Development of V.cholerae FLEX Protein Chip

描述（由申请人提供）：霍乱弧菌基因组序列的完成为应用高度平行的方法分析基因表达和人类对14种霍乱感染的免疫反应提供了机会。该项目的长期目标是确定对霍乱弧菌在人类感染期间表达的关键蛋白的免疫反应，这可能为霍乱弧菌的发病机制和改进疫苗的开发提供见解，包括潜在的无细胞霍乱疫苗。本建议有三个具体目标。在SPECIFIC AIM #1中，我们将构建一个包含约140个关键基因的霍乱弧菌FLEXgene库，每个基因克隆为一个完整的开放阅读框（从起始密码子到最后一个密码子），两侧是重组位点。每个基因都将被克隆到一个“主载体”中并进行序列确认。选择纳入储存库的基因将包括那些被认为与霍乱弧菌定植有关的蛋白质，产生毒性活性，调节毒力基因表达，趋化性和运动性，群体感应，关键外膜蛋白，霍乱弧菌II型分泌系统中的蛋白质，以及先前体内筛选检测到的基因。在SPECIFIC AIM #2中，我们将开发这些选定基因子集的霍乱弧菌柔性蛋白库。每个ORF将通过定向重组克隆移动到各种表达载体上，根据需要在编码蛋白上引入氨基和/或羧基末端亲和标签。我们将使用两种不同的方法来表达和净化FLEX存储库中尽可能多的orf：a)在大肠杆菌中表达单个蛋白质，并将纯化的蛋白质定位到微阵列上；b)利用哈佛蛋白质组学研究所开发的一种新方法，称为核酸可编程蛋白质阵列（NAPPA），在适当包被的微阵列上定位表达质粒本身，并利用体外转录/原位翻译，通过融合的亲和标签直接在该点捕获翻译的蛋白质。在SPECIFIC AIM #3中，我们将利用14种霍乱弧菌FLEXprotein芯片筛选孟加拉国霍乱弧菌感染患者恢复期血清中针对这些关键蛋白的免疫应答。此外，我们将收集霍乱病人的米水粪便

DESCRIPTION (provided by applicant): The completion of the Vibrio cholerae genome sequence has provided the opportunity to apply highly parallel methods to the analysis of gene expression and human immune responses to 14 cholerae infection. The long-range goal of this project is to identify immune responses to key proteins of V. cholerae expressed during human infection, that may provide insights on V. cholerae pathogenesis and the development of improved vaccines, including a potential acellular cholera vaccine. There are three SPECIFC AIMS in the present proposal. In SPECIFIC AIM #1, we will construct a V. cholerae FLEXgene repository of ca. 140 key genes, each cloned as a complete open reading frame (from the initiation codon to the last codon) and flanked by recombination sites. Each gene will be cloned into a "master vector" and sequence-confirmed. Genes selected for inclusion in the repository will include those proteins thought to be involved in colonization by V. cholerae, production of toxic activity, regulation of virulence gene expression, chemotaxis and motility, quorum sensing, key outer membrane proteins, proteins in the type II secretion system of V. cholerae, and genes detected by previous in vivo screens. In SPECIFIC AIM #2, we will develop a V. cholerae FLEXprotein repository of a subset of these selected genes. Each ORF will be moved by directional recombinational cloning into a variety of expression vectors, introducing amino- and/or carboxy-terminal affinity tags to the encoded proteins as needed. We will utilize two different approaches to express and purify as many of the ORFs in the FLEX repository as possible: a) expressing individual proteins in Escherichia coli, and spotting purified proteins onto a microarray, and b) utilizing a novel approach being developed at the Harvard Institute for Proteomics, termed Nucleic-Acid Programmable Protein Array (NAPPA), to spot the expression plasmids themselves on an appropriately coated microarray and utilize in vitro transcription/translation in situ, with capture of the translated protein directly at that spot via a fused affinity tag. In SPECIFIC AIM #3, we will utilize the 14 cholerae FLEXprotein microarray to screen for immune responses against these key proteins in convalescent sera from patients infected with V. cholerae in Bangladesh. In addition, we will collect rice-water stool from patients with cholera in Bangladesh directly into formalin, to preserve proteins expressed in vivo by V. cholerae in the stool. For comparison, V. cholerae will be grown in vitro and also mixed with formalin. Rabbits will be immunized with protein preparations from these two groups of organisms and the rabbit sera will then be used to detect proteins on the microarray that react with sera from rabbits immunized with 14 cholerae recovered directly from stool but not after in vitro growth, suggesting that those specific proteins are expressed in vivo during human infection.