Gene Cloning and Testing

该计划项目的总体目标是开发一种用于T. cruzi感染，并确定分发这种疫苗的最佳手段和方法。在上一个资助期间，重点放在了把基础设施放在一起，以测试大多数T。cruzi基因的疫苗效力。这一基础设施现已建立，并正在高效率地运作。项目1的目标：基因注释和克隆，是保持基因鉴定和克隆的机器运行，继续微调过程，克隆所有T。cruzi基因的兴趣。我们将依靠生物信息学核心和T。cruzi基因组测序联盟（TcGSC）的基因鉴定，也将使用的信息 源自项目2中描述的蛋白质组学研究，用于识别和优先排序基因， 克隆和测试。选择用于克隆的基因的主要标准包括：1）基因组中存在相对低数量的等位基因或变体，2）在哺乳动物宿主中存在的寄生虫阶段中表达，和3）存在于适当的区室中以充当免疫识别的有效靶标。识别为感兴趣的基因将被传递到克隆管道，其中通过自动化引物设计以及初始和二次PCR反应克隆推定基因，以产生克隆到Gateway进入载体中所需的衔接子位点。然后将基因转移到疫苗载体中，用于在小鼠中进行测试。我们每年将克隆和测试至少1000个基因，我们预测，以这个速度，我们可以覆盖T。cruzi基因组将在大约96个基因库中测试基因保护小鼠免受致命感染的能力，并将进一步解析保护性库，以最终确定提供最佳保护的单个基因。在这一过程中克隆的基因将用于生产和测试项目3所述疫苗接种研究中的基因保护性鸡尾酒。

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.