ISOLATION OF SFVS THAT REGULATE TRANSCRIPTION

描述：(改编自申请人的摘要)此第二阶段SBIR 应用程序讨论了进一步发展的理由和方法 智能试剂系统。第一阶段在酵母中建立了一种改进的双杂交系统 用于分离具有高亲和力的单链抗体(SFV) 体内的抗原。一个人源性SFV文库被克隆到酵母中 编码融合蛋白的表达载体，将SFV与 组成型反式激活结构域(VP16)。最初研究的目标是 为了开发这项技术并从这个展示了 适当的抗原结合特异性，并能够靶向抗原 在活体内。为了扩大这一新系统的实用性，第二阶段的目标是 主要有：1)构建带有Zeocin选择的最佳文库载体 促进SFV/VP16重组质粒的分离及人SFV亚克隆 库添加到新的库载体中。2)筛选新的SFV/VP16zeo 带有各种新诱饵(包括转录)的表达文库 因子、细胞内信号分子和核激素受体)。3) 为了使用人类开放阅读框架HORF激酶家族，已经 克隆和表达作为诱饵分离中和抗体。 建议的商业应用：不可用

DESCRIPTION: (Adapted from the applicant's abstract) This Phase II SBIR application discusses the rationale and approach for further development of the SMART reagent system. Phase I developed a modified two-hybrid system in yeast for isolating single-chain antibodies (sFv's) with high affinity for desired antigens in vivo. A library of human-derived sFv's was cloned into a yeast expression vector that encodes fusion proteins linking sFv's with a constitutive transactivation domain (VP16). The goal of the initial studies was to develop this technology and isolate sFv's from this library that exhibit the appropriate antigen binding specificity and are capable of targeting antigens in vivo. To expand the utility of this novel system, the aims for phase II are: 1) To construct an optimal library vector with zeocin selection to facilitate the isolation of the sFv/VP16 plasmids and to subclone a human sFv library into the new library vector. 2) To screen the new sFv/VP16zeo expression library with a variety of new baits (including transcription factors, intracellular signaling molecules and nuclear hormone receptors). 3) To use the family of human open reading frame HORF kinases that have been cloned and expressed as baits to isolate neutralizing antibodies. PROPOSED COMMERCIAL APPLICATION: Not available