十字花科黑腐病菌XpsL與XpsE蛋白之表現,純化及抗體分析

Abstract

在十字花科黑腐病菌（Xanthomonas）中，XpsL 蛋白是被預測為一N端 點朝向胞內的胞膜蛋白，而其蛋白之角色則幾乎未有相關研究報告出現， 只有Sandkvist在1995所提出關於霍亂弧菌(Vibrio cholerae)其 EpsL 與 EpsE 蛋白角色之研究，認為Eps蛋是藉由EpsL蛋白的交互作用而附著於胞 膜上執行其功能。本實驗在大腸桿菌中大量表現 XpsE與 XpsL的融合蛋白 後以凝膠電泳純化，以用為抗原製造抗體，得到抗體後進行抗體效價之檢 測此外以次細胞分離法製備黑腐病菌的胞外漿、胞膜和細胞質蛋白，隨後 以鑑定過之抗體進行西方墨點法分析，初步結果顯示大部分之XpsL蛋白出 現在胞膜部份，而XpsE蛋白則在胞膜和細胞質有接近等量的分佈。

XpsL ,a protein member of extracellular protein secretion complex in Xanthomonas, is predicted to be a transplasmic membrane protein with its N-terminus facing inward. In 1995, Sandkvist have reported that EpsE, a cytoplasmic protein in Vibrio cholerae, is associated with and stabilized by the cytoplasmic membrane via interaction with EpsL. Furthermore, such interaction between EpsE and EpsL is required for protein translocation accross outer membrane. This report is first one revealing the role of XpsL prvealing the role of XpsL protein in the secretion complex. Nevertheless, the protein-protein interaction between XpsL and XpsE have never been examined by biochemical experiments. In this study, XpsL and XpsE fusion protein are overexpressed in E. cloi. and electrophoresis purified. Antibodies against XpsL and XpsE fusion proteins are raised and characterized. Perplasmic, membrane and cytosolic proteins of Xanthomonas are prepared by subcellular fractionaction and analysed by western blot. The preliminary results show that XpsL is mainly associated with membrane, and XpsE is evenly distributed between membrane and cytoplasm.