DEVELOPMENT OF A MONOVALENT PHAGE DISPLAY VECTOR

描述(改编自申请者摘要)：该项目为 可用于研究的单价噬菌体展示载体的研制 抗体-抗原、蛋白质-配基和蛋白质- 感受器。向量的可用性可以显示单个 融合蛋白将比目前基于M13的噬菌体具有优势 向量。新的载体可能最终导致一种 用于研究高亲和力和低亲和力抗体的体外系统。 该提案将解决现有ml3核聚变系统中存在的一个弱点。 展示载体，其中与基因III蛋白(5)融合 每病毒颗粒拷贝数)或基因产品(每噬菌体2700拷贝 粒子)。因为这些融合蛋白有多个副本， 具有低亲和力表位的噬菌体通常被分离出来，而不是 大概是更稀有的高亲和力表位。该建议是使用 枯草芽孢杆菌噬菌体phi29代替M13。多肽和蛋白质 将与GP3蛋白进行融合，GP3蛋白与 成熟噬菌体的每个5‘端都是双链DNA，是噬菌体所必需的 复制。DNA将从噬菌体颗粒或细胞中分离出来 裂解产物，用适当的限制性内切酶切割以去除 一端，但保留含有蛋白-GP3融合蛋白的一端 和编码它的基因(以第二阶段形式)。这个系统，在其中 DNA带有附着的单价蛋白质探针，可用于 多肽文库的构建及其抗体GP3的制备 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.