汉逊酵母表达乙肝表面抗原的结构研究

目的解析汉逊酵母表达重组乙肝表面抗原（HBsAg）的空间结构，给出其颗粒大小、结构对称性方面的信息。方法整合adr亚型HBsAg的基因到汉逊酵母染色体上，建立重组汉逊酵母菌。经过发酵扩菌、诱导表达、精制纯化等步骤得到纯化的HBsAg。滴加5μl浓度为80μg／ml HBsAg溶液到亲水处理的纯碳膜铜网上，用2％醋酸铀染色，放置24h后，通过透射电镜得到汉逊酵母表达HBsAg颗粒的二维电镜图像。运用EMAN软件包完成图像选择、颗粒框取、颗粒全同性处理、初始模型构建和结构精修等HBsAg重构步骤，获得HBsAg颗粒的三维结构。结果汉逊酵母表达HBsAg颗粒大小服从高斯分布（P=0．45），均值为（28．4±2．6）nm；汉逊酵母表达HBsAg是～中空的“鱼笼”状结构，蛋白亚基分布在球状结构的表面；蛋白的表面分布大小为1～2nm的孔状结构和围绕孔状结构的突起；不具备八面体对称性。结论汉逊酵母表达的HBsAg与血源性的HBsAg在大小和结构对称性方面都有显著差异。

Structure analysis of HBsAg produced by recombinant yeast cells Hansenula polymorpha

Objective To reconstruct the tridimensional structure of HBsAg produced by recombinant yeast cells Hansenula polymorpha, and to present its size distribution and symmetric properties.Methods DNA sequence of HBsAg was intergrated with yeast cells to produce HBsAg. After inducible expression and purification, highly purified HBsAg was gained. 5 μl HBsAg suspension （80 μg/ml） was placed on a perforated carbon film that was treated by glow discharge, and then the film was stained with 2% uranyl acetate. 24 hours later, the film was inserted in a transmission electronic microscope to acquire the images. Reconstruction was accomplished by EMAN software packages, including images chosen, particles collected and selected, initial model construction and refinement. Results The size distribution of HBsAg produced by recombinant yeast cells Hansenula polymorpha obey Gaussian distribution and the average size is （28.4 ±2.6） nm. With small pores and bulges on its envelope, HBsAg is not octaherdral. Conclusion HBsAg expressed by recombinant yeast cells Hansenula polymorpha is different from HBsAg in human serm on both size distrubtion and symmetric properties.