恶性疟原虫FCC-1/HN株环子孢子蛋白基因片段的亚克隆及表达

Cloning and expression of circumsporozoite protein of FCC-1/HN from Plasmodium falciparum

目的 构建恶性疟原虫FCC 1/HN株CSP基因的重组真核表达质粒 pBK CSP ,在大肠杆菌中进行表达 ,并进行鉴定。方法 采用限制性内切酶法从重组的大肠杆菌 分枝杆菌穿梭质粒pBCG5 .6 /CSP中分离出经过测序鉴定的CSP基因片段 ,将其亚克隆于 pBK CMV真核表达载体 ,构建重组真核表达质粒 pBK CSP。经IPTG诱导 ,重组质粒在大肠杆菌DH5α中进行表达 ,并进行SDS PAGE及免疫印迹分析。结果 从 pBCG5 .6 /CSP中分离出CSP基因片段 ,成功构建 pBK CSP重组质粒 ;SDS PAGE及免疫印迹分析结果显示特异性蛋白条带的相对分子质量约为 42 0 0 0。结论 从pBCG5 .6 /CSP中成功分离出CSP基因片段 ,并成功构建 pBK CSP重组质粒 ,诱导表达CSP非融合蛋白 。

Objective To construct the eukaryotic expression recombinant plasmid containing a gene encoding circumsporozoite protein (CSP) of FCC 1/HN isolate of Plasmodium falciparum , express in E.coli DH5α and identify its expressing products. Methods Using restriction enzymes, the sequenced and identified CSP gene fragment was isolated from the recombinant E.coli Mycobacteria shuttle plasmid pBCG5.6/CSP, then subcloned into eukaryotic expression vector pBK CMV, and recombinant eukaryotic expression plasmid pBK CSP was constructed. Induced by IPTG, the recombinant plasmid was expressed in E.coli DH5α, and its expressing products was identified by SDS PAGE and Western blot. Results The CSP gene fragment was isolated from pBCG5.6/CSP, and a recombinant plasmid pBK CSP was successfully constructed. The results of SDS PAGE and Western blot revealed that the molecular weight of recombinant protein was approximately 42 kDa and can be specially recognized by positive serum of malarial patients from Hainan Province. Conclusion The gene encoding CSP was isolated from pBCG5.6/CSP and pBK CSP recombinant plasmid was successfully constructed. The recombinant protein was expressed in E.coli DH5α and its product was specific for antiserum of Plasmodium falciparum . The recombinant plasmid could be used to carry out further study for DNA vaccine of Plasmodium falciparum.