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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。
OBJECTIVE: To efficiently express Pichia pastoris expression system and to obtain a highly purified recombinant protein of the major merozoite surface protein 1 C-terminal fragment (MSP-119) of Plasmodium falciparum. Methods The MSP-119 gene sequence with 6-his gene was inserted into the pichia pastoris expression vector pPIC9k. The target gene was transformed into GS115 yeast by high-pressure electroporation and the high copy transformants were screened. Optimization of expression conditions, the use of methanol induced expression. The expression product was detected by SDS-PAGE and Western blotting. Results MSP-119 protein was secreted by Pichia pastoris. Western blotting showed that the MSP-119 protein was recognized by anti-MSP-119 monoclonal antibody and showed a specific band. After the culture supernatant was purified by Ni-NTA column, The expression level of MSP-119 protein was estimated to be 1.0 g / L. Conclusion The yeast cell expression system can express MSP-119 recombinant protein with high immunogenicity.