兔抗人MiR-338抗体制备与鉴定  

Preparation and Identification of Rabbit Anti-Human Antibody of MiR-338

作　　者：吴大英[1] 张秀艳 李霞[1] 郭予武[1] WU Daying;ZHANG Xiuyan;LI Xia;GUO Yuwu(Longang Central Hospital;Chinese Traditional Medicine Hospital Of Longgang District,Shenzhen,Guangdong 518116,China)

机构地区：[1]深圳市龙岗中心医院 [2]深圳市龙岗区中医院,广东深圳518115

出　　处：《九江学院学报（自然科学版）》2020年第2期96-99,共4页Journal of Jiujiang University：Natural Science Edition

基　　金：深圳市龙岗区科技计划医疗卫生项目(编号2017040518421728)的成果之一。

摘　　要：目的制备兔抗人MiR-338分子多克隆抗体。方法PCR法扩增出MiR-338基因,将其克隆入原核表达载体pET28中,转化大肠杆菌BL21(DE3)、IPTG诱导重组蛋白的表达,Ni^2+-NTA Agarose亲和层析法纯化重组蛋白,以之为免疫原免疫家兔,制备多克隆抗体,并用ELISA、Western blot和免疫组织化学法检测抗体。结果成功构建了MiR-338原核表达载体pET28/MiR-338,高效表达并纯化MiR-338蛋白,间接ELISA检测所制备抗体的效价为1:6400,Western blot显示该抗体能与MiR-338特异结合,免疫组织化学法检测结果表明该抗体识别人卵巢癌组织中的天然的MiR-338。结论成功地制备了效价高、特异性较强兔抗人MiR-338抗体,为进一步建立MiR-338的ELISA检测方法和探讨MiR-338在卵巢癌的发生发展中所起的重要作用奠定了良好的基础。Objective To prepare the rabbit antibody against the human MiR-338.Methods The gene fragment of MiR-338 was amplified by PCR and cloned into prokaryotic expression vector pET28,then expressed in E.coli BL21(DE3).The recombinant protein MiR-338 was purified with Ni 2+-NTA agarose column and used as immunogen to immunize the rabbit.The titer and specificity of the MiR-338 antibody from the rabbit were analyzed by ELISA,Western blot and immunohistochemistry,respectively.Results The recombinant MiR-338 was successfully expressed and purified and the polyclonal anit-human MiR-338 antibody was successfully prepared.The titer of the antiserum was 1:6400 by ELISA.Western blot analysis showed this antibody reacted specifically with MiR-338.Immunohistochemistry analysis showed the antibody could recognize the native MiR-338 in the human ovarian carcinomas tissue.Conclusion The anti-MiR-338 antibody from the rabbit with high titer and specificity was prepared with purified recombinant MiR-338 as immunogen,which laid a foundation for further research on the detection of MiR-338 by ELISA as well as exploration of the important role of the MiR-338 on the development of ovarian cancer.

关 键 词：卵巢癌 MiR-338 抗体 制备 

分 类 号：R737.31[医药卫生—肿瘤]