T CELL REPERTOIRES SPECIFIC FOR DEFINED VIRAL EIPTOPES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 以前，我们描述了我们在使用我们最初的策略从T7载体在E.杆菌去年，我们提出了一个计划，将趋化因子基因融合到葡萄球菌蛋白A的合成结构域中，称为“Z”。我们生产并测试了设计用于表达细胞内和分泌型Z-趋化因子融合的载体;尽管这两类载体都产生重组蛋白，但具有分泌信号的构建体不能分泌，因此我们选择放弃它们，并专门使用设计用于细胞内表达的载体。这些构建体的表达是以包涵体的形式进行的，我们现在正在开发蛋白A融合标签的折叠、纯化、切割和表征的条件

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Previously, we described unexpected difficulties we encountered in expression of chemokine genes using our initial strategies for expression from T7 vectors in E. coli. Last year, we outlined a plan for expression of the chemokine genes as fusions to a synthetic domain of staphylococcal protein A, termed "Z". We produced and tested vectors that were designed to express both intracellular and secreted Z-chemokine fusions; though both classes of vectors produced recombinant proteins, the constructs with the secretion signals failed to be secreted, so we have chosen to abandon them and to work exclusively with the vectors designed for intracellular expression. Expression from these constructs is in the form of inclusion bodies, and we are now developing conditions for folding, purification, cleavage of the protein A fusion tag, and characterization