Chicken anemia virus (CAV) causes severe anemia and immunosuppression in chickens. VP1 is the main capsid protein, and is suitable for diagnostic kit development, however, it has 24 arginine residues in the first forty N-terminal amino acids of the protein causing toxicity to bacteria leading to reduced prokaryotic expression. In this study, a 60 amino acid N-terminally truncated VP1 (Δ60VP1) which removes the toxic region was expressed in Escherichia coli and the resultant insoluble recombinant protein was purified by Ni-NTA affinity chromatography with anionic denaturing detergents. The high amounts of purified Δ60VP1 produced (150 mg/L) retained appropriate antigenicity and the antigen was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of CAV. One hundred fifty-two chicken serum samples (n = 152) were evaluated using the newly developed Δ60VP1 indirect ELISA (cut off value = 7.58 % S/P). The sensitivity and specificity of the Δ60VP1 indirect ELISA were 87.50% and 95.31%, respectively, while the agreement between the Δ60VP1 indirect ELISA and the commercial IDEXX CAV ELISA was 90.79% (kappa = 0.814). In this study, we have developed an alternative VP1 production platform in E.coli by truncating the N-terminal 60 amino acids (Δ60VP1) and using anionic denaturing detergents during the purification to successfully solubilize the insoluble Δ60VP1. The antigen was purified with high yield and good immunoreactivity, and an indirect ELISA was developed. The assay could potentially be applied to large-scale CAV serosurveillance.

中文翻译：

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在大肠杆菌中有效生产重组Δ60VP1鸡贫血病毒蛋白及其在血清诊断间接ELISA中的应用。


鸡贫血病毒（CAV）会导致鸡严重贫血和免疫抑制。 VP1是主要的衣壳蛋白，适合用于诊断试剂盒的开发，但是，它在该蛋白的前40个N末端氨基酸中具有24个精氨酸残基，对细菌具有毒性，从而导致原核表达减少。在本研究中，去除毒性区域的60个氨基酸N端截短的VP1（Δ60VP1）在大肠杆菌中表达，所得不溶性重组蛋白通过Ni-NTA亲和层析与阴离子变性去污剂纯化。产生的大量纯化 Δ60VP1 (150 mg/L) 保留了适当的抗原性，并且该抗原用于开发用于 CAV 血清学诊断的间接酶联免疫吸附测定 (ELISA)。使用新开发的 Δ60VP1 间接 ELISA（截止值 = 7.58 % S/P）对 152 个鸡血清样品（n = 152）进行了评估。 Δ60VP1 间接 ELISA 的灵敏度和特异性分别为 87.50% 和 95.31%，而 Δ60VP1 间接 ELISA 与商业 IDEXX CAV ELISA 之间的一致性为 90.79%（kappa = 0.814）。在本研究中，我们在大肠杆菌中开发了另一种 VP1 生产平台，通过截短 N 端 60 个氨基酸 (Δ60VP1) 并在纯化过程中使用阴离子变性去污剂，成功溶解不溶性的 Δ60VP1。纯化的抗原产量高、免疫反应性好，并建立了间接ELISA方法。该测定法有可能应用于大规模 CAV 血清监测。