ISOLATION AND EXPRESSION OF COCCIDIOIDES T CELL ANTIGENS

球孢子菌 T 细胞抗原的分离和表达

• 批准号: 6657468
• 负责人: GARRY Thomas COLE
• 金额: $ 15.71万
• 依托单位: VETERANS MEDICAL RESEARCH FDN/SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6657468
• 关键词: Coccidioides immitis; T lymphocyte; beta glucosidases; cellular immunity; coccidioidomycosis; disease /disorder model; epitope mapping; fungal antigens; fungal proteins; fungal vaccines; gene expression; glycoproteins; laboratory mouse; leukocyte activation /transformation; recombinant proteins; serine proteinases; urease; vaccine development

Coccidioidomycosis is a disease in which T-cell mediated immunity has been shown to play a critical role in host defense. Both clinical and experimental data support this conclusion. An unusual feature of this mycosis is that high titers of antibody, as detected by complement fixation, are a poor prognostic sign. Therefore, determination of which C. immitis antigens stimulate T-cell responses rather than antibody responses is essential for subsequent isolation of macromolecules of the organism that elicit immunoprotection. In the proposed research, we have used a novel molecular approach to the systematic identification of the recombinant T-cell reactive proteins (RTPs), an evaluation of the immunoprotective properties of these macromolecules in a murine model of coccidioidomycosis. Results of our earlier studies have indicated that potent T-cell reactive proteins are expressed during 1) transition of the saprobic to parasitic phase, 2) isotropic growth of spherules, and 3) endosporulation. On this basis, we will construct three corresponding cDNA expression libraries with mRNA isolated from the above parasitic phases. The libraries will be screened with existing antiserum raised against cell wall and whole cell preparations which have been shown to be T-cell reactive in immune lymph node proliferation (ILNP) assays. Reactive clones are isolated and the C. immitis cDNA of each is ligated into the pCMV mammalian expression vector. BALB/c mice are immunized subcutaneously with the pCMV plus cDNA insert which expresses the C. immitis protein. Mice are monitored by ELISA for production of the antibody against crude C. immitis antigen and then sacrificed for evaluation of their T-cell reactivity in ILNP assays. The cDNA of reactive clones is subcloned into a prokaryotic expression vector (e.g., pSE40), and the RTP is purified by immunoaffinity chromatography using the corresponding specific murine antiserum obtained as sacrifice, as above. The RTP is further tested for reactivity in murine ILNP assays and T-cell lines, an in patient lymphocyte proliferation assays. The selected cDNAs are used to screen the original expression library, or a C. immitis genomic library, to isolate the full-length gene. The cDNA from that gene will be used for expression o the RTP for further evaluation of its reactivity as above. Ultimately, this approach will yield multiple RTPs which can be evaluated for immunoprotection in mice against C. immitis challenge. Immunoprotective recombinant proteins obtained by this approach are qualified candidates for a human vaccine against coccidioidomycosis.

球孢子菌病是一种T细胞介导的免疫系统被破坏的疾病。 在宿主防御中起着关键作用。 临床和 实验数据支持这一结论。 这其中一个不寻常的特点是 真菌病是由补体检测到最高滴度抗体 固视是预后不良的标志。 因此，确定哪种C. 免疫性抗原刺激T细胞应答而不是抗体应答 是随后分离生物体大分子所必需的 引发免疫保护。 在拟议的研究中，我们使用了 一种新的分子方法来系统地鉴定 重组T细胞反应蛋白（RTPs）， 这些大分子在小鼠模型中的免疫保护特性 球孢子菌病 我们早期的研究结果表明， 有效的T细胞反应蛋白在以下过程中表达：1） 腐殖到寄生阶段，2）小球的各向同性生长，以及3） 内孢子形成 在此基础上，我们将构建三个相应的cDNA， 用从上述寄生阶段分离的mRNA构建表达文库。 将用针对细胞的现有抗血清筛选文库。 已被证明是T细胞的壁和全细胞制剂 在免疫淋巴结增殖（ILNP）测定中具有反应性。 反应性克隆 分离得到C.将每一种的猴cDNA连接到pCMV中， 哺乳动物表达载体。 BALB/c小鼠皮下免疫 表达C.免疫蛋白。 小鼠 通过ELISA监测针对粗C.丝虫 抗原，然后处死以评估它们在 ILNP测定。 将反应性克隆的cDNA亚克隆到原核生物中， 表达载体（例如，pSE 40），并通过免疫亲和纯化RTP 使用获得的相应的特异性鼠抗血清进行层析， 作为牺牲，如上所述。 在鼠中进一步测试RTP的反应性。 ILNP测定和T细胞系，患者淋巴细胞增殖 分析。 筛选出的cDNA用于筛选原始表达 图书馆或C。immitis基因组文库，分离全长基因。 来自该基因的cDNA将用于RTP的表达，用于进一步的研究。 如上所述评价其反应性。 最终，这种方法将 产生多个RTP，其可用于评价小鼠的免疫保护 针对C.免疫激发。 免疫保护性重组蛋白 通过这种方法获得的是人用疫苗的合格候选物 对抗球孢子菌病