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目的 :在毕赤酵母中表达 Annexin32并研究其功能。方法 :用 G4 18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、MM 3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 ) 2 SO4 +PI法沉淀 ,过 FPL C- Superdex75分子筛纯化蛋白并以此免疫小鼠。 结果 :有两个表型为Muts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 2 0 0～ 35 0 m g/ 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
OBJECTIVE: To express Annexin32 in Pichia pastoris and study its function. Methods: Nine high copy transformants were rapidly screened by G418 method. After the phenotypes were identified by plate culture (MD and MM) and PCR, they were induced by methanol in three kinds of media of BMMY, BMM and MM respectively. The supernatants were identified by SDS-PAGE and Western blotting, precipitated by (NH4) 2SO4 + PI, and the mice were immunized with the purified protein over FPL C-Superdex75 molecular sieves. Results: There were two high copy transformation strains with high expression of Muts, the BMMY was the best, and the expression product had two bands, the relative molecular masses were about 38,000 and 40,000 respectively, accounting for 80% of the total secreted protein Above, the product concentration of 200 ~ 35 0 mg / L. Western blot confirmed that both of the expressed products have the immunogenicity of native Annexin32 molecules. Animal experiments show that the immunogenicity of yeast-expressing Annexin32 is significantly higher than that of prokaryotic expression. Conclusion: Annexin32 was successfully expressed in Pichia pastoris, which laid the foundation for further research