摘要
目的构建布鲁菌OMP31蛋白原核表达载体,获得OMP31蛋白。方法 PCR扩增OMP31基因,连接入pET-30a(+)表达载体中,构建pET-30a(+)/OMP31质粒,双酶切鉴定、测序正确后,转化入大肠埃希氏菌BL21(DE3)中,以不同浓度的异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达,并通过二十烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析;蛋白质免疫印迹(western blot)初步分析其免疫原性。结果成功构建pET-30a(+)/OMP31表达载体,并表达出OMP31蛋白,分子量约为31 kDa,主要以包涵体形式存在;通过复性获得>95%的高纯度可溶性蛋白;以布鲁菌虎红平板阳性血清检测,显示复性后蛋白具有良好的免疫反应性。结论成功表达出带组氨酸(HIS)标签的OMP31融合蛋白,且具有较好的蛋白抗原性。
Objective To construct a prokaryotic vector for the expression of OMP31 protein of Brucella melitensis. Methods A DNA fragment coding OMP31 of Brucella melitensis was amplified by PCR and inserted into the vector of pET-30a（＋）. The resultant recombinant plasmid,designated as pET-30a（＋）/OMP31, was verified by double digestion using restriction endonucleases Nde I and Xho I and direct DNA sequencing. Then the plasmid was transfeced into BL21 （DE3） competent cells for the expression of the OMP31 protein. After induction with different concentrations of isopropyl β-D-thiogalactopyranoside（IPTG） ,the collected cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-FAGE）. Results We successfully constructed an expression vector of pET30a（＋）/OMP31. An IPTG-induced expression of the OMP31 protein（31 KDa in molecular weight）was identified with SDS-PAGE. Only appearing in the inclusion bodies, the highly pure OMP31 protein could be obtained by refolding. Western blot assay showed that the refolded protein could be recognized by the anti-serum against Brucella melitensis. Conclusion The recombinant protein of OMP31 with a C-terminal hexa-His-tag was successfully expressed in E. coli BL21 as inclusion bodies. The refolded protein is of good immunogenicity.
出处
《中国公共卫生》
CAS
CSCD
北大核心
2013年第4期596-598,共3页
Chinese Journal of Public Health
基金
国家973计划项目(2010CB530204)
国家自然科学基金(31100657)
广东省大学生创新实验项目(1212110032)
关键词
布鲁菌
OMP31
表达载体构建
蛋白表达
抗原性
Brucella melitensis
OMP31
construction of expression vector
protein expression
antigenicity
