再生基因4(RegⅣ)的原核表达、蛋白结构复性及纯化  

Prokaryotic expression,protein structure renaturation and purification of Regenerating Gene Ⅳ

作　　者：徐杨[1] 张学良[2] 杜惠芬[1] 柴丹丹[1] 连晓雯[1] 张瑞君[1] 李克生[1] Xu Yang;Zhang Xueliang;Du Huifen;Chai Dandan;Lian Xiaowen;Zhang Ruijun;Li Kesheng(Gansu Academy of Medical Science,Lanzhou 730050,China;Gansu Provincial Cancer Hospital,Lanzhou 730050,China)

机构地区：[1]甘肃省医学科学研究院,甘肃兰州730050 [2]甘肃省肿瘤医院,甘肃兰州750050

出　　处：《甘肃医药》2020年第7期577-579,588,共4页Gansu Medical Journal

基　　金：兰州市科技发展指导性计划项目(项目编号:2019-ZD-131)。

摘　　要：目的:构建再生基因4(Reg Ⅳ)的原核表达载体,获得重组人Reg Ⅳ蛋白。方法:将已构建好的pEGFP-C1-Reg Ⅳ质粒进行酶切纯化获得目的基因,并构建到pET-30a原核表达载体上,经测序鉴定后将序列正确的重组质粒转化E.coli BL21(DE3),IPTG诱导表达,SDS-PAGE凝胶电泳鉴定,并用凝胶层析对表达产物进行复性纯化。结果:构建了pET-30a-Reg Ⅳ原核表达重组质粒,IPTG诱导表达出约22KD的重组蛋白,表达产物经复性纯化后获得纯度较高的目的蛋白。结论:成功构建了Reg Ⅳ原核表达载体,并成功表达了重组人Reg Ⅳ蛋白。Objective:To construct the prokaryotic expression system of regenerating gene Ⅳ(Reg Ⅳ)and obtain recombinant human Reg Ⅳ protein. Methods:The constructed pEGFP-C1-RegⅣ plasmid is digested and purified to obtain the target gene,and constructed into the pET-30a prokaryotic expression vector. After sequencing and identification,the recombinant plasmid with the correct sequence was transformed into E.coli BL21(DE3),and was induced by isopropyl-beta-D-thiogalactoside(IPTG)to expression. The expressed product was identified by SDS-PAGE gel electrophoresis,and was renatured and purified by gel chromatography. Results:The recombinant expression vector pET-30a-RegⅣ was constructed and about 22 KD recombinant protein was induced. The target protein with higher purity was obtained after renaturation and purification. Conclusion:The prokaryotic expression vector pET-30a-RegⅣ was successfully constructed,and the recombinant human Reg Ⅳ protein was successfully expressed in prokaryotic expression system.

关 键 词：再生基因4 原核表达 纯化 

分 类 号：R784[医药卫生—口腔医学]