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首页> 外文期刊>Comparative and functional genomics >Mimotopes and Proteome Analyses Using Human Genomic and cDNA Epitope Phage Display
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Mimotopes and Proteome Analyses Using Human Genomic and cDNA Epitope Phage Display

机译：使用人类基因组和cDNA表位噬菌体展示的拟态和蛋白质组分析

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In the post-genomic era, validation of candidate gene targets frequently requires proteinbasedstrategies. Phage display is a powerful tool to define protein-protein interactions bygenerating peptide binders against target antigens. Epitope phage display libraries have thepotential to enrich coding exon sequences from human genomic loci. We evaluated genomicand cDNA phage display strategies to identify genes in the 5q31 Interleukin gene clusterand to enrich cell surface receptor tyrosine kinase genes from a breast cancer cDNAlibrary. A genomic display library containing 2 ×106clones with exon-sized inserts wasselected with antibodies specific for human Interleukin-4 (IL-4) and Interleukin-13. Thelibrary was enriched significantly after two selection rounds and DNA sequencing revealedunique clones. One clone matched a cognate IL-4 epitope; however, the majority of cloneinsert sequences corresponded toE. coligenomic DNA. These bacterial sequences act as‘mimotopes’ (mimetic sequences of the true epitope), correspond to open reading frames,generate displayed peptides, and compete for binding during phage selection. The specificityof these mimotopes for IL-4 was confirmed by competition ELISA. OtherE. colimimotopes were generated using additional antibodies. Mimotopes for a receptor tyrosinekinase gene were also selected using a breast cancer SKBR-3 cDNA phage display library,screened against an anti-erbB2 monoclonal antibody. Identification of mimotopes ingenomic and cDNA phage libraries is essential for phage display-based protein validationassays and two-hybrid phage approaches that examine protein-protein interactions. Thepredominance ofE. colimimotopes suggests that theE. coligenome may be useful togenerate peptide diversity biased towards protein coding sequences.Abbreviations used: IL, interleukin; ELISA, enzyme linked immunoabsorbant assay;PBS, phospho-buffered saline; cfu, colony forming units.
机译：在后基因组时代，候选基因靶标的验证经常需要基于蛋白质的策略。噬菌体展示是一种强大的工具，可通过生成针对靶抗原的肽结合剂来定义蛋白质-蛋白质相互作用。表位噬菌体展示文库具有丰富来自人类基因组基因座的编码外显子序列的潜力。我们评估了基因组和cDNA噬菌体展示策略，以鉴定5q31白介素基因簇中的基因，并丰富乳腺癌cDNA库中的细胞表面受体酪氨酸激酶基因。用特异于人白细胞介素4（IL-4）和白细胞介素13的抗体选择含有2×106个克隆的外显子大小插入片段的基因组展示文库。经过两轮选择后，库显着丰富，DNA测序揭示出独特的克隆。一个克隆与相关的IL-4表位匹配;然而，大多数克隆插入序列对应于E。大肠菌基因组DNA。这些细菌序列充当“模拟表位”（真实表位的模拟序列），对应于开放阅读框，生成展示的肽，并在噬菌体选择过程中竞争结合。通过竞争ELISA证实了这些模拟表位对IL-4的特异性。其他使用其他抗体可产生大肠杆状抗原。还使用乳腺癌SKBR-3 cDNA噬菌体展示文库，针对抗erbB2单克隆抗体筛选了受体酪氨酸激酶基因的拟表位。鉴定拟态基因组和cDNA噬菌体库对于基于噬菌体展示的蛋白质验证测定和检查蛋白质-蛋白质相互作用的两杂交噬菌体方法至关重要。 E的优势。大肠杆菌拟表位提示。大肠菌群基因组可能有助于产生偏向于蛋白质编码序列的肽多样性。 ELISA，酶联免疫吸附测定; PBS，磷酸缓冲盐溶液; cfu，菌落形成单位。

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