Development of V.cholerae FLEX Protein Chip

Massachusetts General Hospital, Boston MA

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Abstract

[unreadable] 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 [unreadable] 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. [unreadable] [unreadable] [unreadable]