OS-9基因的融合表达、纯化及多克隆抗体制备  

The Fusion Expression and Purification of OS-9 Gene and Preparation of Its Polyclonal Antibody

作　　者：韩月恒[1] 张文红[1] 沈岚[1] 季少平[1] 刘新平[1] 药立波[1] 

机构地区：[1]第四军医大学生物化学与分子生物学教研室,西安710032

出　　处：《中国生物化学与分子生物学报》2003年第3期327-331,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目 (No .3 9870 718No .3 982 5 113 )~~

摘　　要：OS 9基因广泛表达于人体多种组织 ,该基因的表达产物可能与肿瘤的发生相关 .为获得可溶性表达的OS 9蛋白 ,制备多克隆抗体 ,深入了解OS 9基因的功能 ,将OS 9基因片段克隆入组氨酸标签融合的表达载体pET2 8a中 ,IPTG诱导 ,利用金属螯合亲和层析 (MCAC)进行纯化 ,薄层扫描及Bradford法检测纯化蛋白的纯度与含量 .免疫家兔制备多克隆抗体 ,利用间接ELISA法检测抗体效价 ,Western印迹检测抗体特异性 .经表达形式分析证明 ,融合蛋白大部分可溶 .薄层扫描分析纯度可达 90 %以上 ,Bradford法检测蛋白浓度约 0 1mg ml.抗血清效价可达 1∶32 0 0以上 ,Western印迹检测证明抗体特异性良好 .经诱导表达及纯化制备出可溶的纯度较高的OS 9蛋白产物 。OS 9 gene is ubiquitously expressed in human tissues and frequently coamplified with CDK4 gene in human sarcomas, which suggests that the protein may be involved in cell growth or tumor genesis. To obtain soluble protein of OS 9 and its polyclonal antibody for studying the function of OS 9,the fragment of OS 9 gene obtained in the yeast two hybrid system was amplified and cloned into the expression vector pET28a.Induced by IPTG,soluble protein of interest could be obtained. The protein was purified by metal chelate affinity chromatography and its purity and content were detected by thin layer scan analysis and Bradford assay. Rabbits were immunolized by OS 9 protein.The titer and specificity of polyclonal antibody were detected by indirect ELISA assay and Western blotting. Thin layer scanning showed that its purity reached at least 90%.The titer of antiserum was as high as 1∶3200 ,with very high spcificity detected by Western blotting. Through IPTG induction and metal chelate affinity chromatography purification,soluble OS 9 protein with high purity and its specific antibody have been obtained.

关 键 词：OS-9基因 融合表达 纯化 多克隆抗体 肿瘤 

分 类 号：R730[医药卫生—肿瘤] Q786[医药卫生—临床医学]