血管紧张素Ⅱ相关新基因BC097361融合蛋白的表达、纯化及多克隆抗体的制备  

Expression, purification and polyclonal antibody preparation for a novel gene BC097361

作　　者：王琦[1] 梁赟磊[1] 魏红山[1] 邢卉春[2] 成军[1] 兰孟东[3] 张斌[1] 

机构地区：[1]北京地坛医院传染病研究所,100011 [2]北京地坛医院肝病中心,100011 [3]北京地坛医院病理科,100011

出　　处：《中华肝脏病杂志》2009年第8期589-593,共5页Chinese Journal of Hepatology

基　　金：国家自然科学基金（30671875、30872243）

摘　　要：目的构建血管紧张素Ⅱ相关新基因BC097361的原核表达载体，诱导融合蛋白的表达，并对其进行纯化；制备兔抗BC097361蛋白多克隆抗体并进行鉴定。方法应用RT—PCR技术，以LX02细胞总RNA为模板，扩增BC097361目的基因片段，构建原核表达载体pET-32a（＋）-BC097361。转化大肠埃希菌BL21（DE3），异丙基-β-半乳糖苷诱导并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析、Western blot分析证实融合蛋白表达的特异性。大量表达后利用Ni^＋亲和柱对表达蛋白进行纯化及柱上复性。纯化蛋白免疫新西兰兔，获得抗BC097361蛋白的多克隆抗体。以纯化的BC097361蛋白为抗原，分别以免疫前后的新西兰兔血清作为第一抗体，利用Western blot和酶联免疫吸附法对多克隆抗体进行特异性分析及效价检测。结果扩增获得BC097361基因片段，成功表达了BC097361相关蛋白，经十二烷基硫酸钠聚丙烯酰胺凝胶电泳和Western blot分析得到证实。成功获得融合蛋白及兔抗BC097361多克隆抗体。酶联免疫吸附法检测证实多克隆抗体效价〉1：320000，Western blot、免疫组织化学检测证明多克隆抗体的特异性良好。结论利用大肠埃希菌BL21（DE3）能够成功表达BC097361蛋白，获得高特异性、高效价兔抗BC097361蛋白的多克隆抗体，为今后研究BC097361蛋白的生物学特性奠定了基础。Objective To express and purify of the BC097361 recombinant protein, and to prepare the BC097361 specific rabbit polyclonal antibody. Methods BC097361 cDNA was ligated into the prokaryotic expressive vector pET-32a （＋）, and the resulting plasmid was transformed into E.coli BL21 （DE3）. The protein expression was induced with IPTG and the protein was analyzed with SDS-PAGE and western blotting. The expressed product was purified using Ni^＋ affinity column chromatography.Tben the purified pET-32a （＋） -BC097361 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by Western blot and ELISA. Results The BC097361 fusion protein was highly expressed.The protein was mainly in the inclusion body. ELISA indicated the titer of polyclonal antibody 〉 1 ： 320 000. The high specificity was comfirmed with Western blot. Conclusions The recombinant BC097361 fusion protein and the BC097361 specific polyclonal antibody will be valuable tools for the investigation on the biological function of BC097361.

关 键 词：肝纤维化 血管紧张素Ⅱ 基因 原核表达 蛋白纯化 

分 类 号：R392[医药卫生—免疫学] R575.2[医药卫生—基础医学]