鼠伤寒沙门菌PagC蛋白单克隆抗体的制备及其特性分析

[目的]本试验旨在制备抗鼠伤寒沙门菌PagC蛋白的单克隆抗体并初步分析其特异性和识别的抗原表位，为沙门菌抗体阻断ELISA检测方法的建立奠定基础。[方法]原核表达并纯化PagC蛋白，免疫BALB/c小鼠，采用杂交瘤技术制备单克隆抗体；用生物信息学分析法获得PagC蛋白在沙门菌属内保守的区段，合成相应多肽P1及P2用于单克隆抗体筛选和结合表位鉴定；最后采用免疫印迹方法对单克隆抗体与肠杆菌科其他细菌的交叉反应进行检测，评价其特异性。[结果]纯化后的PagC蛋白相对分子质量约23×103；免疫小鼠后经2次亚克隆最终得到稳定分泌抗体的细胞株6株，标记为A、B、C、D、I、J；单克隆抗体的反应性试验结果显示6株单克隆抗体均能够识别PagC蛋白；单克隆抗体的结合表位分析显示，J细胞株分泌的单克隆抗体可特异性识别P1序列；采用免疫印迹方法对J细胞株上清液进行特异性检测，证实该株单克隆抗体与变形杆菌、大肠杆菌O1和宋内志贺菌PagC蛋白均无结合反应。[结论]成功获得1株与PagC蛋白有良好结合活性且具有高度特异性的单克隆抗体，该株单克隆抗体的识别表位位于PagC中沙门菌属内保守区段，可作为建立沙门菌抗体阻断ELISA检测方法的候选单克隆抗体。
[Objectives]In order to establish the blocking ELISA(bELISA) Salmonella antibody detection kit,we select and identify a monoclonal antibody(mAb) production hybridoma cell line,which can secret mAb against conserved motif of protein PagC in Salmonella. Moreover,we have detected the specificity and binding epitope of this mAb.[Methods]Immunize BALB/c mouse with purified recombination PagC protein,and produce hybridoma cell line. Identify the conserve domain of PagC and synthesize peptide P1 and P2 according to these domains. Screen mAb with the conserve domain peptides and identify their binding epitope. Western blot was used for the mAb specificity identification.[Results]The relative molecular weight of the recombination PagC protein is 23×103. Six mAb secret hybridoma cell lines were identified(hybridoma cell line). The mAbs of these 6 cell lines were all able to bind to PagC. We coupled peptides P1 and P2 with KLH separately and found that mAb from cell line J could bind to P1. The mAb J was very specific to PagC of Salmonella. It cannot react with PagC from Proteus species,Escherichia coli O1,Shigella sonnei.[Conclusions]The mAb identified in this research has great reaction activity and specificity,and the recognized epitope is located in conserve domain of PagC protein. This mAb is an excellent candidate for construction of blocking ELISA(bELISA) Salmonella antibody detection kit