果蝇再生蛋白(rgn)多克隆抗体的制备及鉴定  

Development and characterization of polyclonal antibody against regeneration( rgn) of Drosophila

作　　者：李哲[1] 柳宗琳 金丽华[1] 

机构地区：[1]东北林业大学生命科学学院遗传学科,黑龙江哈尔滨150040

出　　处：《细胞与分子免疫学杂志》2015年第10期1404-1407,1412,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31270923)

摘　　要：目的制备具有高度特异性的抗果蝇再生蛋白(rgn)的多克隆抗体。方法利用PCR技术从果蝇W1118c DNA中获得rgn基因片段,构建重组质粒;将其转入JM109(DE3)菌株中诱导rgn融合蛋白表达,再利用Ni-NTA superflow层析柱法将其纯化;经Western blot法检测后,利用制备的抗原免疫SD大鼠,从血清中获得高纯度的抗rgn多克隆抗体,利用Western blot法和免疫组织化学染色法对抗体特异性进行鉴定。结果构建的pRSETA-rgn表达载体成功表达6×His-rgn蛋白,融合蛋白在大肠杆菌中大量表达并纯化后,作为抗原免疫SD大鼠,获得了抗rgn的多克隆抗体;免疫组织化学染色技术证实rgn定位在果蝇三龄幼虫血细胞的细胞质。结论成功获得了特异性较高的大鼠抗rgn多克隆抗体。Objective To prepare and characterize rat polyclonal antibodies highly specific for Drosophila regeneration （rgn）. Methods Gene fragments rgn were cloned from Drosophila W1118 cDNAs by PCR and then subcloned into vector pRSETA. Its expression was induced in E. coli JMI09 （DE3）. Then the fusion protein was purified by Ni-NTA superflow chromatography. After the products was identified by Western blotting, SD male rats were immunized with the rgn fusion protein. The polyclonal antibodies were obtained from the rat serum. The specificity and Uter of the polyclonal antibodies were detected by Western blotting and immunohistochemistry. Results Through the recombinant plasmid pRSETA-rgn, 6 x His-rgn fusion protein was expressed abundantly in E. coli JMI09 （ DE3 ）. After purification, it was used to immunize SD rats as antigen to generate the polyclonal antibodies against rgn. Immunohistochemistry showed that rgn is located in the cytoplasm of hemocytes of the third instar larvae of Drosophila. Conclusion The experiment has prepared high-specificity rat polyclonal antibodies against rgn.

关 键 词：rgn蛋白 基因克隆 原核表达 多克隆抗体 

分 类 号：Q969.462.2[生物学—昆虫学] R392.11[医药卫生—免疫学]