人ATG4B的表达及蛋白纯化和鉴定  

The expression,purification and characterization of human His-ATG4B protein

作　　者：黄蓉[1,2] 徐小洁[1] 梁迎春[1] 张立 王涛[1] 周丽英[1] 杜楠[2] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]解放军总医院第一附属医院,北京100048 [3]总参警卫局卫生保健处,北京100017

出　　处：《细胞与分子免疫学杂志》2015年第2期173-176,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31100604);院创新基金(ZHYX003)

摘　　要：目的构建含His标签的自噬相关蛋白4同源物B(ATG4B)重组质粒以得到His-ATG4B蛋白,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ATG4B基因的编码序列,插入p ET-28a(+)载体中得到重组质粒,经酶切鉴定后转化Rossate菌,挑选小量诱导出的His-ATG4B菌液进行His-ATG4B蛋白的纯化,SDS-PAGE和Western blot法检测His-ATG4B蛋白的纯化效果,GST pull-down技术对蛋白生物学功能进行鉴定。结果用PCR技术从人乳腺文库中扩增得到大小约1100 bp的目的片段,插入p ET-28a(+)载体中构建出His-ATG4B重组质粒,酶切鉴定及测序结果均表明His-ATG4B质粒构建成功;转化Rossate菌并小量诱导,表达鉴定成功后纯化蛋白,得到相对分子质量(Mr)约为47 000的His-ATG4B蛋白,且SDS-PAGE检测蛋白纯化效果较好;GST pull-down实验证实His-ATG4B蛋白可以和LC3B蛋白在体外相互作用,生物学活性良好。结论本实验成功得到His-ATG4B蛋白,为研究ATG4B在自噬中的作用机制奠定实验基础。Objective To construct the prokaryotic expression vector of human autophagy-related protein 4 homolog B （ATG4B） labeled with His-tag, obtain the purified His-ATG4B protein, and identify its activity preliminarily. Methods ATG4B coding region was amplified from human mammary gland cDNA library by PCR, and was cloned into the prokaryotic expression vector pET-28a（＋）. After verification by enzyme digestion, the correct recombinant plasmid His-ATG4B was introduced into E. coli Rossate. The expressed recombinant plasmid was purified by Ni-NTA beads and identified by SDS-PAGE and Western blot analysis. The function of the purified protein His-ATG4B was detected by GST pull-down assay. Results The DNA fragment of about 1100 bp was successfully amplified from human mammary gland cDNA by PCR, and inserted into pET-28a vector correctly. The results of double digestion and sequencing suggested that the His-ATG4B recombinant plasmid was successfully obtained. His-ATG4B protein of about Mr 47 000 was induced and identified by SDS-PAGE analysis. GST pull-down assay showed that His-ATG4B could interact with LC3B in vitro, suggesting that it has a good biological function. Conclusion The prokaryotic expression protein of His-ATG4B has been obtained successfully, which lays a foundation for further research on the function of ATG4B in autophagy.

关 键 词：自噬相关蛋白4同源物B 原核表达 纯化 自噬 

分 类 号：R392.12[医药卫生—免疫学] Q786[医药卫生—基础医学]