A Monoclonal Antibody Toolkit for Functional Genomics of Plant Cell Walls

用于植物细胞壁功能基因组学的单克隆抗体工具包

• 批准号: 0421683
• 负责人: Michael Hahn
• 金额: $ 387.6万
• 依托单位: University of Georgia Research Foundation Inc
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0421683&HistoricalAwards=false
• 关键词: Monoclonal; Antibody; Toolkit; Functional; Genomics

A Monoclonal Antibody Toolkit for Functional Genomics of Plant Cell WallsProject Summary List of Senior PersonnelPI: Michael G. Hahn, CCRC and Department of Plant Biology, University of Georgia (UGA)Co-PI: Geert-Jan Boons, CCRC and Department of Chemistry, UGACo-PI: Christopher S. King, College of Veterinary Medicine, UGACo-PI: Malcolm A. O'Neill, CCRC, UGACo-PI: William S. York, CCRC and Department of Biochemistry and Molecular Biology, UGAIntroductionPlant cell walls are composed primarily of polysaccharides, many of whose structures are complex due to multiple sugar components and branching between the sugars. Available evidence suggests that over 2000 genes in a given plant may encode enzymes involved in cell wall synthesis and modification. To date, only a handful of these genes have been functionally identified and characterized. This project will provide a large, diverse, well-characterized library of monoclonal antibodies against plant cell wall polysaccharide structures as tools for the functional identification of genes that encode cell wall biosynthetic and modifying enzymes; genes for which there are limited alternative assays. The availability of diverse antibodies against plant cell wall polysaccharide structures will provide additional tools that are complementary to existing genetic and biochemical experimental approaches for studies of plant cell walls. Project Description and Expected OutcomesThe specific goals are: 1) To isolate monoclonal antibodies that recognize plant cell wall carbohydrate structures either from hybridoma cell lines or from phage display libraries. Mice will be immunized with diverse neoglycoproteins prepared using cell wall extracts or purified poly/oligosaccharides isolated from diverse plants, and hybridoma cell lines will be generated from splenic lymphocytes of the immunized animals. Resulting hybridoma cell lines, or alternatively, antibody phage display libraries, will be screened for production of antibodies reactive with plant carbohydrate structures. 2) To characterize in detail the carbohydrate structure (epitope) recognized by the cell wall-reactive antibodies. The antibodies obtained will be grouped by their abilities to bind to a set of purified wall polysaccharides from diverse plants. The epitope structure(s) recognized by selected antibodies in each reactivity group will be characterized in detail. The ~100 unique antibodies generated during the course of this project will be instrumental to those interested in the functional characterization of genes involved in the biosynthesis and modification of plant cell walls, and will also serve as valuable tools for chemical and biological investigations of plant cell wall structure and function. Outreach and TrainingAn explicit effort will be made to involve undergraduate students in the research carried out under this grant. The participation of historically underrepresented groups in the research will be fostered by recruiting undergraduate students through the UGA Summer Undergraduate Research Program (SURP) (www.gradsch.uga.edu/rr/) and through a newly established interaction between the Department of Plant Biology, the CCRC and Morehouse College in Atlanta that brings Morehouse undergraduates to the Department/Center for summer research projects. Access to Expected Outcomes Information about all antibodies generated through this project, including details about the polysaccharide structures recognized by each antibody, will be made available immediately to the scientific community via posting to an antibody database on the NSF-funded Plant Cell Wall Biosynthesis Research Network web site (http://xyloglucan.prl.msu.edu/). A link to this database will also be posted on the website of the Complex Carbohydrate Research Center (CCRC) (http://www.ccrc.uga.edu/). The antibodies will be made available to the research community without restriction through antibody stock centers established at the CCRC at UGA (Athens, GA) and the Centre for Plant Sciences at the University of Leeds (UK). Antibodies may be ordered from and will be distributed by CarboSource (http://www.ccrc.uga.edu/web/services/carbosource/CSS_home.html) and Plant Probes (http://www.plantprobes.co.uk/). The distribution of the antibodies will be self-supported by modest fees charged to those ordering the antibodies. The fee structure will be posted to the CarboSource and Plant Probes web sites. The hybridoma cell lines themselves will be replicated and stored in three physically separated facilities: 1) the CCRC and 2) the Monoclonal Antibody Facility of the College of Veterinary Medicine, both at UGA; and 3) with Dr. Paul Knox at the University of Leeds..

