人细胞骨架调节蛋白基因Nelin的原核表达与分离纯化  

Prokaryotic expression and purification of human cytoskeleton regulation gene Nelin

作　　者：尤昕[1] 时娜[1] 柳明洙[2] 曹慧青[1] 刘冬青[1] 孟宪敏[1] 

机构地区：[1]中国医学科学院中国协和医科大学阜外心血管病医院分子医学中心,北京100037 [2]延边大学医学院生物化学与分子生物学教研室,延吉133000

出　　处：《中国免疫学杂志》2006年第12期1128-1131,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金资助项目(No.30500200和No.30470724)

摘　　要：目的:原核表达并分离纯化人细胞骨架调节蛋白基因Nelin。方法:利用DNAstar软件预测Nelin的B细胞表位,选择Nelin基因B细胞表位较多、特异性最强的片段。用DNA重组技术,将此片段插入原核表达载体pET28a(+),构建C末端带有His6标签的原核表达载体pET28a-Nelin,转化大肠杆菌BL21(DE3),IPTG诱导,产物用Ni2+金属螯合吸附层析柱纯化。结果:成功构建了pET28a-Nelin表达载体,IPTG诱导后表达分子量约为17000的可溶性蛋白,Nelin-His6蛋白的表达量占菌体总蛋白的18%,经Ni2+金属螯合层析柱纯化,得到了电泳纯的Nelin-His6蛋白。结论:建立了稳定的Nelin基因的表达体系,为其功能研究奠定了基础。Objective：To purify recombinant protein of human cytoskeleton regulation gene Nelin expressed in Escherichia coll. Methods：The B cell epitopes of human Nelin gene were predicted by DNAstar software. A cDNA fragment of Nelin gene with two epitopes was amplified by PCR, and expression plasmid vector pg128a（ ＋ ） was ligated together with T4DNA ligase after digested by corresponding restriction endonucleases respectively. The new constructed vector pET28a-Nelin was identified by endonuclease digesting and confirmed by DNA sequencing. The BL21 （DE3） containing pET28a-Nelin plasmid was induced by IPTG, and the expressed protein were purified with Ni^2＋ metal chelate affinity chromatography columns. Results ：The recombinant expression vector pET28a-Nelin was successfully constructed. After induction, a new protein band of approximately 17 000 appeared on SDS-PAGE as soluble protein, which accounted for 18% of the total bacterial protein. Purified protein was obtained by Ni^2＋ metal chelate affinity chromatography. Conclusion：The stability expression method of Nelin gene is set up, and the result lays a foundation for the study of Nelin gene function.

关 键 词：Nelin基因 原核表达 分离纯化 B细胞表位 

分 类 号：Q786[生物学—分子生物学]