荧光纳米颗粒标记技术快速检测产enterotoxin B金黄色葡萄球菌的实验研究

Research on a rapid detection of enterotoxin B Staphylococcus aureus with fluorescent nanoparticles labeling technology

目的通过制备SA肠毒素B(enterotoxin B)荧光纳米颗粒单克隆抗体,探讨产enterotoxin B的SA快速检测方法。方法将已构建好的重组质粒载体PET-28a-enterotoxin B转化入E.coli BL21(DE3),进行株中诱导表达,获得表达蛋白,免疫新西兰大白兔获得多克隆抗体。再免疫Bal b/c小鼠,制备单克隆抗体荧光纳米颗粒偶联物。最后将多克隆抗体包被,制成免疫层析检测试纸条,并设质控线。将不同浓度的标准菌株混入样品中,收集提取液,在反应杯中与荧光纳米颗粒-单抗偶联物和试纸条共同反应5 min,紫外光下肉眼观察结果。结果实验克隆分子量为798 bp的enterotoxin B基因片段,构建的重组质粒载体能高效表达,获得分子量为34.5 k D的目的蛋白质。单克隆抗体荧光颗粒偶联物与阳性菌株特异性反应,阴性对照菌株无反应。结论研制的荧光纳米颗粒-enterotoxin B单克隆抗体偶联物特异性强、稳定性好且灵敏度高,实现短时间内对产enterotoxin B的SA快速检测。

Objective Through the preparation of Staphylococcus aureus （ SA ） enterotoxin B monoclonal antibody with fluores- cent nanoparticles, to establish a rapid detection method for SA. Methods The structured recombinant plasmid PET - 28a - Enterotoxin B was transferred into E. coli BL21 （ DE3 ） strains, and then induced and expressed to obtain expression proteins. Then those proteins were used to immune the New Zealand white rabbit to get the polyclonal antibody. Then we used the target proteins to immune Bal b/c mice to obtain monoclonal antibody coupled with fluorescent nanoparticles. At last, the immuno- chromatographic test strip was manufactured by coating the polyclonal antibody and the quality control line was set up. We mixed positive standard strains of different diluted concentration into sample, collected extracting solution after simple processing, left it in a reaction vessel to react with the fluorescent nanoparticles - monoclonal antibody conjunction and the test strip for 5 rain, observed results under ultraviolet light with naked eyes. Results This test successfully cloned 798 bp fragment of Staphylococ- cus aureus enterotoxin B gene, the constructed recombinant plasmid was efficiently expressed and we obtained proteins with the molecular weight of 34.5 kD. These made monoclonal antibody fluorescent particle conjugation had a specific reaction with positive strains, but had no specific reaction with negative control stains. Conclusion The fluorescent nanoparticles - Staphylococcus aureus Enterotoxin B monoclonal antibody conjunction has well specificity, good stability and high sensitivity, they can detect enterotoxin B Staphylococcus aureus rapidly and lay a solid foundation for the further commercialized detection.