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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：1331 下载量：191
| 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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