Hodgetts Jennifer, Johnson Gaynor, Perkins Kate, Ostoja-Starzewska Sioban, Boonham Neil, Mumford Rick, Dickinson Matthew
School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK,
Mol Biotechnol. 2014 Sep;56(9):803-13. doi: 10.1007/s12033-014-9759-8.
Partial recombinant secA proteins were produced from six different phytoplasma isolates representing five 16Sr groups and the expressed, purified recombinant (partial secA) protein from Cape St. Paul wilt disease phytoplasma (CSPWD, 16SrXXII) was used to immunise mice. Monoclonal antibodies (mAbs) were selected by screening hybridoma supernatants for binding to the recombinant proteins. To characterise the binding to proteins from different phytoplasmas, the antibodies were screened by ELISA and western blotting, and epitope mapping was undertaken. Eight different mAbs with varying degrees of specificity against recombinant proteins from different phytoplasma groups were selected. Western blotting revealed that the mAbs bind to proteins in infected plant material, two of which were specific for phytoplasmas. ELISA testing of infected material, however, gave negative results suggesting that either secA was not expressed at sufficiently high levels, or conformational changes of the reagents adversely affected detection. This work has shown that the phytoplasma secA gene is not a suitable antibody target for routine detection, but has illustrated proof of principle for the methodology.
从代表五个16Sr组的六种不同植原体分离物中制备了部分重组secA蛋白，并用来自圣保罗角枯萎病植原体（CSPWD，16SrXXII）的表达并纯化的重组（部分secA）蛋白免疫小鼠。通过筛选杂交瘤上清液与重组蛋白的结合来选择单克隆抗体（mAb）。为了表征与不同植原体蛋白质的结合，通过ELISA和蛋白质印迹对抗体进行筛选，并进行表位作图。选择了八种对来自不同植原体组的重组蛋白具有不同程度特异性的不同单克隆抗体。蛋白质印迹显示，这些单克隆抗体与受感染植物材料中的蛋白质结合，其中两种对植原体具有特异性。然而，对受感染材料的ELISA检测给出了阴性结果，这表明secA要么没有以足够高的水平表达，要么试剂的构象变化对检测产生了不利影响。这项工作表明，植原体secA基因不是常规检测的合适抗体靶标，但已说明了该方法的原理证明。
