铜绿假单胞菌外膜蛋白OprM原核表达及抗体制备

The prokaryotic expression and antibody preparation of outer membrane protein OprM of Pseudomonas aeruginosa

目的制备铜绿假单胞菌(PA)主动外排系统外膜蛋白OprM及抗OprM多克隆抗体,为深入研究主动外排系统奠定基础。方法从PA基因组中克隆oprM基因,构建pET28 a-c-OprM表达载体,在大肠埃希菌中经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达并用免疫转印试验(W estern b lot)验证,建立重组OprM纯化方案,免疫动物制备多克隆抗体。结果扩增出带有EcoRⅠ和NotⅠ酶切位点及保护碱基的oprM基因序列,构建了pET28 a-c-OprM表达载体,诱导表达的重组OprM蛋白为带有H is标签的不可溶包涵体,建立并优化了重组OprM的纯化方法,免疫小鼠获得抗OprM蛋白的多克隆抗体。结论成功制备了OprM蛋白和抗OprM多克隆抗体,为深入研究该蛋白及相关的主动外排系统提供了有效手段。

Objective To obtain an active efflux system outer membrane protein OprM of Pseudomonas aeruginosa（PA） and the OprM polyclonal antibodies,and promote the studies on active efflux system of PA.Methods The oprM gene was amplified from PA genome.The expression of pET28a-c-OprM was constructed,expressed and induced in Escherichia coli by isopropyl-beta-D-thiogalactopyranoside（IPTG）,and the results were verified by Western bolt.The purification of recombinant OprM was established.It was used as the immunogen to produce polyclonal antibodies in mice.Results The oprM gene with EcoR Ⅰ and Not Ⅰ enzyme sites and basyl protection was amplified,and the expression vector of pET28a-c-OprM was constructed.The recombinant expression-induced OprM was insoluble inclusion body with His tag.The protocols to purify recombinant OprM were established and optimized,and OprM polyclonal antibodies were produced in immunized mice.Conclusions The recombinant OprM protein is successfully purified,and OprM polyclonal antibodies are obtained.They will be useful to deeply explore the biological functions of OprM and relative active efflux system.