Department of Biology, Shahed University, Tehran, Iran.
Trans R Soc Trop Med Hyg. 2014 Feb;108(2):92-8. doi: 10.1093/trstmh/trt114.
Acinetobacter baumannii, an important nosocominal pathogen, causes various human infections like meningitis, bacteremia and pneumonia. The aim of this study was to produce nanobodies derived from camel heavy chain antibodies (HcAb) against a conserved region of the biofilm associated protein (Bap) of A. baumannii, by phage display technique.
A camel was immunized with the purified recombinant protein expressed from the conserved region of Bap and polyclonal antibody production was confirmed by ELISA. After RNA extraction from peripheral lymphocytes, cDNA was prepared and a phagemid library was constructed.
Phage particles encoding nanobodies were produced by infecting transformed cells with a M13K07 helper phage. A total of six panning rounds were performed to select high affinity clones. Screening of high affinity monoclonal nanobodies was performed using phage-ELISA. A clone with the highest absorption in monoclonal phage-ELISA was selected for soluble expression of the desired nanobody.
This is the first report on the expression and production of nanobodies against Bap. Increasing trends of drug resistance have shifted the focus to the role of antibodies in diagnosis and treatment of human diseases. Similarities of the produced VHH to human VH, makes the role of this nanobody promising in immunotherapy.
鲍曼不动杆菌是一种重要的医院获得性病原体，可引起脑膜炎、菌血症和肺炎等各种人类感染。本研究旨在通过噬菌体展示技术，从骆驼重链抗体（HcAb）中产生针对鲍曼不动杆菌生物膜相关蛋白（Bap）保守区域的纳米抗体。
用从 Bap 的保守区域表达的纯化重组蛋白对骆驼进行免疫，通过 ELISA 确认多克隆抗体的产生。从外周淋巴细胞中提取 RNA 后，制备 cDNA 并构建噬菌粒文库。
用 M13K07 辅助噬菌体感染转化细胞，产生编码纳米抗体的噬菌体颗粒。进行了六轮淘选，以筛选高亲和力的克隆。使用噬菌体-ELISA 筛选高亲和力的单克隆纳米抗体。在单克隆噬菌体-ELISA 中吸收最高的克隆被选择用于期望的纳米抗体的可溶性表达。
这是首次报道针对 Bap 的纳米抗体的表达和生产。耐药性的增加趋势使人们将注意力转向抗体在人类疾病诊断和治疗中的作用。所产生的 VHH 与人类 VH 的相似性，使得这种纳米抗体在免疫治疗中具有广阔的应用前景。
