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目的构建p ET28a-PTD-Ag85B原核表达载体,并对其在E.coli BL21(DE3)中表达条件进行优化。方法将TAT-PTD序列插入到用限制性内切酶Nhe I和Bam H I双酶切的p ET28a质粒中,获得p ET28a-PTD重组质粒;RT-PCR法扩增Ag85B基因并将其克隆至p ET28a-PTD质粒中,获得p ET28a-PTDAg85B重组质粒。将重组质粒p ET28a-PTD-Ag85B转化至大肠杆菌,异丙基硫代半乳糖苷(IPTG)诱导PTD-Ag85B融合蛋白表达,聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定表达产物。筛选最适诱导剂浓度、诱导温度和时间,尿素复性包涵体,并用His-tag离子亲和层析柱分离纯化融合蛋白。结果成功构建p ET28aPTD-Ag85B重组质粒,在E.coli BL21中能高效表达PTD-Ag85B融合蛋白。结论成功表达及纯化了PTD-Ag85B蛋白,为获得大量PTD-Ag85B融合蛋白提供了技术储备。
Objective To construct p ET28a-PTD-Ag85B prokaryotic expression vector and optimize its expression in E. coli BL21 (DE3). Methods The TAT-PTD sequence was inserted into the pET28a plasmid digested with restriction enzymes Nhe I and Bam HI to obtain p ET28a-PTD recombinant plasmid. The Ag85B gene was amplified by RT-PCR and cloned into p ET28a-PTD plasmid to obtain p ET28a-PTDAg85B recombinant plasmid. The recombinant plasmid pET28a-PTD-Ag85B was transformed into Escherichia coli, and IPTG induced the expression of PTD-Ag85B fusion protein. The expressed product was identified by SDS-PAGE. The optimal inducer concentration, induction temperature and time, urea refolding inclusion body were screened, and the fusion protein was purified by His-tag ion-affinity chromatography. Results The p ET28aPTD-Ag85B recombinant plasmid was successfully constructed and the PTD-Ag85B fusion protein was highly expressed in E. coli BL21. Conclusion The PTD-Ag85B protein was successfully expressed and purified, providing a technical reserve for obtaining a large amount of PTD-Ag85B fusion protein.