Gene Cloning and Testing

该计划项目的总体目标是开发一种针对克鲁兹旋毛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。在之前的赠款期间，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得很好，并且正在以高效率运作。项目1：基因注释和克隆的目标是保持基因鉴定和克隆的机器运行，继续微调这一过程，并克隆所有感兴趣的克氏锥虫基因。我们将依靠生物信息学核心和克氏锥虫基因组测序联盟(TcGSC)进行基因鉴定，并将使用这些信息 源自项目2中描述的蛋白质组学研究，用于识别和优先处理基因 克隆和测试。选择克隆基因的主要标准包括：1)在基因组中存在数量相对较少的等位基因或变异体，2)在哺乳动物宿主中存在的寄生虫期表达，以及3)在适当的间隔中作为免疫识别的有效靶标。被确定为感兴趣的基因将被传递到克隆管道，在该管道中，通过自动引物设计和初始和二次聚合酶链式反应来克隆假定的基因，以产生克隆到Gateway入口载体所需的适配区。然后，基因被转移到疫苗载体中，在小鼠身上进行测试。我们每年将至少克隆和测试1000个基因--我们预测，按照这个速度，我们可以覆盖克氏锥虫基因组中的大多数低拷贝基因。将在大约96个基因池中测试保护小鼠免受致命感染的能力，并将进一步分析保护池，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中描述的疫苗接种研究中的保护性基因鸡尾酒。

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.