摘要
【目的】对大肠杆菌AcrAB外排泵系统acrA、acrB基因进行原核表达,获得的表达产物AcrA、AcrB蛋白分别免疫兔,制备相应的抗体,建立大肠杆菌外排泵表达水平的ELISA检测方法。【方法】扩增AcrAB外排泵系统acrA、acrB基因片段,扩增片段分别与原核表达载体pET-41a(+)连接构建重组质粒,于BL21(DE3)中进行诱导表达。表达产物AcrA、AcrB纯化后分别免疫新西兰大白兔,获得抗AcrA、AcrB抗血清,并用Western blot方法对表达产物进行鉴定分析。纯化的AcrA、AcrB蛋白分别按不同的浓度包被酶标板,与不同稀释倍数的抗血清反应,通过ELISA检测确定最佳抗原包被浓度及抗体稀释度,初步建立大肠杆菌外排泵表达水平的ELISA检测方法。【结果】成功克隆和表达了acrA、acrB基因片段,经SDS-PAGE检测表明两种表达产物AcrA、AcrB均以可溶性蛋白形式存在。AcrA、AcrB抗原蛋白最佳包被浓度分别为1.0μg.ml-1和0.5μg.ml-1,抗AcrA、AcrB血清的最佳稀释度分别为1﹕800和1﹕400。用初步建立的ELISA方法检测8株大肠杆菌多重耐药菌株外排泵表达水平,结果表明分别用两种蛋白作包被抗体检测外排泵表达水平的结果一致。【结论】本研究通过原核表达的方法获得大肠杆菌AcrAB外排泵系统AcrA、AcrB蛋白并制备了相应的抗体,分别用两种抗体包被酶标板,并建立了大肠杆菌外排泵表达水平的ELISA检测方法,对8株多重耐药菌株的检测结果表明该方法可行、可靠。
[Objective] In the present study, the prokaryotic expression of acrA and acrB genes of AcrAB effiux system in E. coli was conducted and then AcrA, AcrB polyclonal antibody was prepared after inoculated rabbits with the two expression products, respectively. ELISA method for detecting expression level of AcrAB efflux system in multi-drug resistant E. coil was established. [ Method ] Partial fragments of acrA and acrB genes were amplified. By using the recombinant DNA technology, the recombinant protein of pET-AcrA and pET-AcrB was obtained. After sequencing and assaying, positive recombinant plasmids pET-AcrA and pET-AcrB were transformed into E. coli BL21 （DE3） to express under the induction of IPTG. Subsequently the expression products were examined. The above two expression products were purified and then injected subcutaneously into rabbits respectively to prepare polyclonal antibody. Western blot was performed to confirm that the expressed fusion protein could specifically react with antiserum. ELISA method was developed with either AcrA or AcrB as coated antigen for detecting AcrAB efflux system expression level. [Result] About 567bp, 516bp of acrA, acrB partial fragments were successfully cloned and expressed in E. coli BL21 （DE3）. Two expression products were confirmed to be fusion protein by SDS-PAGE, and showed excellent antigenicity with anti-AcrA, anti-AcrB rabbit antibodies in the Western blotting. Polyclonal antibodies of AcrA and AcrB were successfully obtained. The optimal coated concentration of AcrA and AcrB were 1.0 μg·ml^-1 and 0.5 μg·ml^-1, respectively. The best dilution of the sera to be tested was 1 ： 800 and 1 ： 400, respectively. The results from using each fusion protein as coated antigen for detecting expression level were highly coincident, demonstrating high expression in efflux pump in most detected multi-drug resistant E. coli. [ Conclusion ] The present study purified the AcrA, AcrB fusion protein, prepared the polyclonal antibody of the two proteins, and established ELISA methods for detecting AcrAB efflux system expression in multi-drug resistant E. coli.
出处
《中国农业科学》
CAS
CSCD
北大核心
2008年第12期4238-4243,共6页
Scientia Agricultura Sinica
基金
教育部高等学校博士学科点专项科研基金(20050564012)
国家自然科学基金--广东省自然科学基金联合基金重点项目(U0631006)
