| Summary: | 碩士 === 國立屏東科技大學 === 獸醫學系 === 92 === Aeromonas hydrophila is pathogenic to soft-shell turtle. It could cause hemorrhagic lesions in gastric tract, liver, spleen and kidneys and body surface of the soft-shell turtle as well. A. hydrophila could be isolated from the infected soft-shell turtle. Since hemolysin ( hly ) gene is a distinguished exotoxin of A. hydrophila, we design a pair of specific primers for the amplification of hly gene by using polymerase chain reaction ( PCR ). The amplificon was 1866 bp to be used as an inserted DNA. After the inserted DNA fragment and vector, pGEX6P-1, were digested with Eco RI and Xho I respectively, and then T4 ligase was employed for the ligation and construction of the recombinant plasmid. The recombinant plasmid was transferred to DH5α E.coli for expression. The purified expressed recombinant fusion protein was obtained by using GST affinity column and was strongly reactive to the specific antibody arisen from rabbit by using Western blotting assay in terms of antigenicity. However, in vivo, the purified fusion protein was toxic to BALB/c mice. After intraperitoneal injection, the mice were suffering from congestion and edema in intestines showing intestinal goblet cells massive infiltration and necrosis in the epithelial cells of stomach, multifocal necrosis in liver. In the other hand, this recombinant protein capable of inducing lesions in mice indicates that it is a functional protein and it’s pathogenicity against mice was demonstrated. However, it could be potential used as a candidate antigen for the development of subunit vaccine against A. hydrophila if the recombinant fusion protein could be appropriately attenuated in the near future.
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