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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。
Objective To express and purify Ibp A protein of Cronobacterium and test its immunogenicity. Methods The genomic DNA of Cronobacter strain CMCC45402 was extracted. The ibp A gene fragment was obtained by PCR and ligated into expression vector p ET30a (+) to construct recombinant plasmid p ET30a (+) - ibpA. The recombinant plasmids were transformed into E.coli BL21 (DE3) strain and induced by IPTG (0.1,0.2,0.5,0.8,1.0 mmol / L) at different final concentrations for 30, 37, and 37 ℃ for different times (2.5, 4, 5 h ), SDS-PAGE and Western blot analysis. BALB / c mice were immunized intraperitoneally once every 2 weeks for 2 times. After the last immunization, blood was collected from the eyeballs of mice and the serum was separated. ELISA France test serum titer. The effect of heat shock on the expression of Ibp A protein was also examined. Results The recombinant plasmid p ET30a (+) - ibp A was confirmed by double enzyme digestion. The best final induction concentration of IPTG was 0.2 mmol / L, the best induction temperature was 30 ℃, and the best induction time was 5 h. The relative molecular mass of the expressed product was about 24 000, which existed mainly in soluble form and could specifically bind to anti-His-Tag monoclonal antibody. The purity of the purified protein was up to 96%. The titer of immune serum in mice reached 1:25 000 or more. Expression of Cronobacter Ibp A after heat shock was detectable with mouse serum. Conclusions We successfully constructed a highly efficient prokaryotic expression system of ibp A gene, obtained high purity recombinant protein and prepared high titer antiserum, which lays the foundation for the study on the relationship between ibp A gene and Corynebacterium thermo-resistance.