Richardson Nastassia C, Sargentini Neil J, Singh Vineet K, Stuart Melissa K
Department of Microbiology/Immunology, A.T. Still University, Kirksville College of Osteopathic Medicine, 800 W. Jefferson Street, Kirksville, MO 63501, USA.
Hybridoma (Larchmt). 2012 Feb;31(1):25-31. doi: 10.1089/hyb.2011.0075.
The RadA/Sms protein facilitates DNA repair in Escherichia coli cells damaged by UV radiation, X-rays, and chemical agents. However, the precise mechanism by which RadA/Sms aids DNA repair is unknown. Here we report the production of monoclonal antibodies (MAbs) specific for RadA/Sms for use in biochemical and physiological investigations. Histidine-tagged RadA/Sms (RadA-6xHis) was overproduced in E. coli BL21 cells transformed with the radA/sms coding region in plasmid pRSET A and purified by nickel affinity chromatography. Splenocytes from female BALB/c mice hyperimmunized with the purified protein were fused to SP2/0-Ag14 myeloma cells, and the resultant hybridomas were selected in HAT medium. MAbs were detected in hybridoma culture supernatants by indirect ELISA and Western blot analysis against purified RadA-6xHis. MAbs from four cell lines were further evaluated by Western blotting against peptide maps generated by endoproteinase Glu-C digestion of RadA-6xHis. Each of the four MAbs recognized a unique epitope on the fusion protein. Two of the MAbs (6F5 and 2A2) also detected wild-type (tagless) RadA/Sms produced from the pJS003 plasmid in E. coli K-12 cells. We anticipate that these antibodies will prove useful for the detection, isolation, and functional analysis of RadA/Sms.
RadA/Sms蛋白可促进受紫外线、X射线和化学试剂损伤的大肠杆菌细胞中的DNA修复。然而，RadA/Sms辅助DNA修复的确切机制尚不清楚。在此，我们报告了用于生化和生理学研究的针对RadA/Sms的单克隆抗体（MAb）的制备。在质粒pRSET A中用radA/sms编码区转化的大肠杆菌BL21细胞中过量表达了带组氨酸标签的RadA/Sms（RadA-6xHis），并通过镍亲和层析进行纯化。用纯化蛋白对雌性BALB/c小鼠进行超免疫，取其脾细胞与SP2/0-Ag14骨髓瘤细胞融合，在HAT培养基中筛选所得杂交瘤。通过间接ELISA和针对纯化的RadA-6xHis的蛋白质印迹分析检测杂交瘤培养上清液中的MAb。通过针对由内切蛋白酶Glu-C消化RadA-6xHis产生的肽图的蛋白质印迹进一步评估来自四个细胞系的MAb。四种MAb中的每一种都识别融合蛋白上的独特表位。其中两种MAb（6F5和2A2）还检测到了大肠杆菌K-12细胞中由pJS003质粒产生的野生型（无标签）RadA/Sms。我们预计这些抗体将被证明可用于RadA/Sms的检测、分离和功能分析。
