CORE B - FSH Glycoform Production, Purification, and Analysis

项目摘要： 核心B：FSH糖型生产、纯化和分析 核心B提供关键和必要的试剂、分析和专业知识，以支持所有项目的实施 他们的具体目标。具体而言，核心B将继续产生重组hFSH糖型。程序 已经建立了从培养的条件培养基中产生毫克量的糖型 用人FSH α和FSH β亚基基因转染的GH3细胞。结合项目3，核心B将 产生突变hFSH糖型，其模拟已知的重组低糖基化hFSH糖型， 为所有三个项目提供服务。核心B将继续与项目1合作生产单克隆抗体 其特异性识别完整hFSH、hFSH α亚基以及单个糖型β亚基，用于 糖型分离和检测。核心B将提供各种电池，用于项目1和2，以评估 糖型受体结合和信号转导以及糖型对细胞分化的影响。细胞被 提供的细胞包括天然表达FSH受体的原代猪颗粒细胞、KGN细胞、 表达hFSH受体的人颗粒细胞系，表达人FSH受体的CHO细胞， 和用hFSH基因转染的GH3细胞，用于生产重组hFSH。核心B将 为项目1、2和3提供各种分析，包括FSH、LH和TSH RIA，FSH和LH受体 结合、E2受体和P4 RIA、尿FSH ELISA和单克隆抗体ELISA开发 筛选核心B将协助项目1分析重组hFSH糖型循环半衰期 给药至FSH3缺失小鼠后。核心B将协助项目1分析FSH糖型 因为它与月经周期和衰老的影响有关。此外，关系 糖型丰度和不育之间的关系将通过快速和 直接测定尿中hFSH糖型丰度的方法。目标是发展泌尿评估 作为临床诊断工具的hFSH糖型诊断。

Project Summary: Core B: FSH Glycoform Production, Purification and Analysis Core B provides critical and requisite reagents, analyses, and expertise to support all of the Projects in fulfilling their Specific Aims. Specifically, Core B will continue to produce recombinant hFSH glycoforms. Procedures have been established to produce milligram quantities of glycoforms from the conditioned medium of cultured GH3 cells transfected with the human FSH α and ß subunit genes. In conjunction with Project 3, Core B will generate mutant hFSH glycoforms, which mimic known recombinant hypoglycosylated hFSH glycoforms and supply them to all 3 Projects. Core B will continue to work with Project 1 to generate monoclonal antibodies that recognize specifically intact hFSH, hFSH α subunit, as well as individual glycoform ß subunits, for use in glycoform isolation and detection. Core B will provide a variety of cells for use in Projects 1 and 2 to assess glycoform receptor binding and signal transduction and glycoform impact upon cell differentiation. Cells to be furnished include primary porcine granulosa cells, which naturally express the FSH receptor, KGN cells, a human granulosa cell line that expresses the hFSH receptor, CHO cells that express the human FSH receptor, and GH3 cells transfected with hFSH genes for use in the production of recombinant hFSH. Core B will provide a variety of analyses for Projects 1, 2, and 3 including FSH, LH and TSH RIA, FSH and LH receptor binding, E2ß and P4 RIA, and ELISAs for urinary FSH and ELISA development for monoclonal antibody screening. Core B will assist Project 1 in the analysis of recombinant hFSH glycoform circulating half-life following administration to FSHß null mice. Core B will assist Project 1 in the analysis of FSH glycoform abundance change as it relates to the menstrual cycle and the impact of aging. In addition, the relationship between glycoform abundance and infertility will be studied through the continued development of rapid and direct methods to determine hFSH glycoform abundance in urine. The goal is to develop urinary assessment of hFSH glycoforms diagnostic as a clinical diagnostic tool.