Zhang Yan, Wang Zhanfeng, Zhan Yang, Gong Qian, Yu Wanting, Deng Zhibang, Wang Aibing, Yang Yi, Wang Naidong
Laboratory of Functional Proteomics (LFP) and Research Center of Reverse Vaccinology (RCRV), College of Veterinary Medicine, Hunan Agricultural University, Furong District, Changsha, 410128, People's Republic of China.
Arch Virol. 2016 Jun;161(6):1485-91. doi: 10.1007/s00705-016-2816-9. Epub 2016 Mar 14.
Porcine circovirus type 2 (PCV2) causes increased mortality and poor growth or weight loss in apparently healthy swine. Therefore, methods to detect PCV2-specific antibodies in swine serum are important for prevention, diagnosis, and control of PCV2-associated diseases (PCVAD). In this study, PCV2 virus-like particles (VLPs) were used to develop a rapid, simple and economical indirect enzyme-linked immunosorbent assay to detect (with high sensitivity) PCV2-specific antibodies in swine serum. The PCV2 capsid protein (Cap) was overexpressed in E. coli after optimizing the cap gene. Subsequently, the soluble Cap was rapidly purified in one step by automated fast protein liquid chromatography (FPLC). The purified PCV2 Cap was shown by transmission electron microscopy and gel filtration chromatography to be capable of self-assembling into VLPs in vitro. Using the purified VLPs as antigens, optimal operating conditions for the VLP ELISA were determined. The concentration of PCV2 VLPs was 1 µg/ml per well, and the dilution factors for swine serum and horseradish peroxidase (HRP)-labeled goat anti-pig antibody were 1:150 and 1:4000, respectively. Out of 241 serum samples tested with this assay, 83.4 % were found to be positive. Importantly, the VLP ELISA had a total coincidence rate of 97.4 % (74/76) compared to an Ingezim PCV2 ELISA IgG assay. In summary, this rapid, inexpensive VLP ELISA has the potential to greatly facilitate large-scale investigations of PCV2-associated serotypes.
猪圆环病毒2型（PCV2）可导致看似健康的猪死亡率增加、生长缓慢或体重减轻。因此，检测猪血清中PCV2特异性抗体的方法对于预防、诊断和控制PCV2相关疾病（PCVAD）至关重要。在本研究中，PCV2病毒样颗粒（VLP）被用于开发一种快速、简单且经济的间接酶联免疫吸附测定法，以（高灵敏度地）检测猪血清中的PCV2特异性抗体。在优化衣壳基因后，PCV2衣壳蛋白（Cap）在大肠杆菌中过表达。随后，通过自动快速蛋白质液相色谱（FPLC）一步快速纯化可溶性Cap。透射电子显微镜和凝胶过滤色谱显示，纯化的PCV2 Cap能够在体外自组装成VLP。以纯化的VLP作为抗原，确定了VLP ELISA的最佳操作条件。PCV2 VLP的浓度为每孔1μg/ml，猪血清和辣根过氧化物酶（HRP）标记的山羊抗猪抗体的稀释倍数分别为1:150和1:4000。用该测定法检测的241份血清样本中，83.4%被发现为阳性。重要的是，与Ingezim PCV2 ELISA IgG测定法相比，VLP ELISA的总符合率为97.4%（74/76）。总之，这种快速、廉价的VLP ELISA有潜力极大地促进对PCV2相关血清型的大规模调查。
