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目的构建志贺毒素1-A亚单位(Stx1-A)原核表达质粒,使其在大肠埃希菌中表达,对表达的重组蛋白进行纯化,并探讨其抗原性及应用价值。方法以大肠埃希菌O157DNA为模板扩增Stx1-A基因,重组克隆入原核表达载体pET32a(+),转化大肠埃希菌BL21(DE3),异丙基硫代-半乳糖苷(IPTG)诱导表达,包涵体经镍柱柱上复性和纯化,并对纯化产物进行Western印迹鉴定。结果重组质粒经双酶切鉴定和测序分析后证实构建成功。重组蛋白经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示,其表达量占细菌总蛋白量的15%左右,主要以包涵体形式存在,通过镍柱柱上复性获得纯化的可溶性Stx1-A蛋白,纯度达95%以上,Western印迹检测显示特异性条带。结论成功构建了pET32a(+)/Stx1-A重组质粒,并获得了具有抗原特异性的可溶性Stx1-A蛋白,为进一步制备抗Stx1-A抗体和研究其生物学功能奠定了基础。
Objective To construct the prokaryotic expression plasmid of Shiga toxin 1-A subunit (Stx1-A) and express it in Escherichia coli. The expressed recombinant protein was purified and its antigenicity and application value were discussed. Methods Stx1-A gene was amplified from E. coli O157 DNA and cloned into prokaryotic expression vector pET32a (+). The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and induced by isopropylthio-galactoside (IPTG) The expressed and inclusion bodies were refolded and purified by nickel column, and the purified products were identified by Western blotting. Results The recombinant plasmid was identified by double enzyme digestion and sequencing analysis confirmed the successful construction. The recombinant protein was expressed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which accounted for about 15% of the total bacterial protein content, mainly in the form of inclusion bodies, Purified soluble Stx1-A protein was obtained with a purity of more than 95%. Western blotting showed specific bands. Conclusion The recombinant plasmid pET32a (+) / Stx1-A was successfully constructed and an antigen-specific soluble Stx1-A protein was obtained, which laid the foundation for the further preparation of anti-Stx1-A antibody and its biological functions.