IDENTIFYING CANDIDA GENE IN THRUSH USING IVIAT

描述：(摘要逐字)申请者使用了一本活体小说 微生物表达技术称为体内诱导抗原技术 (IVIAT)研究口咽念珠菌病(OPC)的发病机制 感染艾滋病毒的患者。IVIAT使用血清中的抗白色念珠菌抗体 以确定在体内表达的抗原。 白色念珠菌，但在常规的体外生长过程中不表达。在预赛中 研究证实，IVIAT可以确定C。 白念珠菌。在这项提案中，他们建议修改IVIAT以识别抗原 白念珠菌在OPC期间表达，但在任何一种情况下均不表达 口腔粘膜白念珠菌的定植或在体外生长过程中的定植。他们 假设白念珠菌专属表达的某些抗原 在OPC过程中是重要的毒力因子，识别这些抗原将 洞察白念珠菌从白念珠菌转化为 无害的共生有机体转化为入侵的病原体。此应用程序具有 五个具体目标。在第一个具体目标中，两个单独的批次汇集在一起 血清，一个来自HIV感染的OPC患者，另一个来自HIV感染的患者 患者在体内定植后，会在体外被穷尽的吸附生长 临床分离白色念珠菌以清除所有与抗原反应的抗体 在体外表达。吸附后的血清将用于差异筛查 白念珠菌基因组表达文库。在特定目标2中，质粒DNA 将从与OPC血清反应但不与OPC血清反应的克隆中纯化 克隆血清和开放阅读框(ORF)负责 将测定血清反应性。在具体目标3中，他们将确认 白念珠菌在体外不表达IVIAT抗原。以特定的目标 4，他们将克隆编码IVIAT抗原的白色念珠菌基因，并纯化 由这些基因表达的蛋白质。最后，在具体目标5中，他们将确认 IVIAT抗原存在于从 HIV感染患者抗体的光镜免疫组织化学研究 用提纯的蛋白质来提纯。他们希望这项研究将确定 新的白念珠菌毒力因子，增加了我们对体液的了解 响应OPC，并导致潜在的药物、疫苗或 诊断测试开发。凭借这个项目的经验，IVIAT 应易于适应于研究其他念珠菌感染和其他真菌 病原体。IVIAT相对于现有技术的潜在优势包括 利用人类免疫反应鉴定体内表达的基因 比动物模型，它的相对技术简单，和它的研究能力 不同类型白念珠菌感染中基因表达的差异 人类。

DESCRIPTION: (abstract verbatim) The applicants have used a novel in vivo microbial expression technology called In Vivo Induced Antigen Technology (IVIAT) to study the pathogenesis of oropharyngeal candidiasis (OPC) in HIV-infected patients. IVIAT uses anti-Candida albicans antibodies in the sera of HIV-infected patients to identify antigens that are expressed in vivo by C. albicans but are not expressed during routine in vitro growth. In preliminary studies, they have confirmed that IVIAT can identify virulence factors for C. albicans. In this proposal, they propose to modify IVIAT to identify antigens expressed by C. albicans during OPC but not expressed during either colonization of the oral mucosa by C. albicans or during in vitro growth. They hypothesize that some of the antigens expressed by C. albicans exclusively during OPC are important virulence factors, and identifying these antigens will provide insight into the mechanisms by which C. albicans is transformed from a harmless commensal organism into an invasive pathogen. This application has five specific aims. In the first specific aim, two separate pooled batches of sera, one from HIV-infected patients with OPC and the other from HIV-infected patients with colonization, will be exhaustively adsorbed with in vitro grown clinical C. albicans isolates to remove all antibodies that react with antigens expressed in vitro. The adsorbed sera will be used for differential screening of a C. albicans genomic expression library. In specific aim 2, the plasmid DNA will be purified from clones that are reactive with the OPC sera but not with the colonization sera and the open reading frames (ORFs) responsible for the serum reactivity will be determined. In specific aim 3, they will confirm that the IVIAT antigens are not expressed by C. albicans in vitro. In specific aim 4, they will clone C. albicans genes encoding IVIAT antigens and purify the proteins expressed by these genes. Lastly, in specific aim 5, they will confirm that the IVIAT antigens are present within thrush samples recovered from HIV-infected patients by light microscopic immunohistochemistry using antibody raised against the purified proteins. They hope that this study will identify new C. albicans virulence factors, increase our understanding of the humoral response to OPC, and lead to potential applications for drug, vaccine or diagnostic test development. With the experience from this project, IVIAT should be readily adaptable to study other candidal infections and other fungal pathogens. Potential advantages of IVIAT over existing technologies include the use of the human immune response to identify in vivo expressed genes rather than animal models, its relative technical simplicity, and its ability to study differential gene expression in different types of C. albicans infections in humans.