基于血清4型禽腺病毒fiber-1蛋白间接ELISA检测方法的建立

Establishment of indirect ELISA method based on fiber-1 protein of fowl adenovirus serotype 4

为了获得血清4型禽腺病毒(fowl adenovirus serotype 4,FAdV-4)fiber-1重组表达蛋白,并建立快速鉴别检测方法,本试验将FAdV-4 fiber-1基因连入pET-32a表达载体,转化BL21(DE3)感受态细胞,将测序正确的重组蛋白经IPTG诱导表达、纯化和Western blot鉴定。以纯化的pET-32a-fiber-1重组蛋白作为ELISA包被抗原,优化反应条件,建立了检测FAdV-4抗体的fiber-1-ELISA方法。结果成功表达pET-32a-fiber-1蛋白,证实可与FAdV-4阳性血清特异性结合,具有良好的反应原性。特异性结果显示,除FAdV-4与FAdV-10外,与其他病原的抗体无交叉反应;阳性血清稀释1 600倍仍可检测到,批内和批间最大变异系数均小于10%。检测了176份临床样品,75份为阳性,101份为阴性。fiber-1-ELISA方法易于操作,特异性强,重复性好,为FAdV-4抗体监测提供了有效方法。

In order to obtain the recombinant fiber-1 protein of fowl adenovirus serotype 4(FAdV-4) in serum and establish a rapid differential detection method.In this study,FAdV-4 fiber-1 gene was connected into pET-32 a expression vector,transformed into BL21(DE3) chemically competent cells,and the correctly sequenced recombinant protein was induced and expressed by IPTG,purified and identified by Western blot.It was confirmed that pET-32 a-fiber-1 could specifically bind to FAdV-4 positive serum and had good reactivity.Using purified pET-32 a-fiber-1 recombinant protein as the ELISA coating antigen,a fiber-1-ELISA method for detection of FAdV-4 antibody was established.The specificity results showed that there was no cross-reaction with the antibodies of other pathogens except FAdV-4 and FAdV-10.Positive serum can also be detected after being diluted 1 600 times,intra and interbatch coefficient of variation were less than 10 %.176 clinical samples were tested,75 were positive and 101 were negative.The fiber-1-ELISA method is easy to operate and can be used for mass detection,providing an effective method for FAdV-4 antibody monitoring.