杜仲Dirigent1基因的原核表达及蛋白纯化  

Prokaryotic Expression and Protein Purification of Eucommia ulmoides Dirigent1 Gene

作　　者：邹情 赵懿琛[1] 赵德刚 Zou Qing;Zhao Yichen;Zhao Degang(The 2011 Collaboration Innovation Center for Mountain Ecology and Agro-Bioengineering in Guizhou Province,The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education),Guizhou Key Lab of Agro-Bioengineering,Institute of Ago-Bioengineering and School of Pharmaceutical Sciences,Guizhou University,Guiyang,550025)

机构地区：[1]贵州大学药学院农业生物工程研究院贵州省农业生物工程重点实验室山地植物资源保护与种质创新省部共建教育部重点实验室贵州省山地生态与农业生物工程2011协同创新中心,贵阳550025

出　　处：《分子植物育种》2019年第1期86-91,共6页Molecular Plant Breeding

基　　金：国家863计划子项目(2013AA102605);国家自然科学基金(31660076)共同资助

摘　　要：植物Dirigent蛋白认为指导木脂素和木质素的生物合成,同时在生物防御应答,次生代谢调控和病原体抗性的过程中起到了重要的作用。本研究以实验室前期克隆得到杜仲Dirigent1基因(EuDIR1)为基础,利用基因重组技术构建pET-30a-EuDIR1原核表达载体。重组载体转化大肠杆菌BL21 (DE3)中,在15℃下0.1 mmol/L异丙基硫代β-D-半乳糖苷诱导6 h后,蛋白的沉积量达到最高值,表达模式主要为包涵体。对包涵体进行溶解,使用镍-亚氨基二乙酸亲和层析柱进行纯化。收集纯度较高的洗脱组分进行透析复性,并用15%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Western Blot鉴定重组蛋白。结果表明,成功构建了pET-30a-EuDIR1原核表达载体,表达菌株诱导后在相对分子质量约18 kD处有1条特异性蛋白条带,包涵体纯化、复性后,经Western Blot鉴定,获得可溶性且纯度较高的重组蛋白。实验中得到的EuDIR1蛋白为体外生化实验提供了科学依据。The plant dirigent(DIR) protein are proposed to as an improve role in guiding the biosynthesis of lignan and lignin, as well as in the process of biological defense responses, regulation of secondary metabolism, and pathogen resistance. This study was based on the previous experiments about cloning EuDIR1 gene in laboratory and then the prokaryotic expressing vector of pET-30 a-EuDIR1 was constructed by the gene recombination technique. During the transformation of Escherichia coli BL21(DE3) by recombinant vector, the protein deposition reached the highest level after 6 h induction with 0.1 mmol/L isopropyl β-D-thiogalactopyranoside(IPTG) at 15℃,and the expression pattern was mainly inclusion body. After dissolution, the inclusion bodies were purified by Ni-IDA(Nickel-iminodiacetic acid) affinity chromatography columu. The eluted fractions of high purity were collected for dialysis renaturation, and the recombinant protein was identified by 15% SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western Blot. The results showed that we successfully constructed the pET-30 a-EuDIR1 prokaryotic expressing vector and there was a specific protein band in the relative molecular weight of about 18 kD after the expression strain was inducted. The inclusion bodies were putificated and renaturated, then verified by Western Blot. We obtained the highly pure and soluble recombinant proteins. The EuDIR1 protein obtained in the experiment would provide a scientific basis for biochemical experiments in vitro.

关 键 词：EuDIR1 杜仲 原核表达 蛋白纯化 

分 类 号：S567.19[农业科学—中草药栽培]