PATHOGENESIS OF IMMUNODELICIENCIES IN MAN

人类免疫缺陷的发病机制

• 批准号: 3139247
• 负责人: LAWRENCE K JUNG
• 金额: $ 11.64万
• 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 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3139247
• 关键词: RNA splicing; 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.

我们最近的发现是，CD 7是缺乏的T细胞， 严重联合免疫缺陷病（SCID） 表明CD 7在T细胞的个体发育中起重要作用， 细胞 此外，对该患者的研究表明， CD 7可影响B细胞功能。 因此，CD 7起着关键的作用。 在淋巴发育中的作用以及在早期 淋巴细胞生成导致联合免疫缺陷。 在这 一个CD 7缺乏的SCID的独特例子，我们将有一个很好的 有机会研究的分子和遗传基础， 缺损 由于对这种分子结构知之甚少， 分子，我们将寻求产生CD 7的cDNA探针。 三 将使用的方法。 一种方法是分离CD 7 通过亲和色谱法制备糖蛋白， 获得的单克隆抗体。 纯化的蛋白质将是 进行氨基酸序列分析和合成寡核苷酸 从氨基酸序列推导的氨基酸序列将用于探测 从CD 7 + Jurkat的mRNA产生的λ gt 10文库 细胞系 其次，还将使用针对CD 7的多克隆抗血清 为了筛选lambda gt 11表达式库， 检测CD 7 cDNA克隆。 最后，共转染T细胞 DNA和胸苷激酶阳性（tk+）质粒进入tk-L-细胞 将提供另一种方法来识别和隔离 CD 7的cDNA。 当cDNA探针可用时， 然后将分析这种独特的SCID病例的遗传缺陷。 将通过限制性内切酶检测缺失/插入突变 酶片段多态性 点突变将是 通过序列分析检测。 为了研究 为了研究CD 7在T细胞个体发育中的功能作用， 寻找。 这是基于CD 7的鉴定 在小鼠中的同源物。 抗CD 7 mAb的可用性， CD 7的cDNA探针将提供一种手段， 可以获得CD 7的同源物。 mAB至CD 7， 将测试在仓鼠中制备的疫苗对鼠的交叉反应性。 同源物 这样的抗体将能够更好地表征 小鼠体内的CD 7 此外，它将用于 正常和裸鼠中CD 7+前体T细胞免疫选择。 然后可以分析这些细胞的体内功能作用。 还将尝试诱导免疫缺陷， 这些mAb针对鼠CD 7。 这一成功的结果 方法将提供进一步的机会，探讨的作用， CD 7在个体发育过程中T和B淋巴细胞发育中的作用