植物细胞壁功能基因组学单克隆抗体工具包项目负责人名单。哈恩，CCRC和植物生物学系，格鲁吉亚大学（UGA）共同PI：Geert-Jan Boons，CCRC和化学系，UGA共同PI：克里斯托弗S。King，兽医学院，UGACo-PI：Malcolm A.奥尼尔，CCRC，UGACo-PI：威廉S。约克，CCRC和生物化学和分子生物学系，UGA简介植物细胞壁主要由多糖组成，由于多种糖组分和糖之间的分支，许多多糖的结构是复杂的。 现有的证据表明，在给定的植物中，超过2000个基因可能编码参与细胞壁合成和修饰的酶。 迄今为止，只有少数这些基因已被功能鉴定和表征。 该项目将提供一个大的，多样的，充分表征的单克隆抗体库对植物细胞壁多糖结构作为工具的编码细胞壁生物合成和修饰酶的基因的功能鉴定;基因，有有限的替代测定。 针对植物细胞壁多糖结构的不同抗体的可用性将提供补充现有遗传和生物化学实验方法用于植物细胞壁研究的额外工具。 本研究的具体目标是：1）从杂交瘤细胞系或噬菌体展示文库中分离识别植物细胞壁碳水化合物结构的单克隆抗体。 小鼠将用不同的新糖蛋白免疫，所述新糖蛋白使用从不同植物分离的细胞壁提取物或纯化的聚/寡糖制备，并且杂交瘤细胞系将从免疫动物的脾淋巴细胞产生。 将筛选所得杂交瘤细胞系或抗体噬菌体展示文库以产生与植物碳水化合物结构反应的抗体。2)详细表征细胞壁反应性抗体识别的碳水化合物结构（表位）。 所获得的抗体将根据它们与来自不同植物的一组纯化的壁多糖结合的能力进行分组。 将详细表征每个反应基团中选定抗体识别的表位结构。 在该项目过程中产生的~100种独特抗体将有助于那些对参与植物细胞壁生物合成和修饰的基因的功能表征感兴趣的人，并且也将作为植物细胞壁结构和功能的化学和生物学研究的宝贵工具。 推广和培训将作出明确的努力，使本科生参与这项赠款下进行的研究。 历史上代表性不足的群体参与研究将通过UGA夏季本科生研究计划（SURP）（www.gradsch.uga.edu/rr/）招募本科生，并通过植物生物学系，CCRC和莫尔豪斯学院之间新建立的互动促进亚特兰大莫尔豪斯本科生到部门/中心进行夏季研究项目。 获得预期成果通过该项目产生的所有抗体的信息，包括每种抗体识别的多糖结构的详细信息，将通过张贴到NSF资助的植物细胞壁生物合成研究网络网站（http：//xyloglucan.prl.msu.edu/）上的抗体数据库立即提供给科学界。 该数据库的链接也将张贴在复杂碳水化合物研究中心（CCRC）的网站上（http：//www.ccrc.uga.edu/）。 抗体将通过在UGA（Athens，GA）的CCRC和利兹大学（UK）的植物科学中心建立的抗体储备中心无限制地提供给研究界。 抗体可从CarboSource（http：//www.ccrc.uga.edu/web/services/carbosource/CSS_home.html）和Plant Probes（http：//www.plantprobes.co.uk/）订购并由其分销。 抗体的分发将通过向订购抗体的人收取适度的费用来自我维持。 费用结构将公布在CarboSource和Plant Probes网站上。 杂交瘤细胞系本身将被复制并储存在三个物理上分开的设施中：1）CCRC和2）兽医学院的单克隆抗体设施，都在UGA;和3）与利兹大学的Paul Knox博士。