人内源性博尔纳样核蛋白-1的原核表达及纯化  

Prokaryotic expression and purification of human endogenous Borna-like N element-1

作　　者：吴迪[1] 金戈[1] 刘钊[1] 张亮[1,2] 黄荣忠[1,2] 杨德雨[1] 谢鹏[1,2] 

机构地区：[1]重庆医科大学神经科学研究中心重庆市神经生物学重点实验室,重庆400016 [2]重庆医科大学附属第一医院神经内科,重庆400016

出　　处：《中国生物制品学杂志》2014年第2期172-176,共5页Chinese Journal of Biologicals

基　　金：国家重点基础研究发展计划(973计划;2009CB918300)

摘　　要：目的原核表达人内源性博尔纳样核蛋白-1(endogenous Borna-like N element-1,EBLN-1),并进行纯化及鉴定。方法以人工合成的EBLN-1基因为模板,PCR扩增EBLN-1基因,克隆至载体pET-41a中,构建重组表达质粒pET-41aEBLN-1,转化大肠埃希菌BL21(DE3)和Rosetta中,分别于18℃和37℃下进行IPTG诱导表达。采用包涵体稀释复性法对表达蛋白进行复性,经His亲和层析纯化后,SDS-GAGE和Western blot法鉴定纯化产物。结果重组表达质粒pET-41a-EBLN-1经菌落PCR及测序鉴定构建正确。重组表达蛋白相对分子质量约48 000,以包涵体形式表达。在大肠埃希菌Rosetta中37℃诱导时表达量最高,占菌体总蛋白的30%以上,纯化后蛋白纯度可达90%,可与HRP标记的His探针特异性结合。结论成功表达并纯化了重组EBLN-1蛋白,为后续深入研究EBLN-1在人体中的生理作用及其与精神疾病的相关性奠定了基础。Objective To express human endogenous Borna-like N element-1（EBLN-1）in prokaryotic cells,and purify and identify the expressed product. Methods EBLN-1 gene was amplified by PCR using synthetic EBLN-1 gene as a template and inserted into vector pET-41a. The constructed recombinant plasmid pET-41a-EBLN-1 was transformed to E.coli BL21（DE3）and Rosetta,and induced with IPTG at 18 and 37 ℃,respectively. The expressed protein was refolded by dilution of inclusion body,purified by His affinity chromatography,and identified by SDS-PAGE and Western blot. Results Colony PCR and sequencing proved that recombinant plasmid pET-41a-EBLN-1 was constructed correctly. Recombinant EBLN-1 protein,with a relative molecular mass of about 48 000,was expressed in a form of inclusion body,of which the expression level in E.coli Rosetta reached a maximum of 30% of total somatic protein after induction at 37 ℃. The purified protein reached a purity of 90% and showed specific binding to HRP-labeled His probe. Conclusion Recombinant EBLN-1 protein was successfully expressed and purified,which laid a foundation of further study on physiological role of EBLN-1 in humans as well as its relationship to mental diseases.

关 键 词：人内源性博尔纳样核蛋白-1 原核细胞 基因表达 纯化 

分 类 号：R373.9[医药卫生—病原生物学] Q786[医药卫生—基础医学]