T7 phage libraries displaying random peptides are powerful tools for screening peptide sequences that bind to various target molecules. The T7 phage system has the advantage of less biased peptide distribution compared to the M13 phage system. However, the construction of T7 phage DNA is challenging due to its long 36 kb linear DNA. Furthermore, the diversity of the libraries depends strongly on the efficiency of commercially available packaging extracts. To address these issues, we examined the combination of seamless cloning with cell-free translation systems. Seamless cloning technologies have been widely used to construct short circular plasmid DNA, and several recent studies showed that cell-free translation can achieve more diverse phage packaging. In this study, we combined these techniques to construct four libraries (CX7C, CX9C, CX11C, and CX13C) with different random regions lengths. The libraries thus obtained all showed diversity > 109 plaque forming units (pfu). Evaluating our libraries with an anti-FLAG monoclonal antibody yielded the correct epitope sequence. The results indicate that our libraries are useful for screening peptide epitopes against antibodies. These findings suggest that our system can efficiently construct T7 phage libraries with greater diversity than previous systems.

中文翻译：

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无缝克隆与体外翻译相结合构建T7噬菌体随机肽库

显示随机肽的 T7 噬菌体文库是筛选与各种靶分子结合的肽序列的强大工具。与 M13 噬菌体系统相比，T7 噬菌体系统具有肽分布偏差较小的优点。然而，由于 T7 噬菌体 DNA 具有 36 kb 的长线性 DNA，其构建具有挑战性。此外，文库的多样性在很大程度上取决于市售包装提取物的效率。为了解决这些问题，我们研究了无缝克隆与无细胞翻译系统的结合。无缝克隆技术已广泛用于构建短环状质粒DNA，最近的几项研究表明无细胞翻译可以实现更加多样化的噬菌体包装。在本研究中，我们结合这些技术构建了四个具有不同随机区域长度的文库（CX7C、CX9C、CX11C 和 CX13C）。由此获得的文库均显示出多样性＞1。109 个噬菌斑形成单位 (pfu)。使用抗 FLAG 单克隆抗体评估我们的文库产生了正确的表位序列。结果表明我们的文库可用于筛选针对抗体的肽表位。这些发现表明我们的系统可以有效地构建比以前的系统具有更大多样性的 T7 噬菌体库。