禽腺病毒4型中国流行株Fiber2蛋白的原核表达与间接ELISA抗体检测方法的建立  

Prokaryotic expression of the Chinese fowl adenovirus serotype 4 Fiber2 protein and development of an indirect ELISA for antibody detection

作　　者：田开月 张玉菡 郭慧芳 李宁 杨霞[1] 王增 赵军[1] TIAN Kaiyue;ZHANG Yuhan;GUO Huifang;LI Ning;YANG Xia;WANG Zeng;ZHAO Jun(College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China)

机构地区：[1]河南农业大学牧医工程学院

出　　处：《畜牧与兽医》2019年第9期76-80,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划(2016YFD0500800; 2016YFD0501100)

摘　　要：为建立禽腺病毒4型(FAdV-4)特异性抗体检测方法,将密码子优化合成的FAdV-4中国流行株Fiber2蛋白的编码基因克隆于pET-32a(+)载体,在大肠杆菌BL21(DE3)中诱导表达。用纯化的Fiber2蛋白作为包被抗原,通过一系列条件的优化,建立了一种检测FAdV-4抗体的间接ELISA方法。结果:该方法可以特异地检测抗FAdV-4抗体,与新城疫病毒、传染性法氏囊病病毒、传染性支气管炎病毒、减蛋综合征病毒、H5和H9亚型禽流感病毒等常见禽病毒的抗血清均无交叉反应,表明所建立ELISA具有较好的特异性;FAdV-4阳性血清经1?3 200稀释后检测结果仍为阳性,表明所建立ELISA方法具有较高的敏感性;所建立ELISA的批内和批间重复试验的变异系数分别为1.5%~5.2%和1.1%~5.6%,表明其重复性良好。应用建立的ELISA方法对河南省及周边部分地区养禽场内FAdV-4的感染情况进行了血清流行病学调查,结果显示被检地区养禽场均存在不同程度的FAdV-4型抗体阳性,平均阳性率为59.53%。由此表明本研究所建立的ELISA方法可以用于FAdV-4感染的监测和流行病学调查。To establish a method for detecting specific antibodies against the fowl adenovirus serotype 4(FAdV-4), the codon-optimized Fiber2 gene was cloned into the pET-32 a(+) vector, and the expression of Fiber2 protein was induced in E. coli BL21(DE3). After optimization of a series of conditions, an indirect ELISA for detecting FAdV-4-specific antibodies was established using the purified Fiber2 protein as a coating antigen. The results showed that there was no cross-reaction between the FAdV-4 antigen and antibodies against the Newcastle disease virus, infectious bursal disease virus, infectious bronchititis virus, egg-dropping syndrome virus, H9 and H5 subtype AIV, indicating that the established ELISA had a good specificity. The ELISA had good sensitivity and repeatability, too, because the Anti-FAdV-4 positive serum with 1:3200 dilution was still ELISA positive, and the Intra-and inter-assay coefficients of variation were 1.5%-5.2% and 1.1%-5.6%, respectively. Seroepidemiological prevalence of FAdV-4 infection in some chicken farms in Henan Province and the surrounded area was investigated using the eatablished ELISA. The results indicated that all the tested chicken farms were anti-FAdV-4 antibody positive with average positive rate of 59.53%. The established ELISA in this study might be used for the detection of antibodies against FAdV-4, and might provide a method for the monitoring and epidemiological investigation of FAdV-4.

关 键 词：禽腺病毒4型 Fiber2蛋白 间接ELISA 流行病学调查 

分 类 号：S852.6[农业科学—基础兽医学]