恶性疟裂殖子表面蛋白1合成基因在毕赤酵母中的表达

Production of the Major Merozoite Surface Protein 1(MSP1) of Plasmodium falciparum in Pichia pastoris

恶性疟原虫裂殖子表面蛋白 1是当今疟疾疫苗主要的候选抗原。由于天然MSP1基因AT含量异常高(为 74% ) ,使得克隆全长天然基因无法实现。本文已全合成了msp1基因 (4940bp) ,解决了该天然基因在异源系统中不稳定的问题。为制备大量msp1重组蛋白进行疫苗有效性试验 ,本研究建立了msp1基因在毕赤酵母中的表达 ,将合成的msp1基因克隆到毕赤酵母胞内表达载体 pPIC3 5 ,构建了重组质粒 pPIC3 5 /msp1,用电击转化毕赤酵母得到重组转化子 ,经PCR证实msp1基因已整合于毕赤酵母染色体中。含有重组表达质粒的毕赤酵母菌经甲醇诱导后表达出全长msp1重组蛋白。表达产物能与识别MSP1分子二硫键依赖构象表位的特异性单抗发生很强的反应 ,表明msp1重组蛋白至少在该表位构象上与天然蛋白一致。从毕赤酵母中分离得到大量msp1为开展该蛋白的结构与功能 ,特别是测定其疟疾保护性免疫提供可能。

The major Merozoite Surface Protein 1(MSP1) of Plasmodium falciparum is an important candidate for malaria vaccine.Highly instability of msp1 gene due to its unusual high AT content (74%),however,render cloning of the full\|length of this gene impossible.Synthesis of the entire gene using other codon usage has provided possibility to produce the entire MSP1 in heterogeneous system.In this investigation,the entire MSP1 recombinant protein has been expressed in Pichia pastoris. Insertion of the synthetic msp1 gene into the pichia expression vector pPIC3 5 generated the recombinant plasmid pPIC3 5/msp1 and transformation of the Pichia pastoris SMD1168 by electroporation produced recombinant transformants which were verified by PCR amplification of msp1\|derived fragment from the chromosome.The recombinant MSP1 derived from Pichia was interacted strongly with monoclonal antibody mAb5 2 recognizing the disulfide\|bond\|depended conformational epitope at the C\|terminus of MSP1 molecule,indicating that the protein generated in this system resembled most likely to native protein at least at this conformational epitope.Availability of isolating the recombinant protein from the yeast provides a possibility to examine the protective potential of the molecule as a vaccine candidate.