【摘要】 为制备单核细胞增生李斯特菌SigB蛋白及其多克隆抗体,采用PCR方法扩增参考菌株10403S的sig B基因片段,并克隆至原核表达载体pET-30a构建重组质粒,测序验证后转化至大肠杆菌Rosseta(DE3)感受态细胞进行原核表达,利用Ni-NTA亲和层析纯化Sig B蛋白,通过免疫兔体获得多克隆抗体。结果表明:成功构建SigB重组质粒,并诱导表达出可溶性Sig B蛋白,其相对分子质量约30 ku,纯化后的SigB蛋白质量浓度约为1.5 mg∕mL,获得的多克隆抗体效价为1∶51 200。经Western-blot验证,该多克隆抗体能特异性检测Sig B蛋白,表现出良好的抗体特异性。本研究可为探究单增李斯特菌的应激调控机制奠定基础。更多还原

【Abstract】 To prepare the protein and polyclonal antibody ofListeria monocytogenesSigB,sigBgene fragment of reference strain 10403 S was amplified by PCR. The PCR product was cloned into p ET-30 a expression vector and expressed inEscherichia coliRosseta(DE3) after sequenced. Polyclonal antibody was obtained from immunized rabbits with recombinant SigB protein purified by Ni-NTA affinity chromatography column. The results showed that the recombinant SigB plasmid was successfully constructed,and the soluble protein sigB was induced and expressed,with a relative molecular weight of about 30 ku. The concentration of purified SigB protein was about 1. 5 mg∕mL. The titer of anti-SigB polyclonal antibody was 1∶ 51 200. Western-blot assay result showed that the polyclonal antibody could specifically detect SigB protein, indicating the polyclonal antibody had good specificity. This study could lay a foundation for exploring the stress regulation mechanism ofL. monocytogenes.更多还原