Andrievskaia Olga, Algire James, Balachandran Aru, Nielsen Klaus
Canadian Food Inspection Agency, Ottawa Laboratory Fallowfield, 3851 Fallowfield Road, Ottawa, Ontario K2H 8P9, Canada.
J Vet Diagn Invest. 2008 Mar;20(2):141-6. doi: 10.1177/104063870802000201.
The misfolded form of cellular prion protein (PrP(C)) is the main component of the infectious agent of transmissible spongiform encephalopathies and the validated biomarker for these diseases. The expression of PrP(C) is highest in the central nervous system and has been found in peripheral tissues. Soluble PrP(C) has been detected in cerebrospinal fluid, urine, serum, milk, and seminal plasma. In this study, attempts were made to characterize prion protein in urine samples from normal and scrapie-infected sheep. Urine samples from scrapie-infected sheep and age-matched healthy sheep were collected and analyzed by Western blot following concentration. A protease K-sensitive protein band with a molecular weight of approximately 27-30 kDa was visualized after immunoblotting with anti-PrP monoclonal antibodies to a C-terminal part of PrP(C), but not after immunoblotting with monoclonal antibodies to an N-terminal epitope of PrP(C) or with secondary antibodies only. The amount of PrP(C) in the urine of 49 animals (control group: n = 16; naturally scrapie-infected group: n = 33) was estimated by comparison with known amounts of ovine recombinant PrP in the immunoblot. Background concentration of PrP(C) in urine was found to be 0-0.16 ng/ml (adjusted to the initial nonconcentrated volume of the urine samples). Seven out of 33 naturally scrapie-infected animals had an elevated level (0.3-4.7 ng/ml) of PrP(C) in urine. The origin of PrP(C) in urine and the reason for the increased level of PrP(C) in scrapie-infected sheep urine has yet to be explored.
细胞朊蛋白（PrP(C)）的错误折叠形式是传染性海绵状脑病致病因子的主要成分，也是这些疾病经过验证的生物标志物。PrP(C)在中枢神经系统中的表达最高，并且已在外周组织中发现。在脑脊液、尿液、血清、乳汁和精浆中均检测到可溶性PrP(C)。在本研究中，我们试图对正常绵羊和感染羊瘙痒病的绵羊尿液样本中的朊蛋白进行表征。收集感染羊瘙痒病的绵羊和年龄匹配的健康绵羊的尿液样本，浓缩后通过蛋白质免疫印迹法进行分析。用针对PrP(C) C端部分的抗PrP单克隆抗体进行免疫印迹后，可见一条分子量约为27 - 30 kDa的蛋白酶K敏感蛋白条带，但用针对PrP(C) N端表位的单克隆抗体或仅用二抗进行免疫印迹后则未出现该条带。通过与免疫印迹中已知量的绵羊重组PrP进行比较，估算了49只动物（对照组：n = 16；自然感染羊瘙痒病组：n = 33）尿液中PrP(C)的含量。发现尿液中PrP(C)的背景浓度为0 - 0.16 ng/ml（根据尿液样本初始未浓缩体积进行调整）。33只自然感染羊瘙痒病的动物中有7只尿液中PrP(C)水平升高（0.3 - 4.7 ng/ml）。尿液中PrP(C)的来源以及感染羊瘙痒病的绵羊尿液中PrP(C)水平升高的原因尚待探索。
