| Summary: | 碩士 === 國立屏東科技大學 === 生物科技研究所 === 94 === Verotoxin（VT）is a major virulent factor of enterohemorrhagic E.coli（EHEC）leading to diarrhea and hemolytic uremic syndrome（HUS）in human. It can also cause diarrhea in animals and even death in some serious cases. VT is classified as VT-I and VT-II base on amino acid sequences. All VT-I and VT-II contain subunit A and B which have 55-57% homology in their amino acid sequences. The B subunit of VT-I binds to its host receptor and then this complex is engulfed by endocytosis leading to death of the cells. The purpose of this study is to identify the antigenic regions of subunit B of VT for development of subunit vaccines. The amino acid sequences with high antigenicity were predicted and amplified by PCR. The correct amplicons were cloned into pET32a vectors, transformed into competent cells, and expressed after induction with IPTG. BALB/c mouse were immunized with the expressed proteins purified with HiTrap affinity columns. Sera from immunized mice were used to probe the recombinant proteins in Western blots. However, results indicated sera reacted with all expressed proteins even negative controls. This suggested these antibodies were induced by fusion tags in pET32a vectors were used for expression of subunit B of VT. Results indicated sera from immunized mice specifically reacted with recombinant proteins. Therefore, epitopes of B subunit of VT were included in the recombinant proteins. Exotoxins of EHEC extracted by polymyxin B were added into Vero cells to calculate 50% cytotoxic dose based on cytopathic effects（CPE）. All sera from immunized mice were mixed with dosages of four CD50 of exotoxins to understand their neutralization titers. Results showed sera raised against VT-I-2, VT-II-1, and VT-II-2 proteins exhibited strong neutralization capabilities with titers up to 80 folds. These results offered useful information for further development of subunit vaccines.
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