| 英文摘要 |
At present, only a handful drugs to inhibit the infection of bacteria with New Delhi metallo- beta-lactamase 1 (NDM-1) gene are available in clinical application, especially those against gram negative Enterobacteriaceae such as E. coli and K. pneumonia. Thus, developing the rapid diagnostic agents and new drugs to offer more efficient therapeutic strategies are urgently demanded. In this study, we chemically synthesized the full-length NDM-1 gene (810 bps) and cloned into expression vector. After expression and purification, recombinant GST-NDM-1 protein (~56 kDa) was used to immunize chicken intramuscularly to produce polyclonal IgY antibodies. Purified IgY antibodies specifically recognized the GST-NDM-1 fusion protein immobilized on ELISA wells. In addition to GST-NDM-1, they also showed significant binding activities to individual GST and NDM-1 protein on Western blots. In order to generate specific anti-NDM-1 monoclonal antibodies, we used phage display technology to construct two scFv libraries with short or long linker, which contained 1.2 × 10^7 and 8.4 × 10^6 transformants, respectively. After 4 rounds of panning procedure, we selected 28 clones to express scFv monoclonal antibodies and analyzed their binding activity against GST-NDM-1 or GST-hENO 1 protein. Among these 28 selected clones, 4 clones including one with short linker (S7) and three with long linker (L6, L8, L13) recognized GST-NDM-1 protein alone but not GST-hENO-1 on ELISA and Western blot, suggesting these 4 scFv antibodies possess specific binding activity against NDM-1 protein. We believed that these specific polyclonal IgY antibodies and scFv monoclonal antibodies could be applied in the development of diagnostic agents to prevent and cure the deadly infection of bacteria with NDM-1 gene in the future. |
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