Zebrafish Protein & Antibody Core

描述（由申请人提供）：斑马鱼蛋白质和抗体核心将采取第一个正式步骤，为生产斑马鱼蛋白质及其各自的抗体创建一个国家资源，供斑马鱼研究界使用。工作将集中在三个方面：1)生产斑马鱼蛋白质和蛋白质片段；2)抗体片段的产生；3)阵列蛋白和亲和试剂的制备。用于表达的斑马鱼蛋白包括转录因子、酶、细胞骨架蛋白、细胞信号蛋白、生长因子、受体外结构域等。只要有可能，就会表达蛋白质的全长版本，但由于与某些蛋白质类（例如膜蛋白，多结构域蛋白）一起工作的困难，它们的表达将仅限于可溶性结构域。表达将主要在大肠杆菌中实现，昆虫细胞系统用于高优先目标。在第一年，各种不同的蛋白质类别将成为目标，以便我们可以制定与广泛的蛋白质相关的协议；我们的目标是表达100种蛋白质，产生针对50个目标的抗体。在第二年，我们将把目标和抗体的生产规模提高50%，并分阶段生产由整个社区提名的蛋白质。预计在第三年，用户请求的数量将增加一倍，总共需要200种蛋白质和100种抗体。此外，我们将制造蛋白质和抗体阵列，以便在第三年分发。为了监督这个试点项目，将有一个由斑马鱼科学家组成的科学咨询委员会和一个由生物技术专家组成的技术咨询委员会。我们提出了一个共享计划，包括一个包含公开访问数据的网站，科学会议上的演讲，以及所要求的试剂和方案的分发。

DESCRIPTION (provided by applicant): The Zebrafish Protein & Antibody Core will take the first formal step in creating a national resource for the production of zebrafish proteins and their respective antibodies for use by the zebrafish research community. Effort will center on three efforts: 1) production of zebrafish proteins and protein fragments; 2) production of antibody fragments; and 3) the fabrication of arrayed proteins and affinity reagents. Zebrafish proteins targeted for expression would include transcription factors, enzymes, cytoskeletal proteins, cell signaling proteins, growth factors, ectodomains of receptors, etc. Wherever possible, full-length versions of the proteins would be expressed, but due to the difficulty of working with certain protein classes (e.g., membrane proteins, multi-domain proteins), their expression would be limited to soluble domains. Expression would be achieved primarily in E. coli, with insect cell systems used for high priority targets. For the first year, a variety of different protein classes would be targeted so that we may develop protocols relevant to a wide range of proteins; we would aim for the expression of 100 proteins, and the generation of antibodies to 50 targets. During the second year, we would ramp up the production scale of targets and antibodies by 50%, and phase in proteins nominated by the community at large. It is anticipated that the number of user requests would double during the third year, for a total of 200 proteins and 100 antibodies. In addition, we would fabricate arrays of proteins and antibodies for distribution during the third year. To oversee this pilot project, there will be a scientific advisory committee composed of zebrafish scientists, and a technical advisory committee composed of biotechnologists. We propose a sharing plan consisting of a website containing publicly accessible data, presentations at scientific meetings, and distribution of requested reagents and protocols.