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目的通过原核表达系统制备裂谷热病毒Gn片段主要抗原区蛋白,获取纯化蛋白,并分析抗原性。方法将合成的裂谷热Gn基因用PCR的方法扩增具有保护性抗原表位的两段Gn1和Gn2,并将其定向插入原核表达质粒pColdⅠ中,构建重组表达质粒pColdⅠ-Gn1和pColdⅠ-Gn2,并将重组质粒转化到BL21(DE3)感受态中,以IPTG诱导表达,SDS-PAGE分析表达蛋白和表达量,并利用His-tag Ni柱进行亲和层析纯化,Western-blot鉴定表达蛋白。结果所构建的质粒pColdⅠ-Gn1和pColdⅠ-Gn2序列正确,SDS-PAGE检测到目的蛋白,Western-blot显示截断的Gn蛋白具有很好反应原性。结论成功纯化了裂谷热病毒Gn主要抗原区的表达蛋白。
OBJECTIVE: To prepare the main antigenic regions of Rn of Gn R rivivirus by using prokaryotic expression system and obtain the purified protein, and to analyze the antigenicity. METHODS: Two fragments of Gn1 and Gn2 with protective epitopes were amplified by PCR from the synthesized Rn gene of Rift Valley, and inserted into prokaryotic expression plasmid pColdⅠ to construct recombinant expression plasmids pColdⅠ-Gn1 and pColdⅠ-Gn2 , And the recombinant plasmid was transformed into BL21 (DE3) competent cells and expressed by IPTG. The expressed protein and protein were analyzed by SDS-PAGE and purified by affinity chromatography using His-tag Ni column. The expressed protein was identified by Western-blot . Results The constructed plasmids pColdⅠ-Gn1 and pColdⅠ-Gn2 had the correct sequence. The target protein was detected by SDS-PAGE. Western-blot showed that the truncated Gn protein had good reactivity. Conclusion The expressed protein of the main antigenic region of Gn of Rift Valley fever virus was successfully purified.