克罗诺杆菌IbpA蛋白的表达、纯化及其免疫原性
发布时间：2019-03-17 07:49
【摘要】：目的表达并纯化克罗诺杆菌Ibp A蛋白,检测其免疫原性。方法提取克罗诺杆菌菌株CMCC45402的基因组,经PCR获得ibp A基因片段,连接至表达载体p ET30a(+),构建重组质粒p ET30a(+)-ibp A。将重组质粒转化E.coli BL21(DE3)菌株,用不同终浓度的IPTG(0.1、0.2、0.5、0.8、1.0 mmol/L)分别于30和37℃诱导表达不同时间(2.5、4、5 h),进行SDS-PAGE及Western blot分析。表达产物经Ni凝胶亲和层析纯化后,经腹腔免疫BALB/c小鼠,每2周加强免疫1次,共2次,末次免疫1周后,经小鼠眼球采血,分离血清,ELISA法检测血清效价。同时检测热激对Ibp A蛋白表达水平的影响。结果重组质粒p ET30a(+)-ibp A经双酶切鉴定证明构建正确。最佳IPTG诱导终浓度为0.2 mmol/L,最佳诱导温度为30℃,最佳诱导时间为5 h。诱导表达产物相对分子质量约24 000,主要以可溶性形式存在,可与抗His-Tag单克隆抗体特异性结合;纯化蛋白的纯度可达96%;小鼠免疫血清效价均可达1:25 000以上。用小鼠血清可检测到热激后克罗诺杆菌Ibp A的表达。结论本实验成功构建了ibp A基因的高效原核表达系统,获得了高纯度重组蛋白,制备了高效价抗血清,为后续ibp A基因和克罗诺杆菌热抗性关系的研究奠定了基础。
[Abstract]:Objective to express and purify the Ibp A protein of Clostridium sp., and to detect its immunogenicity. Methods Genome of CMCC45402 was extracted and ibp A gene fragment was obtained by PCR. The recombinant plasmid p ET30a ()-ibp A was constructed by ligating into expression vector p-ET30a (), to construct recombinant plasmid p ET30a ()-ibp A. The recombinant plasmid was transformed into E.coli BL21 (DE3) strain and expressed in different concentrations of IPTG (0.1, 0.2, 0.5, 0.8, 1.0 mmol/L) at 30 鈩，
本文编号：2442079
[Abstract]:Objective to express and purify the Ibp A protein of Clostridium sp., and to detect its immunogenicity. Methods Genome of CMCC45402 was extracted and ibp A gene fragment was obtained by PCR. The recombinant plasmid p ET30a ()-ibp A was constructed by ligating into expression vector p-ET30a (), to construct recombinant plasmid p ET30a ()-ibp A. The recombinant plasmid was transformed into E.coli BL21 (DE3) strain and expressed in different concentrations of IPTG (0.1, 0.2, 0.5, 0.8, 1.0 mmol/L) at 30 鈩，
本文编号：2442079
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