Rig-I蛋白C端Helicase结构域的原核表达、纯化及其多克隆抗体制备  

Prokaryotic expression,purification and polyclonal antibody preparation of C terminal Helicase domain of mouse Rig-I

作　　者：李金鞠[1] 任华[1] 王自强[1] 钱旻[1] 杜冰[1] 

机构地区：[1]华东师范大学生命科学学院,上海200062

出　　处：《中国免疫学杂志》2010年第9期774-777,782,共5页Chinese Journal of Immunology

基　　金：上海市科委生物医药领域重点科技攻关专项(074319104;09JC1405200);中央高校基本科研业务费专项资金资助

摘　　要：目的:为Rig-I蛋白结构与模式识别功能的深入研究提供灵敏、高效的免疫学检测试剂。方法:PCR法克隆小鼠Rig-I基因Helicase区编码序列(726～2240bp,编码241～746位氨基酸),构建带组氨酸标签的原核表达载体pET15b-mRig-I-H,阳性克隆经酶切、测序鉴定后转化E.coliBL21进行诱导表达。目的蛋白电泳分离并割胶纯化后免疫家兔制备抗血清。抗血清用ELISA、Western blot、细胞免疫荧光方法进行鉴定。结果:目的蛋白在大肠杆菌中获得了高效表达,制备的抗Rig-I抗体效价达到1∶100000,Western blot、细胞免疫荧光结果显示该抗体能够有效地对细胞内Rig-I蛋白的表达水平进行检测。结论:成功地对mRig-I-H进行了原核表达、纯化,抗mRig-I-H的多克隆抗体具有较高的特异性,为进一步研究Rig-I尤其是Helicase区的结构与功能奠定了坚实的基础。Objective：To develop a highly efficacious and sensitive immunological reagent for further investigation on the retinoic acid-induced gene I （Rig-I） of mouse .Methods：The Helicase domain coding region （726-2 240 bp） of mRig-I-H was cloned into plasmid pET15b （＋） to construct the recombinant plasmid pET15b（＋）-mRig-I-H.Then the plasmid was transformed into E.coli BL21 for protein expression.Rabbits were immunized with electrophoresis-purified recombinant protein to obtain the polyclonal antibody against mRig-I-H.The titer of polyclonal antibody was detected by ELISA and the specificity was identified by Western blot and Immunofluorescence.Results：The recombinant protein was expressed successfully in E.coli.Western blot analysis showed that target protein was expressed with a molecular weight of 40 kD.Titer of the polyclonal antibody was about 1∶1×105 by ELISA assay.With this antibody,we could detect the expression of Rig-I in RAW 264.7 cell line by Western blot and Immunofluorescence.Conclusion：The high level expression of Rig-I Helicase domain is induced in E.coli expressing system.Anti-mRig-I-H polyclonal antibody with high titer and fine specificity could be a novel tool in future investigation of Rig-I.

关 键 词：RIG-I HELICASE 原核表达 多克隆抗体 

分 类 号：R392.33[医药卫生—免疫学]