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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27kDHaPrP蛋白,Westernblot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scrapie263K毒株仓鼠感染脑组织PrPSc蛋白共同孵育4天后,经蛋白酶K消化,Westernblot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HaPrPsen可以被转化为HaPrPres。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。
PrP extracellular conformational transformation system has been used in some laboratories for prion research, but often requires the use of radioactive isotopes in the reaction system. In order to establish a safe and convenient PrP cell-free conformational transformation system for studying the pathogenesis of TSE, the full-length hamster PrP (HaPrP) gene was obtained by PCR and cloned into the prokaryotic expression vector pQE30. The full-length HaPrP gene was expressed in E.coli and purified As a 27kDHaPrP protein, Westernblot proved to react with PrP-specific monoclonal antibodies. The purified protein was incubated with Biotin-7-NHS in vitro and incubated with PrPSc protein of hamster infected with purified scrapie263K for 4 days. After protease K digestion and Western blotting, it was proved that there was a protease K digestion, A band specifically recognized by avidin has a molecular weight of about 20 kD. This shows that HaPrPsen can be converted to HaPrPres. Experiments show that you can use the prokaryotic system instead of eukaryotic expression PrP, and biotin-labeled 35S labeled purified protein, to establish a more safe and convenient cell-free conformational transformation system for prion research.