MOLECULAR CLONING OF A NOVEL BASEMENT MEMBRANE COMPONENT

候选人劳伦斯·S. Chan是皮肤科助理教授， 在西北大学医学院。 毕业后， 1985年，陈博士获得宾夕法尼亚大学医学院 他在密歇根大学医学院接受的皮肤科住院医师培训 学校由John J. Voorhees博士领导。 他还接受过 作为同一机构的免疫皮肤病学研究员， 凯文·D博士细胞免疫学家Coper和细胞免疫学家James T Eler博士 分子生物学家 候选人的职业兴趣在以下领域 抗皮肤基底膜区（BMZ）成分的人自身免疫。 在住院期间，陈医生发现了一种新的免疫介导的水泡 以抗105 kDa BMZ自身抗体为特征的皮肤病 在较低的椎板的成分。 候选人的短期目标是 为了纯化这种新的BMZ成分并克隆编码这种成分的人类基因， 新组件。 候选人的长期目标是了解 这个新的BMZ组件的功能：它与其他BMZ的关系 成分，其对表皮细胞生物学的影响，其抗原结构域， 及其在诱导自身免疫反应中的病理生理作用。 未来五年的研究目标是净化这105- kD组分，以分离编码该组分的人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受多个层析柱， 包括Mono Q阴离子交换柱和反相柱， 洁净. 将通过二维凝胶色谱法分析纯化的级分。 电泳和免疫印迹。 内部氨基酸序列将 在蛋白水解消化纯化的蛋白质后获得。 单克隆 针对105-kDa组分产生的抗体和多克隆抗体 针对氨基末端肽产生的抗体将用于筛选 人成纤维细胞cDNA表达文库。 阳性斑块将 进行克隆和DNA测序。 一旦整个cDNA 描绘，将产生多个融合蛋白，以检查 抗原结构域、细胞和细胞外附着结构域，以及 影响角质形成细胞生物学的领域。 了解 这种新透明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，以及它们在 表皮-真皮粘连，人类水疱性疾病，妊娠 发育和伤口愈合。 西北大学为陈博士提供的研究环境 大学包括;蛋白质生物技术核心设施 微测序、肽和寡核苷酸合成、单克隆抗体 生产，双向凝胶电泳，流式细胞术，分子生物学 生物用品和动物护理设施，都在同一个研究中， 陈博士的实验室。 皮肤科有 几位可以帮助Nageswararao博士的教职员工，另一位 合作者，是一个蛋白质生物化学家，伍德利博士，候选人的主要 赞助商是一位蛋白质生物化学家，非常熟悉上皮细胞 细胞生物学 皮肤科有一个部门图书馆 其中包含许多主要的科学期刊和最先进的快速 蛋白质液相色谱系统，可以帮助陈博士的蛋白质 洁净. 陈医生已获提供一个设备齐全的660平方英尺 实验室空间，一个200平方英尺的办公空间毗邻他的 实验室、培养室和电子显微镜。 此外该 候选人有80%的时间用于研究。 在一起，这 环境提供了足够的支持，候选人成功地在他的 研究工作。

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.