恶性疟原虫3D7株主要裂殖子表面抗原C—末端基因的全合成及其表达

目的：在毕赤酵母表达系统中获得大量构象正确的3D7／MSP1－42重组蛋白进行疫苗有效性试验。方法：采用不对称PCR法拼接合成了3D7／mps1-42基因（1202bp),用电穿孔法将合成基因导入毕赤酵母并进行诱导表达,用免疫印迹法检测表达产物。结果：按毕赤酵母密码子使用频率重新设计并全合成了3D7/msp1-42基因序列,该合成基因在毕赤酵母系统（Pichia pastoris)中得到高水平分泌表达,产生全长重组蛋白。识别构象表位的特异性单克隆抗体能与该重组蛋白反应。结论：重新设计的基因能在毕赤酵母中高水平分泌表达,并产生构象正确的重组蛋白,从而解决了该天然基因在毕赤酵母中不表达的问题。

Synthesis and expression of 42 kD C-terminal region of the major merozoite surface protein (MSP1 - 42) of P. falciparum 3D7 strain in pichia pastoris

Objective Production of 3D7/MSP1 42 recombinant protein with correct conformation in Pichia pastoris for vaccine efficiency assay. Methods Asymmetric PCR based method was utilized to synthesize the 1 202 bp 3D7/msp1 42 gene.The expressing plasmid containing the synthetic gene was introduced into Pichia pastoris by electroporation. The secreted product was detected by Western Blot. Results The redesigned entire 3D7/msp1 42 gene was generated with error free,and expressed to produce 42 kD recombinant protein in secreted form. Conformational monoclonal antibody specific for MSP1 C terminal can interact with the recombinant protein. Conclusion The redesigned 3D7/msp1 42 gene was expressed in P.pastoris with full length of recombnant protein which resembled most likely to the native protein.