摘要
为建立荧光免疫分析方法对空肠弯曲菌的快速检测,利用基因工程技术构建pET32a-peb1A原核表达载体,并对重组蛋白进行了抗原性分析。利用共价偶联的方法,在水溶性缩合剂EDC和催化剂DMAP的作用下,使水溶性量子点的羧基与重组蛋白的羟基结合,发生酯化反应,并通过磷酸盐缓冲溶液进行透析纯化得到目标偶联物。采用荧光分光光度法对偶联物进行表征,并利用荧光偏振免疫分析方法对空肠弯曲菌阳性血清进行检测。结果表明:空肠弯曲菌黏附蛋白PEB1A具有良好的抗原性,且量子点标记空肠弯曲菌黏附蛋白前后的荧光光谱发射峰由595 nm偏移至592 nm。示踪物与阳性血清混合前后荧光偏振值变化△80mP。由此证明,空肠弯曲菌黏附蛋白成功偶联到水溶性量子点上,且结构未受到破坏。通过荧光偏振法实现了对空肠弯曲菌的检测,所制备的示踪物具有良好的特异性,为建立荧光疫分析方法对空肠弯曲菌的快速检测提供依据。
In order to implement the rapid detection of Campylobacter jejuni by fluorescence immunoassay, the prokaryotic expression vectorpet32a-peb1 A was constructed by genetic engineering technique and induced to express. Then the antigenicity of the recombinant protein wasanalyzed. Using covalent coupling method, under the action of water-soluble condensate EDC and catalyst DMAP, the carboxyl groups of thewater-soluble quantum dots were combined with the hydroxyl groups of the recombinant protein, and the esterification reaction was carriedout and dialyzed through the phosphate buffer solution to obtain the target conjugate. The conjugate was characterized by fluorescence spectro-photometry. The fluorescence polarization immunoassay method was used to detect theCampylobacterpositive serum. The results showed thatthe adhesion protein peb1 A ofC. jejunihad good immunogenicity. When the quantum dot was labeled withC. jejuniadhesion protein, the flu-orescence emission peak shifted from 595 nm to 592 nm. The fluorescence polarization value increased 80 mp after tracer and positive serumwas mixed. The results showed that the adhesion protein ofC. jejuniwas successfully coupled to the water-soluble quantum dots, and thestructure was not damaged. The detection ofC. jejuniwas achieved by fluorescence polarization method, which proved that the prepared tracerhad good specificity. This study provides the basis for the rapid detection ofC. jejuniby fluorescence immunoassay.
出处
《畜牧与兽医》
北大核心
2017年第10期81-85,共5页
Animal Husbandry & Veterinary Medicine
基金
国家重点研发项目(2016YFD0501004)
关键词
空肠弯曲菌
黏附蛋白
原核表达
量子点
荧光免疫分析
Campylobacter jejuni
adhesion protein
prokaryotic expression
quantum dots
fluorescence immunoassay
