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蛋白质错误折叠循环扩增(protein misfolding cyclic amplification,PMCA)技术,是一种具有感染性的朊蛋白(PrPsc)体外扩增的技术,可用于抑制细胞型朊蛋白(PrPc)向PrPsc转化药物的筛选.本研究在Saborio等提供的方法基础上,成功优化了最佳扩增时间,利用优化的PMCA技术实验了不同剂量氮芥(mechlorethamine,MCT)联合二硫苏糖醇(dithiothreitol,DTT)对PrPsc转化的抑制效应.结果发现,MCT联合DTT能体外抑制PrPc向PrPsc的转化,抑制作用具有明显的量效关系.利用针对人PrPc不同结构域的抗体发现,MCT联用DTT可使抗体6H4的抗原表位隐蔽,该表位位于PrPc第1个α螺旋区内,是构象转化的主要部位.对其机制的深入探讨,将有助于新一类可传播性海绵状脑病(transmissible spongiform encephalopathy,TSE)治疗药物的研发.
Protein misfolding cyclic amplification (PMCA) is a technique for in vitro amplification of infectious prion (PrPsc), which can be used to inhibit the screening of PrPc to PrPsc-transformed drugs Based on the method provided by Saborio et al, we successfully optimized the optimal amplification time and optimized the PMCA technique to test the effects of mechlorethamine (MCT) and dithiothreitol (DTT) on PrPsc The results showed that MCT combined with DTT could inhibit the transformation of PrPc to PrPsc in vitro and had a significant dose-effect relationship.Using anti-human PrPc domain antibodies, it was found that MCT combined with DTT could make anti-6H4 antigen The epitope is located in the first α-helical region of PrPc, which is the main part of conformational transformation. The further study on its mechanism will contribute to a new class of transmissible spongiform encephalopathy (TSE Development of therapeutic drugs.