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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(Circumsporozo-ite 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 WG1.3-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 WG1.3-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: To express and purify Circumsporozo-ite protein (CSP) of Plasmodium falciparum by Wheat Germ Cell-Free System for immunological evaluation. Methods: According to the sequence of CSP gene of Plasmodium falciparum retrieved from PlasmoDB, a pair of specific primers was designed. The P. falciparum 3D7 genome DNA obtained from ATCC was used as a template to amplify the target fragment by PCR. The PCR product was double-digested with NcoI and SmaI, ligated with pIVEX 1.3WG vector which was digested with the same sequence, and then transformed into E. coli DH5a. The recombinant plasmid pIVEX WG1.3-CSP was screened. The recombinant plasmids were added to the wheat germ expression kit and expressed according to the instructions. The expressed product was purified by affinity chromatography to finally obtain the CSP protein. Results: The CSP gene sequence was successfully amplified by PCR and the pIVEX WG1.3-CSP recombinant plasmid was successfully constructed. After WB test, it was proved that wheat germ cell-free expression system could successfully express and purify CSP protein. Conclusion: The cell-free expression system of wheat germ cells can efficiently express CSP of Plasmodium falciparum, laying a solid foundation for the research of CSP in the follow-up of diagnostic reagents and vaccine evaluation.