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

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

目的 在毕赤酵母表达系统中获得大量构象正确的 3D7/MSP1 4 2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/msp1 4 2基因 (12 0 2bp) ,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 4 2基因序列 ,该合成基因在毕赤酵母系统 (Pichiapastoris)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 。

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.