人宫颈癌细胞辅酶Ⅱ依赖性视黄醇脱氢/还原酶选择性剪接新亚型的表达及多克隆抗体制备  

Expression of NADP-dependent retinol dehydrogenase/reductase B1 in cervical squamous carcinoma cell line and antibody preparation

作　　者：宋旭红[1] 梁斌[1] 刘戈飞[1] 谢健平[1] 李蕊[1] 黄东阳[1] 

机构地区：[1]汕头大学医学院分子生物学中心,广东汕头515041

出　　处：《解放军医学杂志》2008年第8期983-986,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助项目(30470396;30700474);广东省自然科学基金资助项目(7008212)

摘　　要：目的制备宫颈鳞癌细胞中辅酶Ⅱ依赖性视黄醇脱氢/还原酶(NRDR)选择性剪接新亚型NRDRB1多克隆抗体,为NRDRB1的功能研究奠定基础。方法扩增NRDRB1开放阅读框,重组入Gateway原核表达系统pDEST14,并在大肠杆菌BL21-AI中诱导表达,制备NRDRB1包涵体,使用从SDS-PAGE凝胶中回收的NRDRB1重组蛋白作为免疫原,经肩胛骨下注射免疫新西兰白兔。斑点杂交测定抗血清效价,HiTrapProteinG柱纯化兔IgG,免疫印迹和免疫组织化学鉴定抗体特异性。结果NRDRB1在大肠杆菌BL21-AI中高效表达,回收的NRDRB1蛋白溶液浓度为0.42mg/ml。所有免疫的动物在第2次追加免疫之后都产生了高效价的抗血清。血清效价为l∶2000,对NRDRB1蛋白的检测极限是6.4ng。纯化后的NRDRB1抗体具有较高的免疫活性和特异性。结论NRDRB1原核表达载体的构建、蛋白表达、纯化及多克隆抗体的制备为今后研究该蛋白的功能奠定了良好的基础。Objective The prokaryotic expression vector of NADP（H）-dependent retinol dehydrogenase/reductase（NRDR）B1 was constructed for the detection of NRDRB1 protein,and to prepare its polyclonal antibodies,in order to lay the foundation to study the function of NRDRB1.Methods The coding region of NRDRB1 was constructed to the Gateway-based expression vector（pDEST 14）,which was transformed into the Escherichia coli（BL21-AI）for the native protein expression.Overexpression of the recombinant was induced at mid-log growth phase of BL21-AI（A600=0.6）using 0.2% L-arabinose.After supersonication the inclusion bodies of NRDRB1 were purified.New Zealand rabbits were immunized with NRDRB1 as the immunogen,which was recovered from SDS-PAGE gel and subscapsularly injected.The titer of the antiserum was determined by dot blot assay.The antibody was purified by HiTrap Protein G column,and its activity and specificity were assessed by Western blotting and immunohistochemistry.Results The prokaryotic expression vector pDEST 14 with NRDRB1 was constructed.The constructs were sequenced by dideoxynucleotide method.NRDRB1 was overexpressed in strain BL2l-AI.The concentration of recovered NRDRB1 was 0.42mg/ml with a recovery rate of 52.3%.All the immunized rabbits produced high-titer antisera after the second booster.The titer of the antiserum was 1∶2 000 with a detection limit of 6.4ng NRDRB1.The purified antibody had a high specificity.Conclusion The present study provides an effective method of preparing polyclonal antibody against NRDRB1.The purified NRDRB1 native protein and the specific polyclonal antibody of NRDRB1 would be valuable for the study on the biological function of NRDRB1.

关 键 词：辅酶Ⅱ依赖性视黄醇脱氢/还原酶B1 基因表达 宫颈肿瘤 

分 类 号：Q493.6[生物学—生理学] R392[医药卫生—免疫学]