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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 ,为恶性疟原虫DNA疫苗的研制奠定了基础
Objective To construct recombinant eukaryotic expression plasmid pBK CSP of Plasmodium falciparum CSP gene of Plasmodium falciparum FCC 1 / HN and express in E. coli and identify it. Methods The CSP gene fragment was isolated from recombinant E. coli mycobacterium shuttle plasmid pBCG5.6 / CSP by restriction endonuclease and subcloned into pBK CMV eukaryotic expression vector to construct recombinant eukaryotic Expression plasmid pBK CSP. After induced by IPTG, the recombinant plasmid was expressed in E. coli DH5α, and analyzed by SDS PAGE and Western blotting. Results The CSP gene fragment was isolated from pBCG5.6 / CSP and the recombinant plasmid pBK CSP was successfully constructed. SDS PAGE and Western blot analysis showed that the relative molecular mass of the specific protein band was about 42,000. Conclusion The successful cloning of the CSP gene fragment from pBCG5.6 / CSP and the successful construction of recombinant plasmid pBK CSP induced the expression of non-fusion CSP protein, which laid the foundation for the development of DNA vaccine against Plasmodium falciparum