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目的在大肠埃希菌中表达登革Ⅱ型病毒E蛋白DⅢ结构域,并进行免疫反应性鉴定。方法用C6/36细胞培养病毒,提取病毒RNA,经RT-PCR扩增获得目的基因片段D2E-DⅢ。双酶切D2E-DⅢ和表达质粒pET-30a(+),经回收纯化后连接构建重组质粒pET30a-D2E-DⅢ。筛选阳性菌落,提取质粒转化BL21(DE3)感受态细胞,用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达目的蛋白并进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析和Western blot鉴定。结果 RT-PCR扩增得到约300bp的目的基因片段D2E-DⅢ,双酶切鉴定重组质粒pET30a-D2E-DⅢ位置正确,经测序分析表明插入序列及开放读码框正确。经15%分离胶SDS-PAGE电泳分析,重组菌在相对分子质量为10×103附近有明显的诱导表达带,其大小与预测值一致;Western blot结果显示重组蛋白与确诊登革热Ⅱ型阳性血清存在特异性反应。结论成功地在大肠埃希菌中表达登革Ⅱ型病毒E蛋白DⅢ区,并进行免疫反应性鉴定,结果显示其具有良好的免疫活性。
Objective To express dengue virus E protein D Ⅲ domain in Escherichia coli and identify its immunoreactivity. Methods The virus was cultured in C6 / 36 cells and the viral RNA was extracted. The target gene D2E-DⅢ was amplified by RT-PCR. The double digested D2E-DIII and the expression plasmid pET-30a (+) were recovered and purified to construct the recombinant plasmid pET30a-D2E-DIII. The positive colonies were screened and the plasmids were transformed into BL21 (DE3) competent cells. The target protein was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) and sodium dodecyl sulfate-polypropylene Gel electrophoresis (SDS-PAGE) analysis and Western blot identification. Results The fragment of D2E-DⅢ was amplified by RT-PCR and about 300bp was obtained. The double-digested recombinant plasmid pET30a-D2E-DⅢ was correctly identified. The sequencing result showed that the inserted sequence and open reading frame were correct. The SDS-PAGE analysis of 15% separation gel showed that the recombinant protein had an obvious induction expression band at a molecular weight of 10 × 103, which was consistent with the predicted value. Western blot showed that the recombinant protein was positive with type Ⅱ positive serum Specific reaction. Conclusion The dengue virus E protein DⅢ region was successfully expressed in Escherichia coli and its immunoreactivity was identified. The results showed that it has good immunocompetence.