论文部分内容阅读
人源DNA聚合酶δ(Polδ)是由p125、p50、p68和p12四个亚基组成的异源四聚体,小亚基p12在Polδ参与DNA复制与损伤修复过程中起着至关重要的作用。为了获得具有高度特异性和灵敏性的抗p12抗体,利用PCR技术成功扩增p12基因,通过酶切、连接、转化等常规分子克隆方法构建重组原核表达质粒pGEX-5X-3-p12,经转化大肠杆菌BL21,诱导表达的可溶性融合蛋白经Glutathione Sepharose 4B柱和FPLC Mono Q柱纯化、Factor-Xa酶切,获得不带GST标签的p12蛋白作为抗原;免疫新西兰大白兔制备抗血清并用Protein A/G亲和层析柱纯化多克隆抗体。经Western blot和细胞免疫荧光分析测试,所获得的抗体不但能够特异性识别细胞内源p12,而且观察到p12能够与PCNA共定位到DNA复制叉,首次在亚细胞水平上证明了p12与PCNA的粘连互作反应。高度特异和灵敏的抗p12抗体的获得为深入研究小亚基p12如何调控Polδ的酶学功能、为从人类癌症发病的起因上阐明由于Polδ功能改变而引起遗传基因组不稳定进而导致肿瘤发生的机制提供了重要的手段。
The human DNA polymerase δ (Polδ) is a heterotetramer composed of four subunits of p125, p50, p68 and p12. The small subunit p12 plays a crucial role in the process of Polδ involved in DNA replication and damage repair effect. In order to obtain a highly specific and sensitive anti-p12 antibody, the p12 gene was successfully amplified by PCR and the recombinant prokaryotic expression plasmid pGEX-5X-3-p12 was constructed by restriction enzyme digestion, ligation, transformation and other conventional molecular cloning methods. Escherichia coli BL21 was induced to express soluble fusion protein by Glutathione Sepharose 4B column and FPLC Mono Q column. The fragment was digested with Factor-Xa to obtain p12 protein without GST tag as antigen. Immunized New Zealand white rabbits were used to prepare antiserum, G affinity chromatography purification of polyclonal antibodies. Western blot analysis and immunofluorescence analysis showed that the obtained antibody could not only specifically recognize the endogenous p12, but also p12 was able to co-localize with PCNA to DNA replication for the first time at the subcellular level that p12 and PCNA Adhesion interaction. Obtaining Highly Specific and Sensitive Anti-p12 Antibodies To further investigate how the small subunit p12 regulates the enzymatic function of Polδ, we elucidated the mechanism by which genetic alterations in Polδ function lead to tumorigenesis as a result of the alterations in Polδ function Provide an important means.