【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。更多还原

【Abstract】 The purpose of this study was to prepare monoclonal antibodies against porcine RNA polymerase Ⅱ and 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 PolⅡ monoclonal antibody was IgG 2 A 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 PolⅡ monoclonal 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 PolⅡ monoclonal antibody obtained in this study can provide a theoretical basis for epigenetic research and provide important biological materials.更多还原