PATHOGENESIS OF IMMUNODELICIENCIES IN MAN

人类免疫缺陷的发病机制

• 批准号: 3139249
• 负责人: LAWRENCE K JUNG
• 金额: $ 11.24万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-06-01 至 1991-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3139249
• 关键词: T lymphocyte; affinity chromatography; athymic mouse; biological polymorphism; cell differentiation; congenital blood disorder; disease /disorder model; gene mutation; genetic disorder; genetic library; genetic manipulation; genetic markers; genome; glycoproteins; hamsters; human population genetics; immunogenetics; laboratory mouse; laboratory rabbit; leukocyte activation /transformation; leukopoiesis; lymphoblast; molecular cloning; molecular pathology; monoclonal antibody; nucleic acid probes; point mutation; protein sequence; severe combined immunodeficiency; surface antigens; synthetic nucleic acid; transfection

Our recent finding that CD7 was absent from the T cells of a child with Severe Combined Immunodeficiency Disease (SCID) suggests that CD7 plays an important role in the ontogeny of T cells. Furthermore, studies in this patient have suggested that CD7 can influence B cell function. Therefore CD7 plays a critical role in lymphoid development and its absence during early lymphopoiesis leads to combined immunodeficiency. In this unique instance of SCID with CD7 deficiency, we will have a good opportunity to study the molecular and genetic basis of the defect. As little is known about the molecule structure of this molecule, we will seek to generate a cDNA probe for CD7. Three approaches will be used. One method is to isolate the CD7 glycoprotein by affinity chromatography produced with an available monoclonal antibody. The purified protein will be subject to amino sequence analysis and synthetic oligonucleotides deduced from the amino acid sequence will be used to probe a lambda gt10 library, produced from the mRNA of CD7+ Jurkat cell line. Secondly, polyclonal antisera to CD7 will also be used to screen a lambda gt11 expression library, as an alternative to detect for cDNA clone for CD7. Finally, co-transfection of T cell DNA and thymidine kinase positive (tk+) plasmid into tk- L-cells will provide another mean with which to identify and isolate cDNA for CD7. When a cDNA probe becomes available, the genetic defect in this unique case of SCID will then be analyzed. Deletion/insertional mutations will be detected by restriction enzyme fragment polymorphism. Point mutations will be detected by sequence analysis. In order to investigate the functional role of CD7 in T cell ontogeny, a murine model will be sought. This is predicated on the identification of a CD7 homologue in the mouse. The availability of mAb to CD7 and cDNA probes for CD7 will provide means with which a murine homologue for CD7 can be obtained. mAB to CD7 which are made in hamsters will be tested for cross reactivity to a murine homologue. Such an antibody will enable better characterization of CD7 in the mouse. Furthermore, it will be used to immunoselect of CD7+ precursor T cells in normal and nude mice. In vivo functional role of these cells can then be analyzed. Attempts will also be made to induce immunodeficiency with these mAb to murine CD7. The successful outcome of this approach will provide further opportunities to probe the role of CD7 in T & B lymphocyte development during ontogeny.

我们最近的研究发现，非霍奇金淋巴瘤患者的T细胞中缺乏CD7。 儿童重症联合免疫缺陷病(SCID) 提示CD7在T细胞个体发育中起重要作用 细胞。此外，对这名患者的研究表明 CD7可影响B细胞功能。因此，CD7扮演着至关重要的角色 淋巴发育中的作用及其在早期的缺失 淋巴生成导致联合免疫缺陷。在这 独特的CD7缺乏的SCID病例，我们会有一个很好的 有机会研究这一疾病的分子和遗传学基础 叛逃。因为人们对它的分子结构知之甚少 分子，我们将试图产生一个CD7的cDNA探针。三 将使用各种方法。一种方法是分离CD7 亲和层析法提取糖蛋白 可获得的单抗。纯化的蛋白质将是 进行氨基酸序列分析和合成寡核苷酸 根据氨基酸序列推导出的氨基酸序列将被用来探测 由CD7+Jurkat的mRNA产生的lambda gt10文库 细胞系。其次，还将使用CD7的多克隆抗血清 筛选lambda gt11表达文库，作为 检测CD7的cDNA克隆。最后，共转染T细胞 DNA和胸苷激酶阳性(tk+)质粒导入tk-L细胞 将提供另一种识别和隔离的手段 CD7的cDNA.当一个cdna探针可用时， 然后对这一独特的SCID病例的遗传缺陷进行分析。 将通过限制检测到缺失/插入突变 酶片段多态性。点突变将是 通过序列分析检测到。为了调查 CD7在T细胞个体发育中的功能作用 被追寻。这是基于对CD7的识别 老鼠体内的同源基因。抗CD7和CD7单抗的可获得性 CD7的cDNA探针将提供一种方法，使小鼠 可以获得CD7的同源物。MAb到CD7，分别是 用仓鼠制成的将被测试与一只小鼠的交叉反应 同源。这样的抗体将能够更好地表征 CD7在小鼠体内的表达。此外，它还将被用于 CD7+前体T细胞在正常和裸鼠体内的免疫选择性 然后可以分析这些细胞在体内的功能作用。 还将尝试通过以下方法诱导免疫缺陷 这些抗小鼠CD7的单抗。这一成功的结果是 方法将提供进一步的机会来探索 CD7在T&B淋巴细胞个体发育过程中的作用