猪RNA聚合酶Ⅱ单克隆抗体的制备与应用  

Preparation and Application of Monoclonal Antibody of Porcine RNA PolymeraseⅡ

作　　者：田小欢 刘茹 孙艳[1,2] 任瑞敏 余梅[1,2,3] 赵书红[1,2,3] 曹建华[1,2,3] TIAN Xiaohuan;LIU Ru;SUN Yan;REN Ruimin;YU Mei;ZHAO Shuhong;CAO Jianhua(Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China;Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China;The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China)

机构地区：[1]华中农业大学农业动物遗传育种与繁殖教育部重点实验室,武汉430070 [2]华中农业大学农业农村部猪遗传育种重点实验室,武汉430070 [3]华中农业大学生猪健康养殖协同创新中心,武汉430070

出　　处：《畜牧兽医学报》2021年第10期2783-2791,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31772563);武汉市应用基础前沿项目(2020020601012255)。

摘　　要：旨在制备猪RNA聚合酶Ⅱ的单克隆抗体并进行初步应用。本研究采用生物信息方法预测免疫原,将其化学合成后免疫5只4~8周龄Bal b/c雌性小鼠,对免疫后呈现阳性的小鼠进行细胞融合试验,取其脾细胞与骨髓瘤细胞进行融合并获得能稳定分泌抗RNA PolⅡ的杂交瘤细胞。鉴定结果显示,RNA PolⅡ单克隆抗体的重链为IgG 2A型,轻链为Kappa型。利用间接ELISA方法对杂交瘤细胞进行筛选和亚克隆,获得了8株稳定分泌RNA PolⅡ单克隆抗体的杂交瘤细胞株。将其初步应用到染色质免疫共沉淀(ChIP-seq)技术,并与商业化抗体的富集性进行比较,结果表明,本研究得到的单克隆抗体富集性更强。本研究获得的猪RNA PolⅡ单克隆抗体可为表观遗传学的研究提供理论基础,并且提供重要的生物学材料。The purpose of this study was to prepare monoclonal antibodies against porcine RNA polymeraseⅡand make preliminary applications.The bioinformatics method was used to predict the immunogen,after immunogen was chemically synthesized,5 female Bal b/c mice aged 4-8 weeks were immunized,and cell fusion experiment was performed on the mice that were positive after immunization,spleen cells of these mice were fused with myeloma cells to obtain hybridoma cells that could stably secrete anti-RNA Pol II antibody.The identification result showed that the heavy chain of RNA PolⅡmonoclonal antibody was IgG 2A type,and the light chain was Kappa type.The hybridoma cells were screened and subcloned by the indirect ELISA method,and 8 hybridoma cell lines that stably secreted RNA PolⅡmonoclonal antibody were obtained.It was preliminary applied to chromatin immunoprecipitation(ChIP-seq)technology,and compared with the enrichment of commercial antibodies,the results showed that the monoclonal antibodies obtained in this study were more enriched.The porcine RNA PolⅡmonoclonal antibody obtained in this study can provide a theoretical basis for epigenetic research and provide important biological materials.

关 键 词：RNA PolⅡ 单克隆抗体 猪 杂交瘤细胞 

分 类 号：S852.43[农业科学—基础兽医学]