人源吲哚胺2,3-双加氧酶-2的表达与纯化  

Expression and purification of human indoleamine 2,3-dioxygenase-2

作　　者：李娟娟[1] 李洋[1] 杨青[1,2] 

机构地区：[1]复旦大学生命科学学院生物化学系,上海200438 [2]云南天然产物与生物制药协同创新中心,昆明650091

出　　处：《复旦学报（医学版）》2016年第4期401-408,共8页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金(81373396;81573310);高等学校博士学科点专项科研基金(20130071110037)~~

摘　　要：目的构建人源吲哚胺2,3-双加氧酶-2(human indoleamine 2,3-dioxygenase-2,hIDO2)原核表达载体,表达、纯化获得hIDO2蛋白。方法采用基因工程手段获得hIDO2原核表达载体并转化表达菌株,筛选合适的重组质粒及异丙基-β-D-硫代半乳糖苷(isopropy-β-D-thiogalactoside,IPTG)浓度进行蛋白表达;通过亲和层析纯化重组hIDO2,BCA法测定蛋白浓度并计算蛋白收率;SDS-PAGE电泳检测hIDO2表达情况,通过软件分析得到蛋白纯度;Western blot检测目的蛋白特异性。结果重组质粒经电泳分析、双酶切、DNA测序鉴定表明构建成功;经筛选发现重组质粒pET28a-hIDO2比pGEX-4T-1-hIDO2的hIDO2表达效果好;SDS-PAGE电泳及Western blot均验证了在相对分子质量45 000处的特异性目的条带的存在;经测定及计算得出蛋白纯度为97.1%,蛋白浓度为20 mg/mL,蛋白收率为15 mg/L。结论成功构建重组质粒用于表达及纯化hIDO2,蛋白浓度、纯度、收率及特异性均较高。Objective To construct human indoleamine 2,3-dioxygenase-2 （hIDO2） prokaryotic expression vectors to express and obtain purified hIDO2. Methods Using genetic engineering method to obtain hIDO2 prokaryotic expression vectors and transform Escherichia coli expression strains among which an optimal recombinant plasmid and isopropy-β-D-thiogalactoside （IPTG） concentration were screened and used for massive hIDO2 expressing.Affinity chromatography was used for hIDO2 purification.BCA method was used for protein concentration and yield measurement,SDS-PAGE electrophoresis was used for hIDO2 expression quantity determination and the purity was calculated with relevant software.Western blot was used for specificity detection. Results Electrophoretic analysis,double restriction endonuclease digestion and sequencing confirmed the successful construction of hIDO2 expression vectors.pET28a-hIDO2 was found to be a better recombinant plasmid to express human IDO2 than pGEX-4T-1-hIDO2.A specific target stripe at molecular weight （Mr） of about 45 000,same as predicted,was observed in both SDS-PAGE electrophoresis and Western blot.The purity,concentration and yield of hIDO2 was 97.1%,20 mg/mL and 15 mg/L,respectively. Conclusions We successfully constructed recombinant plasmids using which we expressed and purified hIDO2 with high concentration,purity and yield.

关 键 词：吲哚胺2 3-双加氧酶-2 人源 蛋白表达 蛋白纯化 

分 类 号：Q331[生物学—遗传学] Q786