Mollenkopf H J, Gentschev I, Bubert A, Schubert P, Goebel W
Theodor-Boveri-Institut für Biowissenschaften (Biozentrum), Lehrstuhl für Mikorbiologie, Universität Würzburg, Germany.
Appl Microbiol Biotechnol. 1996 Jun;45(5):629-37. doi: 10.1007/s002530050740.
The outer membrane protein, PagC, of Salmonella typhimurium was converted into a secreted protein by linking the 61-amino-acid long, C-terminal signal sequence of the E. coli hemolysin protein (HlyAs) to the mature PagC peptide. This PagC-HlyAs fusion protein was expressed and efficiently secreted into the culture supernatant by E. coli upon complementation with the hemolysin secretion proteins HlyB and HlyD. Polyclonal antibodies raised against this fusion protein not only recognized PagC in the membrane fraction of all salmonellae by Western blotting, but also reacted with proteins of smaller size in other gram-negative bacteria tested. A monoclonal antibody against the PagC-HlyAs fusion protein recognized only PagC in membrane fractions. The antibody-binding domain was determined using synthetic peptides derived from specific PagC domains. Sera from Salmonella-infected human patients and from a rabbit infected with S. typhimurium did not react with PagC in immunoblots, suggesting that PagC may not be recognized as a major antigen by the humoral immune system.
通过将大肠杆菌溶血素蛋白（HlyAs）61个氨基酸长的C端信号序列与成熟的PagC肽连接，鼠伤寒沙门氏菌的外膜蛋白PagC被转化为一种分泌蛋白。在与溶血素分泌蛋白HlyB和HlyD互补后，大肠杆菌表达了这种PagC-HlyAs融合蛋白并将其有效分泌到培养上清中。针对这种融合蛋白产生的多克隆抗体不仅通过蛋白质印迹法在所有沙门氏菌的膜组分中识别出PagC，还与所测试的其他革兰氏阴性菌中较小尺寸的蛋白质发生反应。一种针对PagC-HlyAs融合蛋白的单克隆抗体仅在膜组分中识别PagC。使用源自特定PagC结构域的合成肽确定了抗体结合结构域。来自感染沙门氏菌的人类患者以及感染鼠伤寒沙门氏菌的兔子的血清在免疫印迹中未与PagC发生反应，这表明PagC可能未被体液免疫系统识别为主要抗原。
