恶性疟原虫AMA-1(Ⅲ)基因在毕赤酵母系统中的高效表达  

High-level expression and purification of the ectodomain Ⅲ in apical membrane antigen-1 of Plasmodium falciparum inPichia pastoris

作　　者：张忠广[1] 赵恒梅[1] 宫玉香[1] 

机构地区：[1]青岛大学医学院人体寄生虫学教研室,青岛266021

出　　处：《中国人兽共患病杂志》2005年第3期240-243,共4页Chinese Journal of Zoonoses

基　　金：青岛市科技计划项目 (2 0 0 1KNS -2E -2 7 2 )

摘　　要：目的　利用毕赤酵母高效表达系统表达用于下游实验的恶性疟原虫AMA - 1(Ⅲ )重组蛋白。方法　将测序正确的AMA - 1(Ⅲ )基因插入毕赤酵母分泌型表达载体 pPIC9k中 ,用高压电穿孔转化法将目的基因转化入酵母感受态细胞GS115 ,筛选高拷贝转化子 ,利用甲醇进行诱导表达。表达产物用SDS -PAGE和免疫印迹进行检测。结果　AMA - 1(Ⅲ )蛋白表达于培养上清 ,蛋白的相对分子质量分别为 16 .3ku ,免疫印迹结果表明AMA - 1(Ⅲ )基因表达蛋白能被抗AMA - 1(Ⅲ )的单抗所识别 ,出现特异条带 ,推算AMA - 1(Ⅲ )蛋白的表达量为 0 .8g/L。 结论　酵母细胞表达系统可高效表达在构象方面接近天然蛋白的AMA - 1(Ⅲ )To obtain enough quantity of the purified ectodomain Ⅲ in apical membrane antigen-1 (AMA-Ⅲ), the gene encoding AMA-Ⅲ was expressed in yeast system of Pichia pastoris at competative level. After the validity of the AMA-1 sequences was confirmed, recombinant expression vector was constructed by ligating the gene with vector pPIC9k. By using electroporation transformation method, recombinant expression vector containing this gene was transformed into susceptible Pichia pastoris GS115 cells, and the multiple inserts were screened. The expressed proteins were analyzed by SDS-PAGE and Western blotting analysis. The experimental results showed that the expressed protein in the culture supernatant was detected with a relative molecular mass of 16.3 ku as demonstrated by SCSD-PAGE, and Western blotting analysis also showed this protein could react with mouse anti-AMA-1 monoclonal antibody. The expression level of the AMA-1(Ⅲ) in Pichia pastoris may reach up to 0.8 g/L. It concludes that the recombinant AMA-1(Ⅲ) protein that approaches the natural proteins in conformation can be expressed at high level in yeast cells.

关 键 词：疟原虫 恶性 裂殖子顶端膜抗原1 疟疾疫苗 毕赤氏酵母 

分 类 号：R382.3[医药卫生—医学寄生虫学]