蓖麻矮化相关RcDof蛋白的原核表达及纯化  

Prokaryotic expression and purification of RcDof protein associated with castor dwarfing

作　　者：张帅 王双[2] 李跃[2,3] 李国瑞 黄凤兰[2,4] 陈永胜[1,2,3,4] ZHANG Shuai;WANG Shuang;LI Yue;LI Guo-rui;HUANG Feng-lan;CHEN Yong-sheng(不详;Agricultural College,Inner Mongolia University for Nationalities,Tongliao,028000,Inner Mongolia Autonomous Region,China)

机构地区：[1]内蒙古民族大学农学院,内蒙古通辽028000 [2]内蒙古民族大学生命科学与食品学院,内蒙古通辽028000 [3]沈阳农业大学食品学院,辽宁沈阳110866 [4]内蒙古民族大学内蒙古自治区高校蓖麻产业工程技术研究中心内蒙古自治区蓖麻育种重点实验室内蒙古自治区蓖麻产业协同创新培育中心,内蒙古通辽028000

出　　处：《中国生物制品学杂志》2022年第4期418-423,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金项目(31460353);内蒙古民族大学科研项目(MDK2019010,MDK2019020)。

摘　　要：目的原核表达并纯化蓖麻矮化相关RcDof蛋白。方法利用SignalP 4.0 server和Iprscan软件分别预测RcDof基因的信号肽和结构域。从pMD-18T-RcDof质粒中PCR扩增RcDof(44-101)基因,克隆入原核表达载体pETMBP-XE,构建原核表达质粒pETMBP-XE-RcDof(44-101),IPTG诱导表达重组蛋白,利用亲和层析、阳离子交换层析以及分子筛层析对目的蛋白进行纯化。结果RcDof基因无信号肽剪切序列,在44~101个碱基之间存在1个锌指保守结构域。重组表达质粒经双酶切及测序鉴定证明构建正确。表达的重组蛋白相对分子质量约43000,以可溶形式存在于上清中。纯化的重组蛋白纯度较高,浓度为10 mg/mL。结论获得了高纯度可溶RcDof蛋白,为解析其晶体结构及探索其在蓖麻中作用的分子机制提供了实验依据。Objective To express RcDof protein associated with castor dwarfing in prokaryotic cells and purify the expressed product.Methods The signal peptide and structural domain of RcDof gene were predicted by using SignalP 4.0server and Iprscan software respectively.RcDof(44-101)gene was amplified from plasmid pMD-18T-RcDof by PCR and cloned into prokaryotic expression vector pETMBP-XE.The constructed recombinant plasmid pETMBP-XE-RcDof(44-101)were induced with IPTG,and the expressed recombinant protein was purified by affinity,cation exchange and molecular sieve chromatography.Results There was no signal peptide cleavage sequence in RcDof gene,while a zinc finger conserved domain between bases 44 and 101.Both restriction analysis and sequencing proved that recombinant plasmid pETMBP-XE-RcDof(44-101)was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 43000,existed in supernatant in a soluble form.The purified recombinant protein reached a high purity,of which the concentration was 10 mg/mL.Conclusion Soluble RcDof with high purity was obtained,which provided an experimental basis for analyzing the crystal structure of RcDof protein and exploring the molecular mechanism of the protein in castor.

关 键 词：蓖麻 RcDof基因 蛋白纯化 

分 类 号：Q816[生物学—生物工程]