无标签人源性脂联素球状结构的表达、纯化与活性检测  

Prokaryotic Expression and Purification of No-Tagged Human Globu lar Domain of Adiponectin and Identification of its Biological Activity

作　　者：张立剑[1] 孙璐[1] 李冬冬[1] 郭云萍[1] 王增禄[2] 刘毅[1] 张英起[2] 陶凌[1] 

机构地区：[1]第四军医大学西京医院,陕西西安710032 [2]第四军医大学药学系生物技术中心,陕西西安710032

出　　处：《生物技术通讯》2013年第2期169-172,共4页Letters in Biotechnology

基　　金：国家重大新药创制(2009ZX09103-673);国家自然科学基金(30670879)

摘　　要：目的:利用基因工程方法构建无标签人源性脂联素球状结构(gAd)基因的原核表达载体,并对重组蛋白进行诱导表达、纯化及鉴定。方法:从正常人脂肪组织里提取总RNA,反转录合成cDNA,经PCR扩增、酶切后连入pET-22b(+)载体构建重组质粒pET-22b(+)-gAd,转化大肠杆菌BL21(DE3)感受态细胞,经低温、低浓度IPTG诱导使其可溶性表达,采用硫酸铵沉淀、凝胶过滤层析和阴离子交换层析三步分离纯化,得到不带任何标签的人源性gAd;运用SDS-PAGE、Western印迹、HPLC对重组蛋白进行鉴定,通过对AMP激活的蛋白激酶(AMPK)的磷酸化水平检测纯化蛋白的生物学活性。结果:构建了原核表达载体pET-22b(+)-gAd,实现了人源性gAd在原核细胞中的可溶性表达,纯化的蛋白经SDS-PAGE和Western印迹分析证实为gAd,HPLC分析蛋白纯度达到95%以上;通过对AMPK磷酸化水平的检测,证明纯化的gAd具有高生物学活性。结论:重组表达和纯化了无标签、高生物学活性的人源性脂联素球状结构,为其进一步的理论研究、生产开发奠定了基础。Objective： To construct prokaryotic expression vector pET-22b（＋）-gAd of human globular domain of adiponeetin（gAd） gene, express and purify no-tagged recombinant human gad protein. Methods： Total RNA was extracted from human adipose tissue. The gad coding sequence was subcloned into the pET-22b（＋） after ampli- fied by PCR. The recombinant plasmids were transformed into E.coli BL21（DE3）, and the gad was soluble ex- pressed in suitable temperature, by IPTG induction. The expressed gAd was purified by three-step procedure： am- monium sulfate precipitation, gel filtration chromatography and anion exchange chromatography. SDS-PAGE, West- ern blot, HPLC and the ability to induce the phosphorylation of AMPK in HUVEC were used for identification, pu- rity and biological activity assay. Results： The human gad coding sequence was cloned into pET-22b（＋） vector. After expression and purification, the purity of gAd was more than 95%. The recombinant human gad significantly induced the phosphorylation of AMPK in HUVEC. Conclusion： The no-tagged recombinant human gad was suc- cessfully expressed and purified by prokaryotic expression system and three-step purification procedure with high purity and biological activity.

关 键 词：重组人脂联素球状结构 表达纯化 活性检测 

分 类 号：Q78[生物学—分子生物学]