DEVELOPMENT OF A MONOVALENT PHAGE DISPLAY VECTOR

描述（改编自申请人的摘要）：该项目是 开发一种单价噬菌体展示载体，可用于研究 抗体-抗原、蛋白质-配体和蛋白质- 受体。 可以显示单个 融合蛋白将具有优于目前基于M13噬菌体的 向量。 新的载体可能最终导致一种 用于研究高和低亲和力抗体的体外系统。 该提案将解决现有M13融合中报告的弱点 展示载体，其中与基因III蛋白进行融合（5 拷贝/病毒颗粒）或基因VIII产物（2700拷贝/噬菌体 粒子）。 由于这些融合蛋白的多个拷贝， 具有低亲和力表位的噬菌体通常是分离的， 推测更罕见的高亲和力表位。 建议是使用 枯草芽孢杆菌噬菌体phi 29代替M13。 肽和蛋白质 将与gp 3蛋白进行融合，gp 3蛋白共价结合到 成熟噬菌体双链DNA的每个5'端，并且是噬菌体 复制的 将从噬菌体颗粒或细胞中分离DNA， 裂解物并用适当的限制性内切酶切割以除去 一端，但保留含有蛋白-gp 3融合蛋白的一端 和编码它的基因（在第二阶段形式中）。 在这个系统中， DNA具有连接的单价蛋白质探针，可用于 肽文库的构建和抗体-gp 3的产生 融合

DESCRIPTION (Adapted from applicant's abstract): The project is to develop a monovalent phage display vector that can be used for studying the interactions of antibody-antigen, protein-ligand, and protein- receptors. The availability of a vector that can display a single fusion protein will have an advantage over the current M13-phage based vectors. The new vector may ultimately lead to the development of an in vitro system for studying high and low affinity antibodies. The proposal will address a reported weakness in the existing Ml3 fusion display vectors, in which fusions are made with the gene III protein (5 copies per viral particle) or gene VIII product (2700 copies per phage particle). Because of the multiple copies of these fusion proteins, phage with low affinity epitopes are generally isolated, instead of the presumably more rare high affinity epitopes. The proposal is to use the Bacillus subtilis phage phi29 instead of M13. Peptide and protein fusions will be made with the gp3 protein, which is covalently bound to each 5' end of mature phage double-stranded DNA and is required for phage replication. The DNA will be isolated from phage particles or cell lysates and cut with an appropriate restriction endonuclease to remove one end, but retaining the end containing the protein-gp3 fusion protein and the gene encoding it (in the Phase II form). This system, in which DNA has an attached monovalent protein probe, can be used for construction of peptide libraries and for generation of antibody-gp3 fusions.