抗结核分枝杆菌RpfB结构域单克隆抗体的制备及鉴定

Production and Characterization of Monoclonal Antibody Against Mycobacterium tuberculosis RpfB Domain

目的:制备抗结核分枝杆菌Rpf B结构域单克隆抗体。方法:将p PRO-EXHT-Rpf B domain原核表达载体接种于大肠杆菌DH5中,用IPTG诱导表达Rpf B结构域蛋白,以纯化的Rpf B结构域蛋白作为免疫原,皮下包埋免疫小鼠3次,每次间隔2周;分离小鼠的脾细胞,与Sp2/0细胞融合,克隆化制备抗Rpf B结构域单抗,ELISA检测其效价,鉴定其特异性和相对亲和力,观察制备的抗Rpf B结构域单抗对Rpf家族其他蛋白的识别能力及其对结核分枝杆菌和藤黄微球菌的生长抑制作用。结果:制备了3株抗Rpf B结构域单抗,特异性高,亲和力较强,均能特异性识别Rpf B结构域。经小鼠腹腔注射制备腹水并纯化,获得了较高纯度的单抗,所制备的抗Rpf B结构域多肽的单克隆抗体可以识别多种Rpf样蛋白及其结构域蛋白。在抗体滴度为1∶1000时可有效抑制Rpf B结构域对结核分枝杆菌H37Ra和藤黄微球菌的生长促进作用,提示抗Rpf B结构域单抗可能会抑制进入机体内生长停滞或潜伏感染的结核分枝杆菌的再次激活,可能具有预防隐性感染复发的作用。结论:抗Rpf B结构域单抗的制备为进一步研究Rpf B结构域的生物学和免疫特性提供了实验工具。

Objective： To prepare monoclonal antibodies（mAb） against Mycobacterium tuberculosis（MTB） RpfB do？main. Methods： pPRO-EXHT-RpfB domain prokaryotic expression vector was transformed into E.coli DH5α strain and induced with IPTG, RpfB domain protein was purified under denaturing conditions. RpfB 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 mAb against RpfB domain. ELISA was used to examine the titer, specificity and relative affinity of the antibody. The ability of produced anti-RpfB mAb to recognize other proteins in Rpf family and to inhibit the growth of M.tuber？culosis and Micrococcus luteus was examined. Results： Our results showed that three anti-RpfB mAb were success？fully generated. All three mAb can recognize RpfB domain specifically and can effectively inhibit the promoting ef？fect of RpfB domain on the growth of MTB H37Ra strain and M.luteus at 1∶1000 dilution, indicating that anti-RpfB domain mAb may inhibit the reactivation of dormant or latent MTB in vivo. Therefore, they may be able to prevent the recurrence of the occult infection. Conclusion： The production of anti-RpfB domain mAb provides a powerful experimental tool to further study the biological and immunological characteristics of the RpfB domain and to evaluate the possibility of using RpfB domain as a candidate component for tuberculosis subunit vaccine.