ZBTB8A多克隆抗体制备及其相关信息的初步研究

发布时间：2018-05-14 09:37

本文选题：亚细胞定位 + 转激活　；参考：《苏州大学》2012年硕士论文

【摘要】：由于ZBTB家族成员主要表现为转录调控因子，通过BTB结构域和ZNF结构域介导的蛋白与蛋白之间的互作及蛋白与特异性碱基序列的结合从而调控基因的转录活性，进而影响细胞的增殖、分化、迁移、凋亡和细胞周期等重要的生理功能。目前关于ZBTB8A功能研究的报道很少，根据NCBI数据库查阅人ZBTB8A相关信息设计相应的引物，通过RT-PCR从胃癌细胞系AGS中克隆到人ZBTB8A基因，，测序证实其为目的基因，构建ZBTB8A全长及其截短的不同重组质粒，为后续实验做准备。使用ZBTB8A全长及其截短的GFP融合表达载体来探索其在亚细胞水平上的分布情况，亚细胞定位显示：GFP呈全细胞分布；GFP-ZBTB8A融合蛋白位于细胞核且呈斑点状分布；GFP-ZBTB8A-BTB融合蛋白显示核周斑点状分布，GFP-ZBTB8A-ZNF融合蛋白呈细胞核均匀分布。这些结果表明，ZBTB8A蛋白是一个转录因子存在于核内，其中这两个结构域在斑点状分布中均起着重要的作用，这可能与它们自身结构域的功能相关。为了探讨ZBTB8A可能参与调控的细胞信号通路，采用双荧光素酶检测试剂盒来检测ZBTB8A对相关信号通路的转录调控作用。双荧光素酶实验显示：ZBTB8A蛋白抑制多个细胞信号通路活性，特别对pCRE-Luc具有显著的抑制效应并表现为剂量依赖性。这说明ZBTB8A蛋白在某些功能方面可能作为一个转录抑制因子，参与多个细胞信号通路活性的调控，进而影响细胞的增殖、分化、凋亡、迁移和细胞周期等重要的生理过程。将原核表达载体pGEX-4T-1-ZBTB8A转化到BL21宿主菌，当菌体生长至对数期，加入终浓度为0.6mM IPTG诱导5小时，将诱导的菌体裂解液经SDS-PAGE电泳后，浸泡于0.25M KCl溶液，切下乳白色的目的条带并切碎，电洗脱和透析，获得GST-ZBTB8A融合蛋白，免疫印迹实验进一步证实所纯化的蛋白为目的蛋白（抗GST标签抗体），BCA法测定其浓度为1.254mg/mL，用纯化的蛋白多次免疫兔子，获得相应的免疫血清。应用免疫印迹实验对免疫血清进行特异性分析，实验表明：免疫血清中既含抗ZBTB8A蛋白的抗体又含抗GST标签的抗体；ZBTB8A蛋白中的BTB结构域和ZNF结构域均为抗原决定簇即多克隆抗体中含抗这两个结构域的抗体；多克隆抗体可用于内源性表达ZBTB8A蛋白的检测。通过琼脂糖双扩散实验测定其效价为1:32；使用ProteinG纯化免疫血清，SDS-PAGE检测其纯化效果，获得相对较纯的抗体，为进一步的研究提供一定的支持。
[Abstract]:Because the members of the ZBTB family are mainly transcriptional regulators, the transcriptional activity of the gene is regulated by the interaction between protein and protein mediated by BTB domain and ZNF domain and the binding between protein and specific base sequence. And then affect cell proliferation, differentiation, migration, apoptosis and cell cycle and other important physiological functions. At present, there are few reports on the functional study of ZBTB8A. According to the relevant information of human ZBTB8A from NCBI database, the corresponding primers were designed and cloned from the gastric cancer cell line AGS by RT-PCR, and the target gene was confirmed by sequencing. The full length and truncated recombinant plasmids of ZBTB8A were constructed to prepare for further experiments. The full length of ZBTB8A and its truncated GFP fusion expression vector were used to explore its distribution at subcellular level. The subcellular localization showed that the GFP-ZBTB8A fusion protein was located in the nucleus and the GFP-ZBTB8A-BTB fusion protein showed that the GFP-ZBTB8A-ZNF fusion protein was evenly distributed in the nucleus. These results suggest that the ZBTB8A protein is a transcription factor in the nucleus, in which the two domains play an important role in the dot-like distribution, which may be related to the function of their own domain. In order to explore the cellular signaling pathway in which ZBTB8A may be involved, double luciferase detection kit was used to detect the transcriptional regulation of ZBTB8A on the related signal pathway. Double luciferase assay showed that the protein of 1% ZBTB8A inhibited the activity of many cell signaling pathways, especially in a dose-dependent manner. These results suggest that ZBTB8A may play a role as a transcription suppressor in regulating the activity of multiple cell signaling pathways, and thus affect cell proliferation, differentiation, apoptosis, migration, cell cycle and other important physiological processes. The prokaryotic expression vector pGEX-4T-1-ZBTB8A was transformed into BL21 host strain. When the cell grew to logarithmic stage, the final concentration of 0.6mM IPTG was added for 5 hours. After SDS-PAGE electrophoresis, the induced lytic liquid was immersed in 0.25 M KCl solution, and the target band of milky white was cut and shredded. The GST-ZBTB8A fusion protein was obtained by electroelution and dialysis. The purified protein was further confirmed by Western blot as the target protein (the concentration of the purified protein was 1.254 mg / mL determined by anti-BCA labeled antibody). Rabbits were immunized with the purified protein several times to obtain the corresponding immune serum. Immunoblotting assay was used to analyze the specificity of immune serum. The results showed that the BTB domain and ZNF domain of ZBTB8A protein contained both anti-ZBTB8A protein and anti-ZBTB8A antibody, which were antigenic determinants, that is, polyclonal antibodies contained antibodies to these two domains. Polyclonal antibodies can be used for the detection of endogenous expression of ZBTB8A protein. The titer was 1: 32 by agarose double diffusion assay, and the purified antibody was obtained by using ProteinG purified immune serum and SDS-PAGE, which provided some support for further research.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【相似文献】