一种新的PrP无细胞构象转化系统的建立

PrP细胞外构象转化系统已经被一些实验室用于朊病毒的研究，但在反应体系中往往需要使用放射性同位素。为了建立一种安全方便的PrP无细胞构象转化系统用于TSE发病机制的研究，通过PCR方法获得仓鼠PrP(HaPrP)全长基因，克隆至原核表达载体pQE30，在大肠杆菌中表达并纯化出分子量为27kD HaPrP蛋白，Western blot证实可与PrP特异性单克隆抗体反应。将纯化蛋白在体外标记生物素(Biotin-7-NHs)，与纯化的scrapie 263K毒株仓鼠感染脑组织PrP^Se蛋白共同孵育4天后，经蛋白酶K消化，Western blot检测，证明存在一条抵抗蛋白酶K消化的、被亲和素特异性识别的条带，其分子量约为20kD。这表明HaPrP^sen可以被转化为HaPrP^res。实验表明，可以利用原核系统取代真核系统表达PrP，并用生物素标记取代^35S标记纯化蛋白，建立一个更加安全方便的无细胞构象转化系统，用于朊病毒的研究。

Establishment of a New Cell Free Conversion System for Prion

Cell-free conversion technique has been used in several laboratories for prion researches, in which radioactivity was commonly used. In order to establish a reliable, easily handling, environmental protecting cell free conversion model for prion and TSE studies, full-length sequence of hamster prp gene was obtained by PCR and the gene was cloned into vector pQE30. A 27kD protein, HaPrP, was expressed in the transformed E. coli and purified with His-tag affinity chromatograph. Western blot revealed that the expressed protein could be recognized by PrP specific monoclonal antibody 3F4. After labeled with biotin (Biotin-7-NHS), the recombinant protein HaPrP was mixed with PrP~(Sc) extracted from the hamsters' brains infected with scrapie strain 263K. A protease resistant band was obviously detected in the preparations of PrP~(Sc) after four-day incubation by using HRP-conjugated streptavidin, illustrating that the biotin-labeled HaPrP~(sen) can be converted into HaPrP~(res) isoform after incubation with PrP~(Sc) in vitro. Our study supplies a new system, using more efficiently prokaryotic expressed PrP instead of that expressed in mammalian or insect cells and more easily handling and environmental protecting biotin instead of isotope in cell free conversion for prion study.