人脑金属硫蛋白3(MT-3)原核载体的构建、表达及纯化  

Construction,Expression and Purification of the Prokaryotic Expression Vector of Metallothionein 3 of Human's Brain (MT-3)

作　　者：卢晓霞[1] 谢海军[1] 赵邑[1] 何永吉[1,2] 

机构地区：[1]山西省生物研究所,山西太原030006 [2]山西省农业科学院农产品加工研究所,山西太原030006

出　　处：《山西科技》2017年第2期30-33,共4页Shanxi Science and Technology

基　　金：山西省应用基础研究项目(201601D102041)

摘　　要：为探讨采用基因工程技术构建小分子人MT-3蛋白原核表达载体的可能性,以MT-3 c DNA序列为模板,采用聚合酶链式反应引入双酶切位点,定向亚克隆至pho A分泌型原核表达载体,转化大肠杆菌BL21(DE3)感受态细胞构建重组工程菌,经0.05 mmol/L低磷培养基培养诱导重组MT-3表达,利用金属螯合柱分离纯化后,利用SDS-PAGE、Western blot、紫外光谱扫描及金属结合能力分析对其进行鉴定。结果成功构建了pho A-MT-3原核表达载体,重组工程菌经诱导后以可溶形式表达重组MT-3蛋白。SDS-PAGE、Western blot和紫外光谱扫描分析证实重组MT-3表达成功。金属结合能力分析结果显示目的蛋白仍具有结合金属离子的生物学功能,实现了小分子MT-3的体外分泌重组表达。本研究成功实现了小分子人MT-3蛋白的重组制备,为MT-3的规模化应用奠定基础。This paper discusses the allothionein of human＇ s brain 3 （ MT - 3 po ）. ssibility of constructing prokaryotic expression vector of small molecule met- It takes MT- 3 cDNA sequence as the template and adopts polymerase chain reaction to introduce the double restriction enzyme cutting site and direction subclone to phoA secretion type prokaryotic expression vector and transfer the competent cells of escherichia coli BL21 （ DE 3） to construct and regroup the engineer- ing bacteria. It uses 0.05 mmol/L low-phosphorous medium to cultivate, induce and regroup MT - 3 expression and uses metal-chelating to separate and refine it and uses SDS-PAGE, Western blot, UV spectrum scan and the binding ability of metal to test it. We successfully construct the prokaryotic expression vector of phoA-MT - 3 and after being induced the regrouped engineering bacteria express and regroup MT -3 protein in soluble form. SDS-PAGE, Western blot, UV spec- trum scan prove that the regrouped MT - 3 expresses successfully. The result of the binding ability of metal shows that the objective protein still has the biological function of binding the metal ion, realizing the regroup and expression of the small molecule MT- 3. This research realizes the regroup and preparation the small molecule MT -3 successfully and lays a foundation for the large-scale application of MT - 3.

关 键 词：人脑金属硫蛋白3 神经生长抑制因子 表达 

分 类 号：Q782[生物学—分子生物学]