Prokaryotic Expression And Immunofluoresecent Localization Of EvpC Of Edwardsiella Tarda

In this study,a strain of bacteria was isolated from the Pelteobagrus fulvidraco,named GD1609211.Based on physiological and biochemical characteristics and 16S rRNA gene sequence analysis,GD1609211 was identified as Edwardsiella tarda.The polyclonal antibody of EvpC protein was prepared by inducing the E.tarda-EvpC prokaryotic expression plasmid.Then,the expression of the EvpC gene and localization in artificial fish infected with E.tarda was detected by immunofluorescence using this antibody,in order to explore EvpC gene function.All the results in this paper will provide an important theoretical basis for understanding the pathogenesis of T6SS.The main results were as follows:1.The main symptoms of sick Pseudobagrus fulvidraco were anal swelling,liver bleeding and redness of the head.A strain was isolated and purified from Pseudobagrus fulvidraco liver,kidney,which was named GD1609211.Results of phenotypic,pHysio-biochemical and molecular characterization showed that GD1609211 was a Gram-negative short rod-shaped bacterium,which formed a gray-white bulge with smooth surface on nutrient agar.The sequence of 16S rRNA gene is 99%homologous with E.tarda,and its phylogenetic analysis showed that it is a cluster of E.tarda.The mainly physiological and biochemical charaacteristics of GD1609211 were producing acid,indole,hydrogen sulfide,and being able to grow on semi-solid agar were the mainly physiological and biochemical characteristics.The results of artificial infection experiment showed that GD1609211 is one of the most important pathogenic bacteria to the sick catfish,belonging to the highly pathogenic strain and the median lethal dose was 1.23×10~5CFU/mL.Drug sensitivity test showed that GD1609211 was sensitive to 12 antibiotics including norfloxacin,cefazolin,cepHalexin and cefoperazone and so on,and moderately sensitive to 6 antibiotics including midecamycin,kanamycin and amikacin and so on.The compound cotrimoxazole,gentamicin,erythromycin and cefradine and other 6 kinds of antibiotics were not sensitive.2.A pair of primer was designed according to the conservative domain of EvpC gene from E.tarda published on GenBank.EvpC was amplified from a strain of GD1609211 by Seamless Cloning PCR and successfully cloned into pET-28b expression vector.Comparison and analysis of the nucleotide and deduced amino sequences was performed with that reported sequence in GenBank(Accession No.AY424360).The results showed that thehomology of the nucleotide and amino acid sequences were 99%and 99%.3.The recombinant plasmid pET-28b-EvpC was double digested by NcoI and Xho I restriction endonucleases and transformed into competent cells of host strain BL21(DE3).The pET-28b-EvpC recombinant protein about 17.86 kDa was obtained by IPTG induction,consistent with the anticipate weight.Rabbits were immunized three times by purified recombinant protein and hyperinmmune serum was obtained.The Pelteobagrus fulvidraco was infected with the virulent bacteria of E.tarda.Then the spleen,liver,kidney,intestine,brain and gill were collected at different times after challenge.The indirect immunofluorescence method mediated EvpC protein antibody was used to detect the expression and loction of EvpC gene in different pHase of the testis.Results showed that the EvpC gene could encode proteins in the spleen,liver,intestine,brain and gill after infection with the virulent E.tarda.Four hours after infection,the protein was detected expression in the kidney,liver and spleen.A positive signal was detected in the intestine at 12 h,while a positive fluorescence of the protein was detected in the brain at 24 h.All the results show that the spleen,kidney,and brain are the major target organs of EvpC gene coding protein,and EvpC protein would selectivity infect and distribut in thses organs.