Core B: FSH Glycoform Production, Purification, and Analysis

项目摘要/摘要： 核心B-卵泡刺激素糖仿的生产、纯化及分析 核心B提供关键和必要的试剂、分析和专业知识，以支持所有项目实现 他们的具体目标。具体地说，Core B将继续生产重组hFSH糖形式和突变体hFSH 糖类。已经建立了程序来生产毫克量的糖形式变体 人FSHa、b亚单位基因CgA、CgA、CgA FSHB或突变的FSHB基因。核心B将继续开发转基因系统，以改进 突变的hFSH糖形生产，为所有四个项目提供激素。这些hFSH变体模仿已知的 重组低糖基化hFSH糖体对卵巢细胞的作用。核心B将继续 与项目4(Bousfield)合作，产生和评估识别特定完整的hFSH的抗体， HFSH a亚基以及单个糖形b亚基，用于糖形分离和检测。核心B 将提供各种单元用于项目2(Davis)、项目3(Jonas)和项目4(Bousfield)以评估 糖型受体结合、FSH受体寡聚、FSH受体介导的信号转导和 糖型对细胞分化的影响。将提供的细胞包括KGN细胞，一种人颗粒细胞系 对hFSH刺激有反应的CHO细胞；表达人FSH受体的CHO细胞；以及原代猪 自然表达FSH受体的颗粒细胞完成正在进行的实验。GH3和HEK293 转导hFSH和突变基因的细胞对重组hFSH糖体的产生至关重要。 核心B将为项目提供分析支持，包括：FSH RIA和ELISA法，以监测hFSH水平 在卵泡刺激素糖形形成过程中以及在包括血清和尿液在内的各种生物液中；小鼠卵泡刺激素酶联免疫吸附试验 对于项目1(Kumar)；FSH和LH受体结合；孕酮ELISA法监测hFSH变异对 靶细胞功能；hFSH变异体抗原ELISA用于单抗筛选。核心B将获得和 开展利用猪卵丘卵母细胞复合体(PCOC)评估潜在差异活性的研究 HFSH糖型变体对COC扩展和基因表达的影响。

Project Summary/Abstract: 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 and mutant hFSH glycoforms. Procedures have been established to produce milligram quantities of glycoform variants from the conditioned medium of cultured GH3 cells transfected with the human FSH a and b subunit genes, CGA and FSHB, respectively, or mutant FSHB genes. Core B will continue to develop transfection systems to improve mutant hFSH glycoform production to supply hormone to all four Projects. These hFSH variants mimic known recombinant hypoglycosylated hFSH glycoforms in terms of their actions upon ovarian cells. Core B will continue to work with Project 4(Bousfield), to generate and assess antibodies that recognize specifically intact hFSH, hFSH a subunit, as well as individual glycoform b subunits, for use in glycoform isolation and detection. Core B will provide a variety of cells for use in Project 2(Davis), Project 3(Jonas), and Project 4(Bousfield) to assess glycoform receptor binding, FSH receptor oligomerization, FSH receptor mediated signal transduction and glycoform impact upon cell differentiation. Cells to be furnished include KGN cells, a human granulosa cell line that responds to hFSH stimulation; CHO cells that express the human FSH receptor; and primary porcine granulosa cells which naturally express the FSH receptor to complete on-going experiments. GH3 and HEK293 cells transfected with hFSH and mutated genes are critical for the production of recombinant hFSH glycoforms. Core B will provide analysis support for the Projects including: FSH RIA and ELISA to monitor hFSH levels during FSH glycoform production and in various biological fluids including serum and urine; mouse FSH ELISA for Project 1(Kumar); FSH and LH receptor binding; progesterone ELISA to monitor hFSH variant impact upon target cell function; and hFSH variant antigen ELISA for monoclonal antibody screening. Core B will obtain and develop studies utilizing porcine cumulus oocyte complexes (pCOCs) to assess potential differential activities of hFSH glycoform variants upon COC expansion and gene expression.