Annexin32的酵母表达及免疫原性分析

目的：在毕赤酵母中表达Annexin32并研究其功能。方法：用G418法快速筛选出的9个高拷贝转化子，经平板培养法（MD和MM）及PCR鉴定表型后，分别在BMMY、BMM、MM3种培养基中以甲醇诱导表达，培养上清行SDS－PAGE和Western印迹鉴定，用（NH4）2SO4＋PI法沉淀，过FPLC－Superdex75分子筛纯化蛋白并以此免疫小鼠。结果：有两个表型为Mut^a的高拷贝转化菌表达量较高，以BMMY为优，表达产物有两条带，相对分子质量分别约为3.8万和4万，占分泌总蛋白的80％以上，产物浓度为200－350mg/L。Western印迹证实两条表达产物均具有天然Annexin32分子的免疫原性。动物实验证明，酵母表达的Annexin32的免疫原性明显高于原核表达者。结论：在毕赤酵母中成功表达了Annexin32，为进一步研究打下了基础。

Expression and immunogenicity of recombinant Annexin32 in methylotrophic yeast Pichia pastoris

Objective:To express and identify Annexin32 in meythylotrophic yeast Pichia pastoris .Methods:Nine high copy number transformants of Pichia pastoris ,whose phenotypes were screened by culture and PCR,were induced by methanol in BMMY,BMM and MM for the expression of Annexin32.Supernatants after induction were analyzed by SDS PAGE and Western blot.The Annexin32 were separated and purified by (NH 4) 2SO 4+PI and Superdex75.The pure Annexin32 were then immunized to mice.Results:Two Mut s transformants gave high level expression in BMMY.The expression product had 2 bands (MW 38 000 and 40 000) and the concentration was 200 350 mg/L,accounting for over 80% of the total secreted protein.Our data showed higher immunogenicity in Annexin32 from yeast than from E.coli .Conclusion:Annexin32 is expressed successfully in mythylotrophic yeast Pichia pastoris ,providing a foundation for further research.