透射电子显微镜观察。结果:HBsAg-gE四拷贝重组质粒经双酶切鉴定正确。表达的重组蛋白经免疫印迹法检测，可以与小鼠抗gE单克隆抗体及抗HBsAg多克隆抗体特异性结合，相对分子质量约90 000。经ELISA检测HBsAg呈阳性。蔗糖密度梯度离心后，重组蛋白聚集在40%至55%区带之间，在透射电子显微镜下观察到直径约20 nm左右的病毒样颗粒结构。结论:成功在毕赤酵母中表达了HBsAg-gE病毒样颗粒，为在酵母系统中表达水痘-带状疱疹重组疫苗奠定了基础。

abstractsObjective:To steadily express HBsAg linked with glycoprotein E(gE) of varicella-zoster virus(VZV) in Picha pastoris and evaluate whether the recombinant protein forms into virus-like particles(VLPs) after preliminary purification. Methods:Four-copy recombinant plasmid for HBsAg-gE was constructed, followed by linearization with endonucleases before transformed to Picha pastoris KM71 by electroporation, and the expression was induced by methanol. The expressed recombinant protein was identified by both Western Blot and ELISA. Sucrose density gradient centrifugation and gel exclusion chromatography were performed to purify the recombinant protein before observed by transmission electron microscopy. Results:The four-copy recombinant plasmid for HBsAg-gE was identified by double enzymatic digestion. The expressed recombinant protein bound to mouse monoclonal antibody against gE and polyclonal antibody against HBsAg specifically and its relative molecular mass was about 90 000. The protein also showed a positive ELISA result of HBsAg and gathered between zones 40% and 55% after sucrose density gradient centrifugation. VLPs with a diameter of 20 nm were observed by transmission electron microscopy.Conclusion:VLPs of HBsAg-gE are successfully expressed in Picha pastoris, laying a foundation for developing the VZV recombinant vaccine in yeast system.