抗草丁膦bar基因的原核表达与多克隆抗体的制备  

Preparation of bar gene prokaryotic expression vector and polyclonal antibody

作　　者：宋贤瑞 李杰 吕玉民[2] 刘玲玲 薛乐勋 

机构地区：[1]郑州大学细胞生物学研究室郑州大学生物工程系,郑州450001 [2]河南省职业病防治研究所毒理科,郑州450052

出　　处：《郑州大学学报（医学版）》2009年第4期739-741,共3页Journal of Zhengzhou University(Medical Sciences)

基　　金：科技部国际科技合作基金资助项目2007DFA01240;国家自然科学基金资助项目30700014

摘　　要：目的:制备抗草丁膦bar基因的原核表达系统和多克隆抗体。方法:以常规基因重组技术,将bar基因片段插入原核表达载体pET28,得到重组质粒pET28-bar,经酶切、测序鉴定正确后,用该重组质粒转化大肠杆菌BL21(DE3),IPTG诱导后进行SDS-PAGE电泳,镍柱亲和纯化目的蛋白,以目的蛋白免疫BALB/c小鼠,ELISA和Western blot法检测抗体的效价和特异性。结果:bar基因在大肠杆菌BL21(DE3)中得到高效表达,占总蛋白的39.2%。以高纯度的目标蛋白作为抗原免疫小鼠,得到抗PAT的抗血清。ELISA检测抗血清效价达到1:51200,Western blot检测证实抗血清能与目的蛋白特异性结合。结论:以纯化的bar基因表达蛋白PAT作为抗原,制备了特异性较高的抗PAT抗体。Aim：To prepare the bar gene prokaryotic expression vector and polyclonal antibody.Methods：The recombinant prokaryotic expression vector pET28-bar was constructed after the bar gene fragments were successfully inserted into the BamHⅠ-HindⅢ sites of pET28 by routine genetic recombinant technology.The total protein of E.coli BL21（DE3） transformed with pET28-bar was analyzed by SDS-PAGE after induction of IPTG.The target protein purified by Ni-His tag column was used to immunize BALB/c mice,the specificity and sensitivity of anti-serum were characterized by Western blot and ELISA, respectively. Results： The bar gene was expressed effectively in E： coli BL21 and the target protein was detected up to 39.2% of the total expressed proteins, and Western blot showed that the anti-serum from the immunized BALB/c mice bound specially with target proteins and the titer of antiserum was 1 ： 51 200 by ELISA. Conclusion： Polyclonal antibody against PAT with high specificity and sensitivity has been prepared using purified PAT as antigens. These results rffake it possible for studying the expression of the bar gene protein in eukaryotic transformants.

关 键 词：BAR基因 原核表达 抗体制备 

分 类 号：Q786[生物学—分子生物学]