(40). revealed that this infected common carp exhibited high resistance to this representative enteropathogenic bacterium upon reinfection, suggesting that IgM plays a key role in the defense mechanisms of the gut against bacterial invasion. Significantly, the second injection of induces strong local mucosal immunity in the gut, which is essential for protection against intestinal pathogens, providing affordable insights for vaccine preparation. Keywords: common carp, are important factors affecting the health and development of the current carp cultivation industry (28C30). is a very common bacteria in aquatic environments worldwide (31). This opportunistic zoonotic bacterium can cause hemorrhagic septicemia, abdominal dropsy, and skin ulceration, and is considered the most devastating pathogen of fish (28, 32). Previous studies have exhibited that induces intestinal inflammation in zebrafish and grass carp (33, 34). Moreover, the IgM expression in the intestine of rainbow trout was significantly increased after contamination with (35). However, the immune response and function of IgM against contamination in the common carp intestine remains largely unexplored. To fill the aforementioned knowledge gaps, here we prepared polyclonal antibodies against the common carp IgM. Afterward, we developed a model of common carp contamination with intraperitoneal injection. In this study, we found that contamination elicited a strong immune response and histopathologic changes Hydrocortisone 17-butyrate in the gut. Notably, bacterial infection induced increases in IgM+ B cells and recovered Hydrocortisone 17-butyrate faster from the pathological changes in their gut. Furthermore, I (Takara, Japan), and ligated to the pET-32a vector. Then the constructed plasmid was transformed into BL21 (DE3) (Vazyme, China), inoculated on Luria-Bertani (LB) solid medium, and cultured at 37C overnight. The Rabbit Polyclonal to MMP23 (Cleaved-Tyr79) expression and purification of recombinant IgM protein (rIgM) expression were performed by referring to the previous method of Wang et?al. with minor modifications (36). Positive clones around the plates were then selected and inoculated in a 500?ml LB liquid medium (with 100 g/ml ampicillin), then cultured at 37C with shaking at 220 rpm. When the medium reached approximately 0.6 at OD600nm, isopropyl–d-thiogalactopyranoside (IPTG; Roche, Switzerland) was added at a final concentration of 1 1 mM and incubated at 28C for 3.5?h. The rIgM was purified according to the manufacturers instructions by a nickel-nitrilotriacetic acid column (Ni-NTA; Qiagen, Germany). 400 g purified rIgM and Freunds Complete Adjuvant (FCA; Sigma, USA) were fully mixed in equal proportions and then used for injection immunization of Japanese white rabbits (2-3 months aged). The rabbits were booster-immunized with 150 g of purified rIgM mixed with Freunds Incomplete Adjuvant (FIA; Sigma, USA) four occasions. After immunization, blood was collected from rabbits, and IgG was purified from rabbit serum with protein A agarose (Thermo Fisher Scientific, USA). Then we purified the obtained rabbit IgG by affinity chromatography to obtain the anti-carp IgM antibody. Briefly, the affinity column was prepared by coupling rIgM to CNBr-activated Sepharose 4B according to the manufacturers instructions (GE Healthcare, USA). For isolation anti-carp IgM pAb, the purified rabbit IgG sample was applied to the column equilibrated in 1 phosphate buffered saline (PBS, pH 7.2). Then incubate in Hydrocortisone 17-butyrate the shaker for 2?h. After several washes of affinity column with PBS, bound IgM were eluted with 0.1 M glycine (pH 2.5), and immediately neutralized with 1 M Tris (pH 9.0). The neutralized antibody was displaced into PBS using a PD-10 Desalting Columns (GE Healthcare, USA) according to the manufacturers instructions and stored at -80C. The specificity of the polyclonal antibody against common carp IgM was detected by western blot and immunofluorescence. Fish maintenance The 10-15?g common carp used for this experiment were obtained from a fish farm in Chongqing, China, and placed in an aquarium containing a recirculating aquaculture system. Fish were fed with 153 commercial fish floating feed pellets (Tongwei Group, China) twice per day and acclimated at 28C for at least 2 weeks. The feeding was terminated 48?h before injection and sampling. Animal procedures were approved by the Animal Experiment Committee of Institute of Hydrobiology, Chinese Academy of Sciences and carried out according to the relative guidelines. strain and challenge test strain AH1 was obtained from the Laboratory of Aquatic Animal Medicine of the College of.