登革Ⅱ型病毒E蛋白第三结构域在大肠杆菌中的表达、纯化及免疫反应性鉴定

Expression, purification and identification of the domain III of DENV II envelop protein in Escherichia coli

目的在大肠杆菌中表达登革Ⅱ型病毒包膜糖蛋白(E)的第三结构域(DⅢ),并进行纯化及免疫反应性鉴定。方法登革Ⅱ型病毒接种乳鼠,提取总RNA,经RT-PCR扩增E蛋白全长基因片段,构建pMD18-T-DV2-E载体。以pMD18-T-DV2-E质粒为模板,PCR扩增E蛋白DⅢ基因片段并纯化。用限制性内切酶双酶切纯化产物和表达质粒pET-32a(+)并连接构建重组质粒。筛选的阳性质粒转化至E.coli BL21(DE3)感受态细胞,IPTG诱导表达,表达蛋白经SDS-PAGE分析和Ni-NTA树脂纯化,Western blot鉴定。结果 RT-PCR扩增获得1.5kb的E蛋白全长基因片段,构建pMD18-T-DV2-E质粒;以pMD18-T-DV2-E质粒为模板,PCR扩增获得320bp的E蛋白DⅢ基因片段,构建pET-32a(+)-DV2-E-DⅢ表达质粒;IPTG诱导后经SDS-PAGE分析,表达相对分子质量约为29000的可溶性重组蛋白,其表达量占细菌裂解液中总蛋白量的52.50%;Western blot鉴定显示重组蛋白与His·Tag单抗和登革病毒(Ⅰ-Ⅳ)单抗均发生反应。结论重组质粒pET-32a(+)-DV2-E-DⅢ在大肠杆菌中可溶性表达登革Ⅱ型病毒E蛋白第三结构域,重组蛋白能被登革病毒(Ⅰ-Ⅳ)单抗识别。

Objective To express the domain III of DENV II envelop protein in Escherichia coli, obtain the purified recombinant protein and identify its immunoreactivity. Methods Suckling mice were inoculated with live DENV II in the brain. The total RNA was extracted from the brain of the infected mice, and the envelope protein DNA fragment was amplified by RT-PCR and ligated into pMD 18-T to construct pMD 18-T-DV2-E. The domain III DNA fragment of the envelope protein was amplified by PCR with pMD 18-T-DV2-E as the template and cloned into pET-32a （＋） to construct the expression plasmid pET-32a（＋）-DV2-E-DIII. The recombinant plasmid was transformed into E.coli BL21（DE3） and induced by IPTG, and the expressed products were analyzed by SDS-PAGE and Western blotting. Results After RT-PCR amplification, a specific DNA fragment of about 1.5 kb was obtained and ligated into pMD 18-T to construct pMD 18-T-DV2-E. With pMD 18-T-DV2-E as the template, the domain III DNA fragment about 320 bp in length was amplified and the expression plasmid pET-32a （＋）-DV2-E-DIII was successfully constructed. After induction with IPTG, a specific soluble protein with a relative molecular mass of 29 000 was obtained and the expression product accounted for 52.50% of the total protein of the cell lysate. Western blotting demonstrated reactivity of the recombinant protein with His·Tag McAb and DENV （Type I-IV） McAb. Conclusion The recombinant plasmid can be highly expressed in E.coli BL21（DE3） in a soluble form and the recombinant protein can react with DENV （Type I-IV） McAb.