凋亡蛋白融合基因在大肠杆菌中的表达及抗体制备  

Expression of apoptin fusion gene in E.coli and preparation of its antibody

作　　者：李士泽[1] 张忠芳[2] 张艳宇[3] 连继勤[3] 张玉静[3] 

机构地区：[1]黑龙江八一农垦大学,黑龙江大庆163000 [2]北华大学医学院,吉林吉林132001 [3]解放军军需大学生化教研室,吉林长春130062

出　　处：《中国预防兽医学报》2004年第4期262-265,共4页Chinese Journal of Preventive Veterinary Medicine

摘　　要：在获得凋亡蛋白融合基因重组质粒pMD18_T_EGF_PⅡ_Apoptin的基础上 ,成功地构建了重组表达质粒pET_2 8a_EGF_PⅡ_Apoptin ,将阳性重组质粒转化表达受体菌BL2 1(DE3)感受态细胞中 ,经IPTG诱导表达 ,进行表达产物的聚丙烯酰胺凝胶电泳 (SDS_PAGE)检测 ,结果表明 ,表达蛋白带位于 33kD处 ,凋亡蛋白融合基因获得高效表达 ,薄层扫描分析表明表达蛋白占菌体蛋白的 4 0 %。表达蛋白经透析袋电洗脱法纯化后 ,对獭兔进行常规免疫 ,应用ELISA检测到较高的多克隆抗体效价 ,表明此蛋白具有良好的抗原性。Westernblot结果显示 ,利用纯化包涵体制备的兔抗血清可以很好地和所表达的蛋白带特异性结合。Recombinant expression plasmid pET-28 a-EGF-PⅡ-Apoptin was constructed according to recombinant plasmid pMD18-T-EGF-PⅡ-Apoptin. Then the recombinant was transformed into the host strain BL21(DE3) induced by IPTG when OD_(600) of the culture was about 0.6.The specific protein expressed (about 33KD) was detected by SDS-PAGE.The fusion protein was expressed at high level,amounting to 40 % of the total bacterial protein analyzed by thin-layer scan.After purified by electroelution in dialysis bags,the fusion protein was used to induce the production of polyclonal antibody in rabbits and ELISA detection showed the antigenicity of the fusion protein was satisfactory.Western blot showed the antiserum raised against the recombinant Apoptin in rabbits could react to the protein expressed specifically.

关 键 词：凋亡蛋白 融合基因 原核表达 

分 类 号：S852.612[农业科学—基础兽医学]