人自噬相关蛋白ATG5的原核表达及纯化  

Prokaryotic Expression and Purification of Human ATG5 with His-Tag

作　　者：晋帅 洪甜[2] 闫志风[1] 马永富[1] 禇健[1,2] 易绍琼[1] 徐小洁[2] 叶棋浓[2] 刘阳[1] 

机构地区：[1]解放军总医院,北京100853 [2]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2016年第2期196-199,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81573026;81502264;81472589;31100604);北京市自然科学基金(7132155);北京市科技新星计划(Z141102001814055);军事医学科学院创新基金转化医学项目(ZHYX003)

摘　　要：目的:原核表达纯化带His标签的自噬相关蛋白ATG5。方法:利用PCR技术从人乳腺文库中扩增出人ATG5基因的编码序列,插入载体p ET-28a(+)中得到重组质粒,经Bam HⅠ和XhoⅠ双酶切鉴定后转化大肠杆菌Ros-sate进行小量诱导,挑选出可以诱导His-ATG5蛋白的菌液进行融合蛋白的纯化,通过Western印迹和SDS-PAGE检测融合蛋白的纯化效果。结果:用PCR技术从人乳腺文库中扩增得到约828 bp的目的片段,插入载体p ET-28a(+)构建出His-ATG5重组质粒并经酶切及测序验证;转化大肠杆菌Rossate后进行小量诱导表达并纯化蛋白,SDS-PAGE检测显示获得相对分子质量约为38×103的融合蛋白。结论:原核表达并纯化获得His-ATG5融合蛋白,为后续研究ATG5在自噬中的作用机制奠定了实验基础。Objective： To prokaryotic express human autophagy related protein 5（ATG5） with His-tag and puri-fy the fusion protein. Methods： ATG5 coding region was amplified from human mammary gland c DNA library byPCR, and was cloned into the prokaryotic expression vector p ET-28a（＋）. The correct recombinant plasmid His-ATG5 was introduced into E.coli Rossate. The expressed recombinant protein was purified by Ni-NTA beads andidentified by SDS-PAGE and Western blot analysis. Results： The DNA fragment of about 828 bp was successful-ly amplified from human mammary gland c DNA library by PCR, and inserted into p ET-28a（＋） vector correctly.The results of double digestion and sequencing suggested that the His-ATG5 recombinant plasmid was obtained.His-ATG5 fusion protein of about Mr 38×10^3was induced and identified by SDS-PAGE analysis. Conclusion：The prokaryotic expression protein of His-ATG5 is obtained successfully, which lays the foundation for further re-search on the fuction of ATG5 in autophagy.

关 键 词：人自噬相关蛋白ATG5 原核表达 纯化 自噬 

分 类 号：Q78[生物学—分子生物学]