全合成纳米抗体噬菌体展示文库的构建及鉴定
首发时间:2020-06-16
摘要:目的:基于M13噬菌体展示技术平台,构建全合成纳米抗体展示文库,用于开发多种具有潜在治疗、诊断等应用价值的纳米抗体。方法:通过对比天然纳米抗体文库大规模测序以及测序结果确定所需构建的全合成纳米抗体文库的骨架区和CDRs区的氨基酸数目及序列。采用NNK随机化方法,以纳米抗体骨架序列为模板,针对三个CDRs区设计简并引物,随机引入突变,通过重叠延伸PCR来获得VHH文库基因,将其构建到噬菌体pCantab 5E载体,经电转化至E.coil TG1构建初级噬菌体抗体库,经辅助噬菌体感染后构成噬菌体展示库,并对噬菌体展示库的库容及多样性进行分析和鉴定。结果:构建的全合成纳米抗体噬菌体展示文库的库容为1 × 107,插入率约为73.3%,多样性良好。结论:成功构建了全合成纳米抗体噬菌体文库,为制备具有治疗及诊断功能的纳米抗体奠定了物质与技术支撑。
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Construction and identification of fully synthetic nanobody phage display library
Abstract:Objective: On the basis of the M13 phage display technology platform, a fully synthetic nanoantibody display library was constructed in order to develop various nanoantibodies with potential therapeutic and diagnostic applications.Methods: The number and sequence of amino acids in the skeletal region and the CDRs region of the fully synthetic nanoantibody library were determined by comparing the large-scale sequencing of the natural nanoantibody library and the sequencing results. Nanobody skeleton sequence as a template for three CDRs area design degenerate primers, NNK randomized methods introduced random mutations, overlap extension PCR to obtain VHH gene library, to build it to phagocytosis pCantab 5E carrier, and then use the electricity conversion to convert carrier to E. coil TG1 to build primary phage antibody library, after helper phage to save the phage display library, finally analyze the diversity and capacity of phage display libraries and identification. Results: The total size of the phage display library is 1 × 107 colony-forming units (CFU). The insertion rate of the fully synthetic nanobody library was about 73.3%. The library has a high diversity.. Conclusion: the fully synthetic nanoantibody library was successfully constructed with good insertion rate and diversity, which provides material and technical support for the preparation of nanobodies with therapeutic and diagnostic functions.
Keywords: Nanobody Phage display Full synthetic library Construction Iditification
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