Han Jun, Han Lu, Shi Qi, Shi Song, Wang Xin, Zhang Bao-Yun, Dong Xiao-Ping
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2007 Sep;21(3):202-4.
To establish a methodology of protein misfolding cyclic amplification (PMCA) and utilize in the detection of PrP(Sc) in brain tissues from prion diseases.
Different amounts of Scrapie 263K agent bulk were mixed with brain homogenates of health hamsters and treated with repeated incubation/sonication for 10 to 15 cycles. The proteinase K-resistant PrP(Sc) was evaluated with Western Blot.
In this experimental situation, 263K agent replicated rapidly in vitro, utilizing hamsters' brains as the medium. With the established PrP(Sc)-PMCA technique, PrP(Sc) signals in the preparations containing less than 10(-5) diluted 263K bulk could be detected. Compared with conveniently used immuno-blot assay, the sensitivity of PrP(Sc)-PMCA for PrP(Sc) was 10(5) to 10(6)-fold increased. It has been also shown that homogenates of cerebellar and brain stem could be used as the medium for PrP(Sc) replication.
A rapidly replicating method for PrP(Sc), PrP(Sc)-PMCA, was successfully established, providing a new approach for early diagnosis of prion diseases and research on the biological features of prion.
建立蛋白质错误折叠循环扩增（PMCA）方法，并用于检测朊病毒病脑组织中的PrP(Sc)。
将不同量的羊瘙痒病263K毒株原液与健康仓鼠脑匀浆混合，经反复孵育/超声处理10至15个循环。用蛋白质印迹法评估蛋白酶K抗性PrP(Sc)。
在本实验条件下，263K毒株能利用仓鼠脑作为介质在体外快速复制。利用建立的PrP(Sc)-PMCA技术，可检测到含有稀释度低于10(-5)的263K原液制剂中的PrP(Sc)信号。与常用的免疫印迹法相比，PrP(Sc)-PMCA对PrP(Sc)的敏感性提高了10(5)至10(6)倍。还表明小脑和脑干匀浆可作为PrP(Sc)复制的介质。
成功建立了一种PrP(Sc)快速复制方法PrP(Sc)-PMCA，为朊病毒病的早期诊断及朊病毒生物学特性研究提供了新途径。
