福氏志贺菌外输调节蛋白基因marA的原核表达载体构建及其鉴定  

Construction and expression of a prokaryotic expression vector encoding efflux pump regulated protein gene marA of Shigella flexneri

作　　者：魏小娟[1] 张继瑜[1] 王松泰[1] 牛建荣[1] 李剑勇[1] 周旭正[1] 吴培星[1] 李金善[1] 

机构地区：[1]中国农业科学院兰州畜牧与兽药研究所,甘肃兰州730050

出　　处：《中国动物检疫》2008年第11期26-28,共3页China Animal Health Inspection

基　　金：国家自然科学基金资助项目(30671582)

摘　　要：目的构建志贺菌外输调节蛋白基因marA的原核表达载体并对其表达的蛋白进行鉴定。方法采用PCR从志贺菌基因组DNA中扩增得到志贺菌外输调节蛋白基因marA,并将其定向克隆到原核表达载体pET-30a,构建原核表达重组质粒pET-30a-marA,重组子经限制性内切酶分析、PCR及测序鉴定后,转化宿主菌BL21,IPTG诱导表达,产物进行SDS-PAGE电泳、免疫印迹分析鉴定。结果经鉴定证实原核表达载体pET-30a-marA构建成功,且在大肠杆菌BL21中获得了MarA与His的融合表达,且表达蛋白产物的分子质量大小与预期值一致,并可被特异性抗体所识别。结论MarA在大肠杆菌中获得了高效的表达,为进一步研究其免疫学特性和功能奠定了基础。Objective To construct a prokaryotic expression vector encoding an efflux pump regulated protein MarA of shigella ,and express the vector in E eoli BL21. Methods The marA gene was amplified from the genomic DNA of Shigella with PCR,and then was inserted into the prokaryotie expression vector pET-30a, to construct the prokaryotic expression recombinant plasmid pET-30a-marA. After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis,the E,coli BL21 containing the recombinant plasmid pET-30a-marA was induced with IPTG.The expression of MarA was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis.Results The prokaryotic expression vector pET-30a-marA was constructed successfully. The E.coli BL21 transformed with recombinant plasmid pET-30a-marA had expressed His-MarA recombinant pro- rein.The expression product could react with the specific antibody,and the relative molecular weight of the expres- sion product was identical to the expected value.Conclusion MarA protein is expressed stably in E.coli BL21.which will provide a basis for the study on function of the orotein and its role in immune responses.

关 键 词：志贺菌 marA 原核表达 

分 类 号：S852.612[农业科学—基础兽医学]