人源DNA聚合酶Pol δ小亚基p12的抗体制备及应用  

Production and Application of Polyclonal Antibody against p12,the Fourth Subunit of Human DNA Polymerase δ

作　　者：李骁[1] 刘留[1] 张磊[1] 周亚竟[1] 

机构地区：[1]江苏大学生命科学研究院,镇江212013

出　　处：《中国生物工程杂志》2012年第5期12-18,共7页China Biotechnology

基　　金：国家自然科学基金资助项目(30970612)

摘　　要：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。DNA polymerase δ（Pol δ） consists of p125,p68,p50 and p12 subunits.The smallest subunit p12 is thought to play a crucial and versatile role in modulating the functions of Pol δ during DNA replication and various DNA repair processes.In order to obtain polyclonal antibody against p12 with high specificity and sensitivity,the recombinant plasmid pGEX-5X-3-p12 was generated by PCR and subcloning of digested PCR product（p12 DNA） into the pGEX-5X-3.GST-tagged p12,expressed in BL21 cells which was transformed with pGEX-5X-3-p12 and induced by IPTG,was purified on Glutathione-Sepharose 4B beads and further purified on FPLC Mono Q column.Non-tagged p12,used as immunogen,was released by proteolysis with Factor Xa.The polyclonal antibody against p12 was produced by immunizing rabbits with highly purified non-tagged p12 protein.The collected antiserums were purified by precipitation with（NH4）2SO4 and followed by the purification on Protein A/G Plus Agarose.Western blotting analysis showed that the obtained antibody recognized endogenous p12 in good specificity and sensitivity.While a cell based immunocytochemistry assay showed a subcellular co-localization of p12 with PCNA to distinct nuclear spots which is the first time to confirm a physiological interaction of p12 with PCNA in vivo.Thus,the study provides a powerful tool for the further research on the p12 modulating Pol δ functions and how alterations in pol δ function could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.

关 键 词：DNA聚合酶Δ 小亚基p12 蛋白纯化 多克隆抗体 PCNA 

分 类 号：Q78[生物学—分子生物学]