一种朊蛋白错误折叠循环扩增方法的建立

目的建立一种类似于PCR的蛋白质扩增方法-蛋白错误折叠循环扩增技术(PMCA),用于朊病毒病脑组织中PrPSc的检测。方法将不同浓度的羊瘙痒因子263K毒株原液与正常仓鼠脑组织匀浆混合,经反复孵育/超声,共10~15个循环。WesternBlot检测扩增产物中蛋白酶K抗性PrPSc信号。结果在本研究试验体系下,263K毒株可以利用仓鼠脑组织为基质在体外迅速复制。所建立的PrPSc-PMCA技术可检测到10-5稀释的毒株原液中的PrPSc。与常规的脑组织免疫印记方法相比,敏感度提高了105~106倍。研究还显示PrPSc还可利用小脑和脑干为基质进行体外扩增复制。结论成功建立了PrPSc-PMCA技术,为朊病毒病的早期诊断和朊病毒生物学特性的研究提供了一种新的手段。

Establishment of a protein misfolding cyclic amplification for PrPSc

Objective To establish a methodology of protein misfolding cyclic amplification (PMCA) and utilize in the detection of PrP Sc in brain tissues from prion diseases. Methods 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. Results 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 105 to 106-fold increased. It has been also shown that homogenates of cerebuller and brain stem could be used as the medium for PrP Sc replication. Conclusion 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.