关键词: 嗜水气单胞菌, 外膜蛋白TolC, 原核表达, 诱导条件, 多克隆抗血清, 抗原性

Abstract: The aim was to explore the immunogenicity and antigenicity of outer membrane protein TolC of fish pathogenic Aeromonas hydrophila(A.hydrophila),evaluate its immunological function,and provide a theoretical basis for the development of Aeromonas hydrophila subunit vaccine. Bioinformatics method was utilized to analyze the physicochemical properties of TolC protein and analyze its homology and evolutionary relationship among bacteria. The molecular cloning method was used to develop a recombinant strain expressing TolC protein, which was purified by inclusion body washing and SDSPAGE,and was used to immunize mice to prepare TolC antiserum.Western blot was used to detect the specificity and titer of the antiserum, and enzyme-linked immunosorbent assay (ELISA) was used to simulate the recognition effect between TolC antiserum and different aquatic pathogens to explore the antigenicity of TolC protein.The results showed that TolC was a porin protein,which had high conservative evolution among Aeromonas.The TolC protein was expressed and purified successfully,and the optimal expression condition for TolC protein was strain OD₆₀₀ of 1.0, final isopropyl-β-d-thiogalactoside(IPTG)concentration of 0.1 mmol/L,inducing temperature of 37℃ and inducing time of 8 hours.Western blot and ELISA analysis showed that the antiserum of TolC held high specificity and titer(1∶3 200),and the TolC antiserum could recognize with Aeromonas hydrophila, Pseudomonas fluorescens and Vibrio alginolyticus.In summary,the outer membrane protein TolC of A.hydrophila had high immunogenicity and antigenicity,and might provide cross immune protection for different aquatic pathogens.

Key words: Aeromonas hydrophila, Outer membrane protein TolC, Prokaryotic expression, Induction condition, Polyclonal antiserum, Antigenicity