Stx1-A亚单位的原核表达、复性及抗原性鉴定

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。

Prokaryotic Expression,Renaturalization and Antigenic Identification of Shiga Toxin I A Subunit

Objective To construct prokaryotic expression plasmid of recombinant Shiga toxin I A(Stx1-A) subunit,express and purify the recombinant protein,and investigate the antigenicity and application of the recombinant protein.Methods The Stx1-A subunit gene was amplified from the genome of E.coli O157:H7 and recombined with expression vector pET32a( ).Subsequently,the recombinant vector was transformed into BL21(DE3).Induced by IPTG,the inclusion bodies of recombinant protein were affiliated to Ni-column,purified and renaturalized with an on-column refolding system.The antigenic activity of purified recombinant protein was assessed with western blot.Results Enzyme digestion analysis and sequencing showed that the target fragment was successfully inserted into the vector pET32a( ).The target fusion protein Stx1-A,most of which was soluble and present in the inclusion bodies,accounted for 15 % of total cellular protein as shown by SDS-PAGE.The soluble Stx1-A was purified and renaturalized during on-column binding to Ni-sepharose,reaching a purity of 95 %.The immunological specificity of Stx1-A was confirmed by Western blot analysis.Conclusion Soluble Stx1-A protein possessing specific antigenicity was expressed and purified successfully,which may facilitate preparation of Stx1-A antibody and future study on the biological function of Stx1-A subunit.