恶性疟原虫环子孢子蛋白在无细胞表达系统的表达及纯化研究

目的: 应用麦胚无细胞表达系统(Wheat Germ Cell-Free System),表达及纯化恶性疟原虫环子孢子蛋白(Circumsporozoite protein,CSP),以便用于免疫学评价。 方法: 根据从PlasmoDB检索获得的恶性疟原虫 CSP基因序列,设计一对特异引物,以从ATCC获得的恶性疟原虫3D7基因组质粒(P.f.3D7 genomic DNA)为模板,经PCR反应扩增目的片段。PCR产物经NcoI与Sma I双酶切后,与经过同样酶切的pIVEX 1.3WG载体连接,随后转化大肠杆菌DH5а,经筛选获得pIVEX WG 1.3-CSP重组质粒。将该重组质粒加入到麦胚表达试剂盒并按照说明书进行表达,表达产物经亲和层析柱纯化,最终获得CSP蛋白。 结果: PCR反应成功扩增CSP基因序列,并成功构建pIVEX WG 1.3-CSP重组质粒;经WB试验证明,麦胚无细胞表达系统可成功表达并纯化CSP蛋白。 结论: 麦胚无细胞表达系统可高效表达恶性疟原虫CSP,为CSP用于后续的诊断试剂和疫苗评价方面的研究打下了坚实的基础。

Objective: Applying Wheat Germ Cell-Free System to express Plasmodium falciparum circumsporozoite protein to meet with the immunology research. Methods: According to the CSP gene sequence downloaded from PlasmoDB,designed two specific primers,and used Plasmodium falciparum 3D7 genomic DNA as template to perform PCR assay;then PCR products were double digests by Nco I and Sma I,and connected with pIVEX 1.3WG vector which were digested by the same enzymes.Ligation products were transformed into E.coli DH5а and screened for recombination plasmid.Put the recombination plasmid into Wheat Germ Cell-Free System Kit and expressed as described by Manufacture's Guide,expression products were purified by affinity chromatography,in the end high purity CSP was acquired. Results: PCR assay could smoothly amplify CSP gene sequence and pIVEX 1.3WG-CSP plasmid was reconstructed successfully.Western Blot test proved the expression of CSP. Conclusion: Using Wheat Germ Cell-Free System can express high level Plasmodium falciparum circumsporozoite protein,this would make a solid foundation for next step research.