克罗诺杆菌IbpA蛋白的表达、纯化及其免疫原性  

Expression, purification and immunogenicity of Ibp A protein of Cronobacter sakazakii

作　　者：赵志晶[1] 梁昊宇[1] 董思国[1] 田万红[1] 王斌[1] 曾明[1] 

机构地区：[1]中国食品药品检定研究院,北京100050

出　　处：《中国生物制品学杂志》2016年第3期242-247,251,共7页Chinese Journal of Biologicals

基　　金：重大新药创制专项课题(1040871310101)

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。Objective To express, purify and determine the immunogenicity of Ibp A protein of Cronobacter sakazakii.Methods The genome of Cronobacter sakazakii CMCC 45402 was extracted, from which the ibp A gene fragment was amplified by PCR and inserted into vector p ET30a（＋）. The constructed recombinant plasmid p ET30a（＋）-ibp A transformed to E. coli BL21（DE3）and induced with IPTG at various concentrations（0. 1, 0. 2, 0. 5, 0. 8 and 1. 0 mmol / L）at 30 and 37 ℃ for 2. 5, 4 and 5 h. The expressed product was identified by SDS-PAGE and Western blot, then purified by Ni gel affinity chromatography. BALB / c mice were immunized i. p. with the purified protein, and boosted twice each at an interval of 2 weeks. Serum samples were collected one week after the last immunization and determined for titer by ELISA. Meanwhile, the influence of heat shock on expression of Ibp A in C. sakazakii was determined. Results Restriction analysis proved that recombinant plasmid p ET30a（＋）-ibp A was constructed correctly. The optimal concentration, temperature and time for induction with IPTG were 0. 2 mmol / L, 30 ℃ and 5 h respectively. The expressed protein, with a relative molecular mass of about 24 000, existed in a soluble form, which showed specific binding to monoclonal antibody a-gainst His-Tag, and reached a purity of more than 96% after purification. The immune sera of mice reached a titer of more than 1 ∶ 25 000, with which the expression of Ibp A was detected in C. sakazakii after heat shock. Conclusion The prokaryotic expression system for ibp A gene was successful constructed, while highly purified recombinant protein was obtained, and high titer antiserum was prepared, which laid a foundation of further study on relationship between ibp A gene and heat-resistance of C. sakazakii.

关 键 词：克罗诺杆菌 Ibp A蛋白 原核表达 纯化 免疫原性 

分 类 号：R378.2[医药卫生—病原生物学] Q786[医药卫生—基础医学]