Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.
Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, Guangzhou 510642, China.
Viruses. 2020 Aug 20;12(9):912. doi: 10.3390/v12090912.
Pseudorabies, also known as disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies and the disease causes huge economic losses in the pig industry. The establishment of a differential diagnosis technique that can distinguish between wild-type infection and vaccinated responses and monitor vaccine-induced immunoglobulin G(IgG) is crucial for the eventual eradication of pseudorabies. The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52-238 and gB codons at aa 539-741 were expressed to obtain recombinant PRV gE and gB proteins via a pMAL-c5x vector. After purification with Qiagen Ni-nitrilotriacetic acid (NTA) agarose affinity chromatography, the two proteins were analyzed via SDS-PAGE and immunoblotting assays. Two single fluorescent-microsphere immunoassays (FMIAs) were established by coupling two recombinant proteins (gE and gB) to magnetic microbeads, and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera, and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection, and 95.74% and 96.3% for the gB IgG antibody detection via dual FMIA. We provide a new method for monitoring PRV protective antibodies in vaccinated pigs and differentiating wild-type PRV infection from vaccinated responses simultaneously.
伪狂犬病，又称猪疱疹病毒病，是由伪狂犬病病毒（PRV）引起的一种急性病毒性感染。猪是伪狂犬病的天然宿主之一，该病给养猪业造成了巨大的经济损失。建立一种能够区分野毒株感染和疫苗接种反应并监测疫苗诱导的免疫球蛋白 G（IgG）的鉴别诊断技术，对于最终根除伪狂犬病至关重要。本研究旨在开发一种具有高特异性和灵敏度的 PRV gE 和 gB 蛋白 IgG 抗体快速双重检测方法。通过 pMAL-c5x 载体表达 PRV gE 氨基酸残基（aa）52-238 处的密码子和 gB 氨基酸残基（aa）539-741 处的密码子，获得重组 PRV gE 和 gB 蛋白。使用 Qiagen Ni-亚氨基二乙酸（NTA）琼脂糖亲和层析法纯化后，通过 SDS-PAGE 和免疫印迹试验分析两种蛋白。通过将两种重组蛋白（gE 和 gB）偶联到磁性微球上，建立了两种单荧光微球免疫分析（FMIAs），并通过整合两种单分析方法，开发了一种有效的双重 FMIA。对每种检测方法的最佳血清稀释度、与其他常见猪病毒阳性血清的相关性以及与 ELISA 检测两种 PRV 抗原的比较进行了验证。与 ELISA 相比，双重 FMIA 检测 gE IgG 抗体的特异性和灵敏度分别为 99.26%和 92.3%，检测 gB IgG 抗体的特异性和灵敏度分别为 95.74%和 96.3%。我们提供了一种监测接种猪 PRV 保护抗体的新方法，同时能够区分野毒株感染和疫苗接种反应。
