Gene Cloning and Testing

该方案项目的总体目标是研制一种克氏锥虫感染的多组分疫苗，并确定分发这种疫苗的最佳手段和方法。在上一个赠款期间，重点放在建立基础设施，以测试大多数克氏锥虫基因的疫苗效力。这一基础设施现在已经建立起来，并在高效率地运作。项目1：基因注释和克隆的目标是保持基因识别和克隆机器的运行，继续微调过程并克隆所有感兴趣的T. cruzi基因。我们将依靠生物信息学核心和克氏T. cruzi基因组测序联盟（TcGSC）进行基因鉴定，并将使用这些信息

The overall goal of the Program Project is to develop a multicomponent vaccine for T. cruzi infection and define the optimal means and methods to distribute such a vaccine. During the previous grant period, a heavy emphasis was placed on putting together the infrastructure to test most T. cruzi genes for vaccine efficacy. That infrastructure is now well established and is functioning at high efficiency. The goals of Project 1: Gene Annotation and Cloning, is to keep the machine for gene identification and cloning running, to continue to fine-tune the process and to clone all T. cruzi genes of interest. We will rely on the Bioinformatic core and the T. cruzi Genome Sequencing Consortium (TcGSC) for gene identification and will also use the information derived from the proteomic studies described in Project 2 for identifying and prioritizing genes for cloning and testing. The primary criteria for selection of genes for cloning include: 1) presence in a relatively low number of alleles or variants in the genome, 2) expression in parasites stages that are present in the mammalian host, and 3) presence in the appropriate compartment to serve as effective targets for immune recognition. Genes identified as being of interest will be passed on to the Cloning Pipeline in which putative genes are cloned by means of automated primer design, and initial and secondary PCR reactions for the production of adapter sites required for cloning into Gateway entry vectors. Genes are then moved into vaccination vectors for testing in mice. We will clone and test a minimum of 1000 genes per year-and we predict that at this rate we can cover the majority of low copy genes in the T. cruzi genome. Genes will be tested in pools of approximately 96 for the ability to protect mice from lethal infection and protective pools will be further parsed to eventually determine the individual genes that provide the best protection. Genes cloned in this process will be used to produce and test protective cocktails of genes in the vaccination studies described in Project 3.