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小鼠重组朊蛋白核心片段PrP23-231的原核表达

发表时间:2018-05-15  浏览量:277  下载量:52
全部作者: 白瑜,何元晨,孙茜
作者单位: 陕西理工大学维生素D生理与应用研究所;陕西理工大学生物科学与工程学院
摘 要: 目的:获取高表达的重组朊蛋白核心片段PrP23-231,研究朊蛋白错误折叠和聚集机理。方法:从C57BL/C小鼠基因组DNA中PCR扩增出PrP23-231基因片段,将该纯化的基因克隆入原核表达载体pET-30a中,构建原核重组表达质粒pET-30a-PrP,并转入Escherichia coli BL21表达菌中,以异丙基硫代半乳糖苷(isopropyl thiogalactoside, IPTG)诱导表达,使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-polyacrylamide gel electrophoresis,SDS-PAGE)和Western blotting对重组蛋白进行鉴定。结果:表达得到的该重组蛋白鉴定为目的朊蛋白PrP23-231,其具有较好的免疫反应特性。结论:利用原核表达方式可以获得高表达的朊蛋白核心片段PrP23-231,为进一步研究朊蛋白错误折叠和聚集机理提供了可应用的材料。
关 键 词: 分子生物学;C57BL/C小鼠;朊蛋白PrP23-231;基因克隆;原核表达
Title: Prokaryotic expression of mouse recombinant prion protein core fragment PrP23-231
Author: BAI YU, HE Yuanchen, SUN Qian
Organization: Vitamin D Research Institute, Shaanxi University of Technology; School of Biological Science and Engineering, Shaanxi University of Technology
Abstract: Objective: To obtain highly expressed mature murine recombinant prion protein PrP23-231 and to understand the mechanism of prion protein conformational misfolding and amyloid fibrillization. Methods: Target gene fragment of PrP23-231 was obtained by PCR amplification from DNA genome of healthy C57BL/C mouse, then the purified target gene was cloned into a prokaryotic expression vector pET-30a. Thus, recombinant pET-30a-PrP plasmid was constructed. The positive recombinant pET-30a-PrP plasmids were transformed into Escherichia coli BL21, and isopropyl thiogalactoside (IPTG) was used to induce the expression of recombinant prion protein PrP23-231. Finally, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were used to identify the recombinant prion protein PrP23-231. Results: It showed that the obtained recombinant protein was exactly prion protein PrP23-231 with good immune characteristics. Conclusion: It indicated that the highly expressed recombinant prion protein PrP23-231 could be obtained by prokaryotic expression system, and be provided as material for prion protein conformational misfolding and amyloid fibrillization.
Key words: molecular biology; C57BL/C mouse; prion protein PrP23-231; gene clone; prokaryotic expression
发表期数: 2018年5月第9期
引用格式: 白瑜,何元晨,孙茜. 小鼠重组朊蛋白核心片段PrP23-231的原核表达[J]. 中国科技论文在线精品论文,2018,11(9):871-874.
 
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