恶性疟原虫主要裂殖子表面蛋白1C末端片段在毕赤酵母表达系统的高效表达与纯化(英文)

High level expression and purification of 1 C-terminal fragment of merozoite surface protein of Plasmodium falciparum in Pichia pastoris

目的利用毕赤酵母表达系统高效表达并获得纯度较为理想的恶性疟原虫主要裂殖子表面蛋白1 C末端片段(MSP-119)重组蛋白。方法将带有6-his基因的MSP-119基因序列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,筛选出高拷贝转化子,优化表达条件,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。结果毕赤酵母分泌表达MSP-119蛋白,免疫印迹结果表明MSP-119基因表达蛋白能被抗MSP-119的单抗所识别,出现特异条带,将培养上清利用Ni-NTA柱纯化后,推算MSP-119蛋白的表达量为1.0g/L。结论酵母细胞表达系统可高效表达可免疫识别的MSP-119重组蛋白。

To obtain an ideal recombinant C-terminal fragment of the merozoite surface protein of Plasmodium falciparum in the Pichia pastoris expression system, the major surface protein-1 19（MSP-119） gene sequence bearing the 6-his gene was inserted into expression vector pPIC9k and the target gene was transformed to the susceptible yeast cells GSll5 by using electroporation. The multiple inserts were screened and the successfully expressed MSP-119 protein with the relative molecular weight of 12kDa in the supernatants of cell cultures could be detected by SDS-PAGE. Meanwhile, Western blot analysis also demonstrated that this protein reacted with mouse anti-MSP-119 monoclonal antibody, and the expression level of MSP-119 was more than 1.0 g/L. It is concluded that this recombinant protein expressed in the Pichia pastoris expression system resembles the native proteins existed.