论文部分内容阅读
目的通过抗体与磁性荧光纳米颗粒标记的免疫层析技术,建立一种快速检测产热稳定直接溶血素(TDH)副溶血性弧菌的检测方法。方法构建副溶血性弧菌TDH基因片段,将p ET-28a-TDH重组质粒载体转化大肠杆菌表达并制备克隆抗体。抗体与磁性荧光纳米颗粒偶联后,制成免疫层析检测试纸条。将混有不同浓度标准菌株样品与荧光纳米颗粒-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。并进行了实验的敏感性、特异性、重复性测试及样品模拟实验。结果转化的重组质粒载体在E.coli BL21(DE3)可稳定高效地表达目的蛋白。并实现了单克隆抗体与磁性荧光颗粒很好的偶联,所得偶联物与阳性菌株反应特异性强、敏感性高,阴性对照菌株无反应。结论实验成功制备了与副溶血性弧菌TDH毒力基因表达产物相结合的磁性荧光纳米颗粒复合物。检测过程操作简单,具有灵敏度高、特异性强、重复性好和检测时间短等特点。
OBJECTIVE: To establish a rapid detection method for thermovariable direct hemolysin (TDH) Vibrio parahaemolyticus by immunochromatography with antibodies and magnetic fluorescent nanoparticles. Methods The TDH gene fragment of Vibrio parahaemolyticus was constructed. The recombinant plasmid p ET-28a-TDH was transformed into E. coli and cloned. Antibody conjugated with magnetic fluorescent nanoparticles, made of immunochromatographic test strip. The samples mixed with different concentrations of standard strains were reacted with fluorescent nanoparticles-MAb conjugate and strip for 5 min, and the results were observed under the naked eye by UV light. The sensitivity, specificity, repeatability and sample simulation of the experiment were also studied. Results The recombinant plasmid vector was expressed stably and efficiently in E. coli BL21 (DE3). The monoclonal antibody and magnetic fluorescent particles are well coupled, and the obtained conjugate has strong reaction specificity with the positive strain and high sensitivity, and the negative control strain has no reaction. Conclusion The experiment successfully prepared a magnetic fluorescence nanoparticle complex with TDH virulence gene expression product of Vibrio parahaemolyticus. Detection process is simple, with high sensitivity, specificity, reproducibility and detection time is short and so on.