人表皮生长因子的原核表达及纯化  

Prokaryotic expression and purification of human epidermal growth factor

作　　者：史飞[1,2] 张国利[2] 李泽鸿[1] 张培培[2] 何苗[1] 赵鑫[1] 付玉和 于亭[1] 

机构地区：[1]吉林农业大学生命科学学院,长春130118 [2]军事医学科学院军事兽医研究所,长春130062

出　　处：《黑龙江畜牧兽医》2015年第12期48-51,共4页Heilongjiang Animal Science And veterinary Medicine

摘　　要：为了利用特殊表达载体在大肠埃希氏菌中表达融合表皮生长因子(EGF)并进行纯化工艺研究,试验采用PCR扩增EGF,克隆至p ET-SUMO-T载体中,构建重组表达质粒p ET-SUMO-TEGF,转化大肠埃希氏菌(E.coil)BL21(DE3),IPTG诱导表达,表达的蛋白经SDS-PAGE分析,利用Celating SepharoseTMFast Flow金属螯合层析介质、Q Sepharose High performance阴离子交换层析介质以及Superdex 200 prep grade分子筛对表达蛋白进行纯化,期间采用SephadexTMG-25 Fine介质进行脱盐、SUMO蛋白酶切割His.tag标签。结果表明:重组质粒经PCR鉴定及测序证明构建正确;表达的EGF蛋白分子质量为6 ku,主要以可溶形式表达,表达量占菌体总蛋白的45%以上;纯化的EGF蛋白纯度达到95%。说明成功在大肠埃希氏菌中表达并经过简易工艺纯化了重组EGF。To utilize a special expression vector to express fused epidermal growth factor （EGF） in Escherichia toll（ E. coil） and conduct the study of purification process, EGF was amplified sing PCR technology, and then was cloned into a pET - SUMO - T vector to construct pET - SUMO -T -EGF recombinant expression plasmid. The vector was transformed into E. coil BL21 （DE3） and induced by IPTG. The expressed protein was analyzed by SDS - PAGE, and then the target protein was purified by Celating SepharoseTM Fast Flow with metal chelate chromatog- raphy medium, Q Sepharose High performance with anion exchange chromatography medium and Superdex 200 prep grade with a molecular sieve. During this process the SephadexTM G -25 Fihe medium was used for desalination, and SUMO protease was also used for cutting His. tag. The results showed that the recombinant plasmid was constructed correctly, which was proven by PCR identification and sequencing. The expressed EGF protein had a relative molecular mass of 6 ku, and mainly expressed in a soluble form. Its accounted for more than 45% of the total bacterial proteins in the expression level. The purity of purified EGF protein reached more than 95%. The results indicate that EGF is suc- cessfully expressed E. coli BL21 （ DE3 ）, and the recombinant EGF is purified using a simple process, which lays a foundation for large - scale preparation of EGF.

关 键 词：表皮生长因子(EGF) 表达 纯化 pET-SUMO-T载体 金属螯合层析 

分 类 号：Q516[生物学—生物化学]