MOLECULAR CLONING OF A NOVEL BASEMENT MEMBRANE COMPONENT

候选人劳伦斯·S·陈是皮肤科助理教授 在西北大学医学院。毕业后的大学 1985年在宾夕法尼亚大学医学院，陈博士获得 他在密歇根大学医学院接受的皮肤科住院医师培训 学校在约翰·J·沃里斯博士的领导下。他还接受过训练 在导师的指导下，在同一家机构担任皮肤病免疫研究员 细胞免疫学家凯文·D·科珀博士和詹姆斯·特勒博士，一位 分子生物学家。候选人的职业兴趣是在以下方面 人体对皮肤基底膜带(BMZ)成分的自身免疫。 在住院期间，陈医生发现了一种新的免疫介导性水泡 以抗105 kDa BMZ自身抗体为特征的皮肤病 组件位于下层的光滑板。候选人的短期目标是 为了提纯这种新的BMZ成分并克隆编码它的人类基因 新组件。候选人的长期目标是理解 这个新的BMZ组件的功能：它与其他BMZ的关系 它对表皮细胞生物学的影响，它的抗原域， 以及其在诱导自身免疫反应中的病理生理作用。 未来五年的研究具体目标是净化这105- KD组分，为了分离编码该组分的人的cDNA， 并确定其功能域。为了实现这些具体目标， 成纤维细胞蛋白将经过多个色谱柱， 包括单Q阴离子交换柱和反相柱，用于 净化。纯化后的组分将用二维凝胶进行分析。 电泳法和免疫印迹法。内部氨基酸序列将 TH纯化蛋白经蛋白水解酶消化后得到。单克隆 针对105 kDa组分的抗体和多克隆抗体 针对氨基末端多肽产生的抗体将被用于筛选 人成纤维细胞c DNA表达文库。阳性斑块将是 进行克隆和DNA测序。一旦整个cdna 描绘出来的，将产生多个融合蛋白来检测 抗原域、细胞和细胞外附着域，以及 影响角质形成细胞生物学的领域。了解 这种新的光板成分的结构和功能将揭示 根据我们对皮肤BMZ复杂结构的理解， 皮肤BMZ不同成分之间的关系及其在血管紧张素转换酶中的作用 表皮-真皮粘连，人类水泡性疾病，妊娠 发展和伤口愈合。 西北大学为陈博士提供的研究环境 大学包括；一个蛋白质生物技术核心设施 微测序、肽和寡核苷酸合成、单抗 生产，双向凝胶电泳，流式细胞术，分子 生物用品和动物护理设施，都在同一项研究中 作为陈博士的实验室。皮肤科有 几名可以帮助Nageswararao博士的教职员工，另一名 合作者，是蛋白质生物化学家，伍德利博士，候选人的初选 赞助商，是一位蛋白质生物化学家，对上皮性很熟悉 细胞生物学。皮肤科有一个系图书馆 它包含了许多主要的科学期刊和最先进的快速 可促进陈博士蛋白质的蛋白质液相色谱系统 净化。陈博士已经被提供了一个设备齐全的660平方英尺 实验室空间，200平方英尺的办公空间，毗邻他的 实验室，一个培养室，和一个电子显微镜。此外， 应聘者有80%的投射时间用于研究。总而言之，这 环境为候选人取得成功提供了足够的支持 研究努力。

The candidate, Lawrence S. Chan, is an Assistant Professor of Dermatology at the Northwestern University Medical School. After graduating form the University of Pennsylvania School of Medicine in 1985, Dr. Chen obtained his Dermatology residency training at the University of Michigan Medical School under the leadership of Dr. John J. Voorhees. He was also trained as an Immunodermatology fellow at the same institution under the mentorship of Dr. Kevin D. Coper, a cellular immunologist, and Dr. James T Eler, a molecular biologist. The candidate's career interest is in the area of human autoimmunity against skin basement membrane zone (BMZ) components. During his residency, Dr. Chan discovered a new immunemediated blistering skin disease characterized by autoantibodies against a 105-kDa BMZ component at the lower lamina lucida. The candidate's short-term goals are to purify this new BMZ component and clone the human gene encoding for this new component. The candidate's long-term goals are to understand the functions of this new BMZ component: its relationship to other BMZ components, its influence on epidermal cell biology, its antigenic domains, and its pathophysiologic role in inducing autoimmune reaction. The research specific aims for the next five years, are to purify this 105- kD component, to isolate the human cDNA that encodes for this component, and to determine its functional domains. To achieve these specific aims, fibroblast protein will be subjected to multiple chromatographic columns, including Mono Q anion-exchange and reverse phase columns, for purification. The purified fractions will be analyzed by 2-dimensional gel electrophoresis and immunoblotting. The internal amino acid sequences will be obtained after proteolytic digestion of th purified protein. Monoclonal antibodies raised against the 105-kDa component and polyclonal antibodies raised against the amino-terminal peptide will be utilized to screen a human fibroblast cDNA expression library. Positive plaques will be subjected to cloning and DNA sequencing. Once the entire cDNA is delineated, multiple fusion proteins will be generated to examine the antigenic domains, the cellular and extracellular attachment domains, and the domains that influence keratinocyte biology. Understanding the structure and function of this new lamina lucida component will shed light on our understanding of the complex structure of the skin BMZ, the relationship between different components of skin BMZ, and their roles in epidermal-dermal adhesion, human blistering diseases, gestational development, and wound healing. The research environment provided for Dr. Chan at the northwestern University include; a Biotechnology Core Facility for protein microsequencing, peptide and oligonucleotide synthesis, monoclonal antibody production, two-dimensional gel electrophoresis, flow cytometry, molecular biology supplies, and an Animal Care Facility, all in the same research building as Dr. Chan's laboratory. The Department of Dermatology has several faculty members who can assist Dr. Nageswararao, another collaborator, is a protein biochemist, Dr. Woodley, the candidate's primary sponsor, is a protein biochemist and is well acquainted with epithelial cell biology. The Department of Dermatology has a departmental library which contains many major scientific journals and a state-of-the-art Fast Protein Liquid Chromatography system that can facilitate Dr. Chan's protein purification. Dr. Chan has been provided a fully equipped 660 square-feet laboratory space, a 200 square-feet office space adjacent to his laboratory, a culture room, and an electron microscope. In addition, the candidate has 80% projective time devoted to research. Together, this environment provides sufficient support for the candidate to succeed in his research effort.