Core B: FSH Glycoform Production, Purification, and Analysis

核心 B：FSH 糖型生产、纯化和分析

• 批准号: 10627090
• 负责人: JEFFREY V. MAY
• 金额: $ 32.34万
• 依托单位: WICHITA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-15 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10627090
• 关键词: Aging; Antibodies; Antigens; Area; Biochemical; Biological; Biological Assay; Capillary Electrophoresis; Cattle; Cell Differentiation process; Cell Line; Cell physiology; Cells; Chinese Hamster Ovary Cell; Complex; Conditioned Culture Media; Cryopreserved Cell; Detection; Development; Enzyme-Linked Immunosorbent Assay; Estradiol; Family suidae; Follicle Stimulating Hormone Receptor; Gene Expression; Genes; Glutamine; Harvest; Hormones; Human; Human Follicle Stimulating Hormone; Hybridomas; Immunologics; In Vitro; Individual; LH Receptors; Liquid substance; Measures; Membrane; Modeling; Monitor; Monoclonal Antibodies; Mus; Mutate; Oocytes; Ovarian; Ovarian Follicle; Pituitary Gland; Preparation; Procedures; Production; Progesterone; Proteins; Quality Control; Radioimmunoassay; Rattus; Reagent; Receptor Mediated Signal Transduction; Recombinants; Role; Serum; Source; Specimen; System; Systems Development; Testing; Testosterone; Transfection; Ultrafiltration; Urine; Validation; Variant; Western Blotting; Work; comparative; design; experimental study; granulosa cell; human model; immunoaffinity chromatography; improved; in vivo; manufacturing run; milligram; mouse model; mutant; programs; quality assurance; receptor; receptor binding; repository; screening; steroid hormone; tool; uptake; urinary; vector

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.

项目摘要/摘要： 核心B - FSH糖型的产生，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，以支持所有项目以实现 他们的具体目标。具体而言，核心B将继续产生重组HFSH糖型和突变HFSH 糖型。已经建立了生产毫克的糖型变体的程序 用人FSH A和B亚基基因，CGA和CGA转染的培养的GH3细胞的条件培养基 FSHB分别或突变的FSHB基因。核心B将继续开发翻译系统以改进 突变的HFSH糖型生产可为所有四个项目供应激素。这些HFSH变体模仿已知 重组降糖性HFSH糖基型的作用在卵巢细胞上。核心B将继续 与项目4（Bousfield）合作，生成和评估识别专门完整HFSH的抗体， HFSH A亚基以及单个糖型B亚基用于糖型分离和检测。核心b 将为项目2（戴维斯），项目3（乔纳斯）和项目4（Bousfield）提供各种细胞以评估 糖基受体结合，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。要提供的细胞包括KGN细胞，人类颗粒细胞系 这对HFSH刺激做出了反应；表达人FSH受体的CHO细胞；和原发性猪 颗粒细胞自然表达FSH受体以完成正在进行的实验。 GH3和HEK293 用HFSH和突变基因翻译的细胞对于重组HFSH糖基型的产生至关重要。 核心B将为项目提供分析支持：FSH RIA和ELISA监视HFSH级别 在FSH糖基型生产和各种生物学液中，包括血清和尿液；鼠标FSH ELISA 对于项目1（库马尔）； FSH和LH受体结合；孕酮ELISA监测HFSH变体对 目标细胞功能；用于单克隆抗体筛选的HFSH变体抗原ELISA。核心将获得并 利用猪积云卵母细胞复合物（PCOC）的研究进行了研究，以评估 COC膨胀和基因表达的HFSH糖型变体。