ISOLATION AND EXPRESSION OF COCCIDIOIDES T CELL ANTIGENS

球孢子菌 T 细胞抗原的分离和表达

基本信息

批准号：
6218779
负责人：
GARRY Thomas COLE
金额：
$ 12.13万
依托单位：
VETERANS MEDICAL RESEARCH FDN/SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-22 至 2000-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6218779
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

项目摘要

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 细胞介导的免疫被破坏的疾病表明在宿主防御中发挥着关键作用。无论是临床还是实验数据支持这一结论。这个的一个不寻常的特点真菌病是补体检测到的高滴度抗体固定，是一个不良的预后标志。因此，确定其中C. 免疫炎抗原刺激 T 细胞反应而不是抗体反应对于随后分离生物体大分子至关重要从而引起免疫保护。在拟议的研究中，我们使用了系统鉴定的新分子方法重组 T 细胞反应蛋白 (RTP)，对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) 腐生期到寄生期，2) 球粒的各向同性生长，以及 3) 内孢子形成。在此基础上，我们将构建三个相应的cDNA 具有从上述寄生期分离的mRNA的表达文库。将使用针对细胞产生的现有抗血清对文库进行筛选已证明是 T 细胞的壁细胞和全细胞制剂在免疫淋巴结增殖（ILNP）检测中呈反应性。反应性克隆分离并将每个的 C.immitis cDNA 连接到 pCMV 哺乳动物表达载体。 BALB/c小鼠皮下免疫 pCMV 加上表达 C.immitis 蛋白的 cDNA 插入片段。老鼠是通过 ELISA 监测粗制粗制 C. immitis 抗体的产生抗原，然后牺牲以评估其 T 细胞反应性 ILNP 测定。将反应性克隆的 cDNA 亚克隆到原核细胞中表达载体（例如pSE40），并通过免疫亲和纯化RTP 使用获得的相应特异性鼠抗血清进行色谱分析作为牺牲，如上所述。进一步测试 RTP 在小鼠中的反应性 ILNP 测定和 T 细胞系，患者淋巴细胞增殖化验。选定的cDNA用于筛选原始表达文库或 C.immitis 基因组文库，以分离全长基因。该基因的 cDNA 将用于 RTP 的表达，以进行进一步的研究。其反应性评价如上所述。最终，这种方法将产生多个 RTP，可用于评估小鼠的免疫保护作用对抗 C. immitis 挑战。免疫保护重组蛋白通过这种方法获得的是人类疫苗的合格候选者对抗球孢子菌病。