恶性疟原虫Pf12基因在大肠杆菌中的表达及其产物的初步鉴定

EXPRESSION OF PLASMODIUM FALCIPARUM PF12 IN ESCHERICHIA COLI AND PRELIMINARY IDENTIFICATION OF THE EXPERSSED PRODUCTS

目的 克隆并表达恶性疟原虫Pf12基因 ,为进一步研究其抗原表位奠定基础。 方法 将恶性疟原虫Pf12基因克隆入高效融合表达载体 pGEX 4T 1,转化大肠杆菌BL2 1(DE3 ) ,2 8℃、IPTG诱导表达 ,SDS PAGE和Western blot免疫印迹分析表达产物。 结果 成功构建重组质粒 pGEX Pf12 ,经诱导后表达出含外源基因的融合蛋白 ,SDS PAGE分析表达产物分子质量约 66ku ,Western blot免疫印迹表明 ,表达的产物能特异地被抗GST多抗 (1∶5 0 0稀释 )识别 ,亦能较特异地与抗疟原虫鼠免疫血清结合。 结论 恶性疟原虫Pf12在原核表达系统 pGEX 4T 1/BL2 1(DE3 )中获得成功表达 ,为下一步表达蛋白纯化 ,以及研究Pf12基因包含的抗原表位提供试验依据。

Objective To cloning and express Pf12 gene of Plasmodium falciparum for its antigenic epitopes studies. Methods Pf12 was recombined and inserted into expression vector pGEX-4T-1, then recombinant plasmid pGEX-Pf12 was transformed into E. coli BL21(DE3).The transformants pGEX-Pf12 were induced by IPTG at the temperature of 28℃ and the expressed protein was analyzed by SDS-PAGE, Western-blot. Results The recombinant plasmid was constructed successfully. SDS-PAGE analysis of bacterial crude extracts showed that the molecular weight of the expressed fused proteins was about 66 ku.Western-blot assay demonstrated that the expressed products could be recognized by antibody-GST and mouse anti- P. falciparum serum. Conclusion The fusion protein harbored the antigenic epitopes of P. falciparum , and the successfully expressed protein could be the foundation of further studies of Pf12.