鲍曼不动杆菌MsrA蛋白原核表达载体的构建、纯化及氧化应激条件下的表达  

Construction,purification and expression under oxidative stress for prokaryotic expression vector of acinetobacter baumannii MsrA protein

作　　者：谭潇[1] 肖非[1] 

机构地区：[1]邵阳学院医学检验系,湖南邵阳422000

出　　处：《微生物学免疫学进展》2017年第1期32-35,共4页Progress In Microbiology and Immunology

基　　金：2015年湖南省教育厅科学研究项目(15C1257)

摘　　要：目的以鲍曼不动杆菌甲硫氨酸亚砜还原酶A(methionine sulfoxide reductase A,MsrA)构建p ET28a-MsrA并表达、纯化原核表达重组质粒,观察在氧化应激条件下MsrA的表达水平,为其抗氧化功能的研究提供依据。方法设计并合成分别带NdeⅠ和XhoⅠ酶切位点的MsrA引物,用PCR方法扩增MsrA基因,构建p ET28a-MsrA重组质粒并转化大肠杆菌,用异丙基-β-D-硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside,IPTG)诱导重组MsrA蛋白表达,经Ni2+亲和层析分离纯化。利用荧光定量PCR方法,观察在氧化应激条件下MsrA蛋白的表达量。结果构建了编码MsrA蛋白的重组质粒p ET28a-MsrA,SDS-PAGE显示在相对分子质量约22 000处有一明显条带的重组MsrA蛋白,并利用Ni2+亲和层析柱纯化获得纯度较高的MsrA蛋白。氧化应激也可快速诱导MsrA蛋白的表达。结论成功构建了p ET28a-MsrA重组质粒及表达纯化系统,且氧化应激条件下MsrA蛋白表达上调。Objective Bauman acinetobacter methionine sulfoxide reductase （MsrA） was used to construct a recombinant prokaryotic expression plasmid pET28a- MsrA, to express and purify the plasmid and observe the expression level of MsrA in oxidative stress, so as to provide a basis for the research on MsrA anti-oxidation function. Methods MsrA primer syn- thesis was designed and constructed with Nde I and Xho I restriction sites and MsrA gene was amplified by PCR, the re- combinant plasmid pET28a-MsrA was constructed and transformed into Escherichia coli. The expression of recombinant MsrA protein was induced by using IPTG （isopropyl thiogalactoside -D- beta） and purified by Ni2＋ affinity chromatography. Fluorescent quantitative PCR was used to observe the expression of MsrA protein under oxidative stress. Results The con- struction of recombinant pET28a-MsrA to encode MsrA protein was completed, there was a clear band with a relative molec- ular mass of about 22 000 by SDS-PAGE analysis, the highly purified recombinant MsrA protein was obtained by a Ni2~ af- finity chromatograph. The rapid expression of MsrA protein could be induced under oxidative stress. Conclusion The ex- pression and purification systems for recombinant plasmid PET28a-MsrA were successfully constructed, and the expression of MsrA protein was up-regulated under oxidative stress.

关 键 词：鲍曼不动杆菌 甲硫氨酸亚砜还原酶A 原核表达 蛋白纯化 氧化应激 

分 类 号：R378[医药卫生—病原生物学]