Zhang Yuyao, Ma Xiuli, Huang Bing, Li Yufeng, Yu Kexiang, Li Jianliang, Liu Cunxia, Han Hongyu, Cui Yanshun
Wei Sheng Wu Xue Bao. 2015 Apr 4;55(4):501-9.
To simultaneously detect antibodies against Duck hepatitis A type 1 (DHAV-1) and type 3 (DHAV-3) viruses, we developed an indirect enzyme-linked immunosobent assay (ELISA) with bacterially expressed recombinant viral protein as antigen in Escherichia coli.
We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into pET-32a expression vector, designated as pET-1VP3-3VP1. The fusion protein DHAV-1VP3-3VP1 expressed correctly and was subsequently used to develop an indirect ELISA assay.
DHAV-1VP3-3VP1 fusion protein expressed in BL21 (DE3) cells following induction by Isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The expressed protein was very antigenic and reactive to virus-specific antibodies in western blot assay. The optimal working concentration for coating antigen was 1.0 microg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650 > OR = 0.38.
The ELISA method based on the prokaryotic expression of VP3 (DHAV-1) and VP1 proteins (DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.
为同时检测1型鸭甲型肝炎病毒（DHAV-1）和3型鸭甲型肝炎病毒（DHAV-3）抗体，我们构建了一种以大肠杆菌中细菌表达的重组病毒蛋白为抗原的间接酶联免疫吸附测定（ELISA）。
我们通过逆转录-聚合酶链反应（RT-PCR）扩增了DHAV-1的全长VP3基因和DHAV-3的全长VP1基因，然后将它们克隆到pET-32a表达载体中，命名为pET-1VP3-3VP1。融合蛋白DHAV-1VP3-3VP1表达正确，随后用于构建间接ELISA检测方法。
异丙基-β-D-1-硫代半乳糖苷（IPTG）诱导后，DHAV-1VP3-3VP1融合蛋白在BL21（DE3）细胞中表达。表达的蛋白具有很强的抗原性，在蛋白质印迹分析中能与病毒特异性抗体发生反应。包被抗原的最佳工作浓度为每孔1.0微克，血清样本的工作浓度为1:200稀释，区分阳性和阴性血清样本的临界值为OD650>或=0.38。
基于VP3（DHAV-1）和VP1蛋白（DHAV-3）原核表达的ELISA方法可有效用于临床检测针对DHAV-1和DHAV-3的抗体。
