人自噬相关蛋白ATG7的原核表达及纯化鉴定  

Prokaryotic Expression and Purification of Human ATG7 with HisTag and the Characterization of the Fusion Protein

作　　者：晋帅 洪甜[2] 张珂[3] 马永富[1] 徐小洁[2] 禇健[1,2] 易绍琼[1] 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 [3]军事医学科学院科技部,北京100850

出　　处：《生物技术通讯》2016年第1期69-72,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81472589,31100604);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);北京市自然科学基金(7132155)

摘　　要：目的:原核表达并纯化自噬相关蛋白ATG7,初步鉴定其生物学活性。方法:利用PCR技术从人乳腺文库中扩增出人ATG7基因的编码序列,插入载体p ET-28a(+)得到重组质粒,经Bam HⅠ和NotⅠ双酶切鉴定后转化大肠杆菌Rossate菌株进行小量诱导,纯化融合蛋白His-ATG7,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2031 bp的目的片段,插入载体p ET-28a(+)后构建出His-ATG7重组质粒,并经酶切鉴定及测序证实无误;转化大肠杆菌Rossate并进行小量诱导,纯化后SDS-PAGE检测显示获得相对分子质量约为78×103的融合蛋白。结论:纯化得到原核系统表达的His-ATG7融合蛋白,为后续研究ATG7在自噬中的作用机制奠定了实验基础。Objective： To purify prokaryotic expressed autophagy related protein 7（ATG7）. Methods： ATG7 cod- ing region was amplified from human mammary gland cDNA library by PCR, and was cloned into the prokaryotic expression vector pET-28a（＋）. The correct recombinant plasmid was introduced into E.coli Rossate. The expressed recombinant protein was purified by Ni-NTA beads and identified by SDS-PAGE and Western blot analysis. Re- sults： The DNA fragment of about 2031 bp was successfully amplified from human mammary gland cDNA library by PCR, and inserted into pET-28a（＋） vector correctly. The results of double digestion and sequencing suggested that the His-ATG7 recombinant plasmid was obtained. His-ATG7 fusion protein of about Mr 78×10^3 was induced and identified by SDS-PAGE analysis. Conclusion： The prokaryotic expression protein of His-ATG7 was obtained successfully, which lays the foundation for further research on the ruction of ATG7 in autophagy.

关 键 词：人自噬相关蛋白ATG7 原核表达 纯化 自噬 

分 类 号：Q78[生物学—分子生物学]