磁性荧光纳米颗粒标记免疫层析技术检测副溶血性弧菌热稳定直接溶血素的试验研究  

Experimental study of fluorescent magnetic nanoparticles labeled immunological assay for Vibrio parahaemolyticus thermostable direct heamolysin

作　　者：向辉[1] 朱海 孙世宏[1] 李颖 王西丽 张汉斌[1] 柳勤[1] 

机构地区：[1]广州市天河区疾病预防控制中心,广东广州510655 [2]深圳市易瑞生物技术有限公司,广东深圳518102

出　　处：《中国食品卫生杂志》2017年第3期302-306,共5页Chinese Journal of Food Hygiene

基　　金：广东省医学科研基金项目(A2013561)

摘　　要：目的通过抗体与标记的磁性荧光纳米颗粒相结合的免疫层析技术,建立一种快速检测产热稳定直接溶血素(TDH)副溶血性弧菌的方法。方法构建产TDH副溶血性弧菌基因片段并进行扩增,扩增产物与质粒载体(p ET-28a)连接,并在大肠埃希菌中表达,制备抗体。抗体与标记的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和阴性对照样品分别与荧光纳米颗粒-单抗偶联物和试纸条共同反应5min,紫外光下肉眼观察结果。对试验的敏感性、特异性、重复性进行测试并进行样品模拟试验。结果重组质粒载体在大肠埃希菌BL21(DE3)可稳定高效地表达分子量为26 k D的可溶形式的目的蛋白,并实现了单克隆抗体与磁性荧光颗粒很好的偶联,所得偶联物与最低浓度10 CFU/ml阳性菌株反应,阴性对照菌株无反应。结论试验制备的产TDH副溶血性弧菌毒力基因表达产物与磁性荧光纳米颗粒有机结合,检测操作过程简单,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 method for rapid detection of Vibrio parahaemolyticus producing thermostable direct haemolysin（ TDH） by immunochromatography technology of the labeled magnetic fluorescent nanoparticles combined with antibody. Methods The gene fragment of Vibrio parahaemolyticus producing TDH was constructed and amplified. The product was ligated with plasmid vector（ p ET-28a） and expressed in Escherichia coli to prepare antibody. The antibody was conjugated with the labeled magnetic fluorescent nanoparticles to prepare an immunochromatographic test strip. The samples were mixed with different concentrations of standard strain and negative control samples respectively with the fluorescent nanoparticle-monoclonal antibody conjugate,incubated on the strip for 5 min,and observed with naked eyes under UV light. Experimental test was conducted for sensitivity,specificity,reproducibility and sample simulation. Results The recombinant plasmid vector could efficiently and efficiently express the soluble protein with molecular weight of 26 k D in Escherichia coli BL21（ DE3）. The monoclonal antibody and the magnetic fluorescent particles were well coupled. The resulting conjugate was reacted with the lowest concentration at 10 CFU/ml positive strain,and the negative control strains had no reaction. Conclusion The magnetic fluorescent nanoparticles coupled with the TDH virulence gene expression product of Vibrio parahaemolyticus was prepared in the experiment. Testing process was simple with high sensitivity,strong specificity,good repeatability and short detection time.

关 键 词：副溶血性弧菌 热稳定直接溶血素 荧光纳米颗粒标记技术 免疫层析 检测 

分 类 号：R155.3[医药卫生—营养与食品卫生学]