Zhang Xuemei, Xiao Gary Guishan, Gao Ying
The Medical College of Dalian University, Dalian Economic & Technical Development Zone, Dalian 116622, China ; Department of Biochemistry and Molecular Biology, Dalian Medical University, 9 Western Section, Lvshun South Street, Lvshunkou District, Dalian 116044, China.
Departments of Medicine and Medical Microbiology, Creighton University, 601 N 30th Street, Omaha, NE 68131, USA.
ScientificWorldJournal. 2013 Nov 7;2013:716289. doi: 10.1155/2013/716289. eCollection 2013.
In this study, the peptide sized 21 kDa covering P-gp transmembrane region was first prepared for generating a novel mouse monoclonal antibody Fab fragment with biological activity against multiple drug resistance protein P-gp21 by phage display technology. Phage-displayed antibody library prepared from mice spleen tissues was selected against the recombinant protein P-gp21 with five rounds of panning. A number of clones expressing Fab bound to P-gp21, showing neutralized activity in vitro, were isolated and screened by enzyme-linked immunosorbent assay based on its recognition properties to P-gp21 and human colorectal cancer tissue homogenate, resulting in identification of an optimal recombinant Fab clone (Number 29). Further characterization by recloning number 29 into an expression vector showed significant induction of the Fab antibody in the clone number 29 by Isopropyl β-D-1-thiogalactopyranoside (IPTG). After purified by HiTrap Protein L, the specificity of the Fab antibody to P-gp21 was also confirmed. Not only was the targeted region of this monoclonal Fab antibody identified as a 16-peptide epitope (ALKDKKELEGSGKIAT) comprising residues 883-898 within the transmembrane (TM) domain of human P-gp, but also the binding ability with it was verified. The clinical implication of our results for development of personalized therapy of colorectal cancer will be further studied.
在本研究中，首先制备了覆盖P-糖蛋白跨膜区域的21 kDa大小的肽段，通过噬菌体展示技术产生一种针对多药耐药蛋白P-gp21具有生物活性的新型小鼠单克隆抗体Fab片段。从小鼠脾脏组织制备的噬菌体展示抗体文库针对重组蛋白P-gp21进行了五轮淘选。通过基于其对P-gp21和人结肠直肠癌组织匀浆的识别特性的酶联免疫吸附测定，分离并筛选出许多表达与P-gp21结合的Fab且在体外具有中和活性的克隆，从而鉴定出一个最佳的重组Fab克隆（编号29）。将编号29重新克隆到表达载体中进行进一步表征，结果显示异丙基β-D-1-硫代半乳糖苷（IPTG）可显著诱导克隆编号29中的Fab抗体表达。通过HiTrap Protein L纯化后，也证实了Fab抗体对P-gp21的特异性。不仅该单克隆Fab抗体的靶向区域被确定为包含人P-gp跨膜（TM）结构域内883 - 898位残基的16肽表位（ALKDKKELEGSGKIAT），而且还验证了其与之的结合能力。我们的结果对结直肠癌个性化治疗发展的临床意义将进一步研究。
