1 College of Marine Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2 Key Laboratory of Marine Biotechnology of Fujian Province, Fuzhou 350002, China
Abstract：TypeⅢsecretion system (T3SS) is ubiquitous transport machinery in Gram-negative bacteria and an important way for bacterial virulence factors to produce pathogenic effects. Ara-C type DNA binding protein ExsA is one of the main activators of T3SS and can regulate the expression of T3SS-related proteins. In this study, ExsA gene of Pseudomonas plecoglossicida PQLYC4 strain was cloned into the prokaryotic expression vector pET28a (+). The recombinant expression plasmid pET-28a-ExsA was transformed into competent cells of Escherichia coli BL21 (DE3) for induced expression. The results showed that the molecular mass of the expressed recombinant ExsA protein was slightly less than 30 kD, which was consistent with the expected molecular mass (29.7 kD). The purified recombinant ExsA protein was used as an antigen to immunize rabbits (Oryctolagus cuniculus) to prepare polyclonal antibodies, and the titer was above 1∶256 000. The prepared antibody were used to detect ExsA protein in P. plecoglossicida and the over expressed ExsA protein in the epithelioma papulosum cyprini (EPC) of carp (Cyprinus carpio) by Western blot and indirect immunofluorescence methods, the results showed specific reactions. This study provides a tool for further exploring the structure of ExsA protein and its function in regulating the virulence factor T3SS system of P. plecoglossicida.
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