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CLONING OF THE CDNA ENCODING A CENTROSOME AUTOANTIGEN

编码中心体自身抗原的 CDNA 的克隆

基本信息

批准号：
2183928
负责人：
RONALD D BALCZON
金额：
$ 10.95万
依托单位：
UNIVERSITY OF SOUTH ALABAMA
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-08-01 至 1995-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (adapted from applicant's abstract): The centrosome, which is composed of a centriole pair and an amphorous cloud of pericentriolar material, is the principal microtubule organizing center in mammalian cells. It has been demonstrated that it is the pericentriolar material, and not the centrioles that contains the microtubule nucleating capacity within the centrosome complex. How the pericentriolar material is able to interact with tubulin and microtubules is unknown, due in part to the fact that the biochemical composition of the pericentriolar region remains largely unknown. In preliminary experiments, a human fetal liver cDNA expression library has been screened using a human autoimmune antiserum that recognized proteins within the pericentriolar region, and two clones have been identified that contained cDNAs encoding a portion of a 185/299 kd centrosome protein. Experiments are detailed that will allow for the structural and functional characterization of this high molecular weight centrosome protein. This will be achieved by cloning and sequencing the full-length cDNA encoding the centrosome protein and then using the cloned cDNA to produce a bacterial expression system. The expressed centrosome protein fragments then will be used to investigate centrosome-microtubule interactions using in vitro microtubule assays, to identify the major autoantigenic epitopes, and to generate monospecific anticentrosome antibodies. The monospecific antibodies, as well as the autoimmune anticentrosome antiserum, then will be microinjected into cultured cells to identify the in vivo function of the centrosome protein. Specific techniques to be used include molecular assembly assays, cell culture, microinjection, SDS-PAGE immunoblotting, and various forms of microscopy. Cell lines to be used include HeLa, CHO, and PtK2. Rabbits will be used for antibody production.

描述（改编自申请人的摘要）：中心体，由一对中心粒和一个中心粒周的两性云组成是哺乳动物微管的主要组织中心细胞已证明它是中心粒周围物质，而不是包含微管成核能力的中心粒在中心体复合体中。中心粒周围物质如何能够与微管蛋白和微管的相互作用是未知的，部分原因是中心粒周围区域的生化成分大部分未知。在初步实验中，用人自身免疫抗血清筛选表达文库识别中心粒周围区域的蛋白质，已经鉴定出含有编码185/299 kd中心体蛋白。详细的实验将允许这种高分子量结构和功能表征中心体蛋白这将通过克隆和测序来实现，编码中心体蛋白的全长cDNA，然后使用克隆的 cDNA以产生细菌表达系统。表达的中心体蛋白质片段将用于研究中心体微管使用体外微管测定的相互作用，以确定主要的自身抗原表位，并产生单特异性抗中心体抗体的单特异性抗体和自身免疫性抗体抗中心体抗血清，然后将其显微注射到培养的细胞中，鉴定中心体蛋白的体内功能。具体使用的技术包括分子组装测定，细胞培养，显微注射、SDS-PAGE免疫印迹和各种形式的显微镜检查。待使用的细胞系包括HeLa、CHO和PtK 2。将使用兔子用于抗体生产。