Development of a Novel Efficient Method To Construct an Adenovirus Library Displaying Random Peptides on the Fiber Knob
Development of a Novel Efficient Method To Construct an Adenovirus Library Displaying Random Peptides on the Fiber Knob
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DOI：
10.1021/mp4005854
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发表时间：
2014-03-01
影响因子：
4.9
通讯作者：
Aoki, Kazunori
中科院分区：
文献类型：
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作者：
Yamamoto, Yuki;Goto, Naoko;Aoki, Kazunori
通过改造衣壳蛋白编码区的腺病毒载体的重定向已经显示出有限的成功，因为适当的靶向配体通常是未知的。为了克服这一局限性，我们构建了一个腺病毒文库展示随机肽的纤维球，它的筛选导致成功的选择几个特定的靶向载体。在先前的文库构建方法中，在转染到生产细胞之前，通过Cre-lox介导的纤维修饰的质粒文库和酶消化的腺病毒DNA/末端蛋白复合物（DNA-TPC）之间的体外重组产生腺病毒基因组的全长。在该系统中，程序复杂且耗时，文库中约30%的载体是缺陷的，没有展示肽。这些可能会阻碍癌症靶向载体的进一步广泛探索。为了解决这些问题，在这项研究中，我们开发了一种新的方法，在表达Cre的293细胞中转染纤维修饰的质粒文库和无纤维腺病毒DNA-TPC。细胞内Cre重组和无纤维腺病毒的使用大大简化了文库制备步骤。无纤维腺病毒可用于抑制不必要的腺病毒载体的扩增。此外，在6孔培养皿中，文库的复杂度在一个孔中超过104水平，这是原始方法的10倍。结果表明，这种新的方法是有用的，在生产高质量的活腺病毒库，这将有助于开发靶向腺病毒载体在医学上的各种应用。
Redirection of adenovirus vectors by engineering the capsid-coding region has shown limited success because proper targeting ligands are generally unknown. To overcome this limitation, we constructed an adenovirus library displaying random peptides on the fiber knob, and its screening led to successful selections of several particular targeted vectors. In the previous library construction method, the full length of an adenoviral genome was generated by a Cre-lox mediated in vitro recombination between a fiber-modified plasmid library and the enzyme-digested adenoviral DNA/terminal protein complex (DNA-TPC) before transfection to the producer cells. In this system, the procedures were complicated and time-consuming, and approximately 30% of the vectors in the library Were defective with no displaying peptide. These may hinder further extensive exploration of cancer-targeting vectors. To resolve these problems, in this study, we developed a novel method with the transfection of a fiber-modified plasmid library and a fiberless adenoviral DNA-TPC in Cre-expressing 293 cells. The use of in-cell Cre recombination and fiberless adenovirus greatly simplified the library-making steps. The fiberless adenovirus was useful in suppressing the expansion of unnecessary adenovirus vectors. In addition, the complexity of the library was more than a 104 level in one well in a 6-well dish, which was 10-fold higher than that of the original method. The results demonstrated that this novel method is useful in producing a high quality live adenovirus library, which could facilitate the development of targeted adenovirus vectors for a variety of applications in medicine.