Production and Characterization of Monoclonal Antibody Against Mycobacterium tuberculosis RpfB Domain
Production and Characterization of Monoclonal Antibody Against Mycobacterium tuberculosis RpfB Domain
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抗结核分枝杆菌 RpfB 结构域单克隆抗体的制备和表征
作者：
Peng, Daorong;Fan, Ailin;Xu, Zhikai;Bai, Yinlan;Ma, Yueyun;Wang, Limei;An, Qunxing;Jian, Wen;Shi, Changhong;Hao, Xiaoke
目的：制备抗结核分枝杆菌（MTB）Rpf B域的单克隆抗体（mAb）。研究方法：将pPRO-EXHT-Rpf B结构域原核表达载体转化大肠杆菌DH 5 α，经IPTG诱导表达，变性条件下纯化Rpf B结构域蛋白。本研究选用结核分枝杆菌Rpf B结构域蛋白，皮下免疫小鼠3次，间隔2周，分离小鼠脾细胞，与Sp2/0细胞融合。用抗Rpf B结构域的mAb筛选杂交菌落. ELISA检测抗体效价、特异性和相对亲和力。检测了抗Rpf B mAb识别Rpf家族其他蛋白的能力以及对结核分枝杆菌和藤黄微球菌生长的抑制作用。结果：成功制备了3株抗Rpf B mAb。3种mAb均能特异性识别Rpf B结构域，并能有效抑制Rpf B结构域对MT B H37 Ra株和黄体分枝杆菌生长的促进作用，表明抗Rpf B结构域mAb在体内可抑制休眠或潜伏的MT B的再激活。因此，他们可能能够预防隐性感染的复发。结论：抗Rpf B结构域单克隆抗体的制备为进一步研究Rpf B结构域的生物学和免疫学特性以及评价Rpf B结构域作为结核亚单位疫苗候选组分的可能性提供了有力的实验工具。
Objective: To prepare monoclonal antibodies(m Ab) against Mycobacterium tuberculosis(MTB) Rpf B do-main. Methods: p PRO-EXHT-Rpf B domain prokaryotic expression vector was transformed into E.coli DH5α strainand induced with IPTG, Rpf B domain protein was purified under denaturing conditions. Rpf B domain protein from M.tuberculosis was selected for this study, mice were subcutaneously immunized 3 times with 2 week interval,mice splenocytes were then isolated and fused with Sp2/0 cells. Hybridoma colonies were screened for m Abagainst Rpf B domain. ELISA was used to examine the titer, specificity and relative affinity of the antibody. Theability of produced anti-Rpf B m Ab to recognize other proteins in Rpf family and to inhibit the growth of M.tuberculosis and Micrococcus luteus was examined. Results: Our results showed that three anti-Rpf B m Ab were success-fully generated. All three m Ab can recognize Rpf B domain specifically and can effectively inhibit the promoting ef-fect of Rpf B domain on the growth of MTB H37 Ra strain and M.luteus at 1∶1000 dilution, indicating that anti-Rpf B domain m Ab may inhibit the reactivation of dormant or latent MTB in vivo. Therefore, they may be able toprevent the recurrence of the occult infection. Conclusion: The production of anti-Rpf B domain m Ab provides apowerful experimental tool to further study the biological and immunological characteristics of the Rpf B domainand to evaluate the possibility of using Rpf B domain as a candidate component for tuberculosis subunit vaccine.