Kim Sang-Hyun, Lee Sang-Rae, Kim Keun-Su, Ko Ara, Kim Ekyune, Kim Yong-Hwan, Chang Kyu-Tae
The National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Cheongwon, Korea.
FEMS Immunol Med Microbiol. 2010 Apr;58(3):412-20. doi: 10.1111/j.1574-695X.2010.00654.x. Epub 2010 Jan 22.
Shiga toxins (STx) are secreted extracellularly through the outer membrane vesicles (OMVs) of Escherichia coli O157:H7. In an attempt to produce STxA-deficient OMVs from E. coli O157:H7, site-specific deletions of the stx1A and stx2A subunit genes were carried out. The STxA-deficient phenotype of the stx1A/stx2A mutant was confirmed by Vero cell cytotoxicity and VTEC-RPLA assay. Western blot analyses showed that the B (STxB) subunits were present without coupling to STxA in the OMVs of the STxA-deficient mutant. Furthermore, STxB was located in its homo-pentameric complexes, as revealed by immunoprecipitation and immunoblotting with anti-STxB antibodies. These results suggest that STxB alone can be oligomerized into the B pentamer in the periplasm, and subsequently entrapped into the OMVs. Determination of the median lethal dose concentration for the OMV preparations suggests that the STxA-deficient OMVs containing STxB complex could be safely used as vaccine delivery vehicles.
志贺毒素（STx）通过大肠杆菌O157:H7的外膜囊泡（OMV）分泌到细胞外。为了从大肠杆菌O157:H7产生缺乏STxA的OMV，对stx1A和stx2A亚基基因进行了位点特异性缺失。通过Vero细胞细胞毒性和VTEC-RPLA测定证实了stx1A/stx2A突变体缺乏STxA的表型。蛋白质印迹分析表明，在缺乏STxA的突变体的OMV中，B（STxB）亚基存在且未与STxA偶联。此外，免疫沉淀和用抗STxB抗体进行的免疫印迹显示，STxB位于其同五聚体复合物中。这些结果表明，单独的STxB可以在周质中寡聚成B五聚体，随后被困在OMV中。对OMV制剂的半数致死剂量浓度的测定表明，含有STxB复合物的缺乏STxA的OMV可以安全地用作疫苗递送载体。
