Molecular characterization, expression and functional analysis of TGFβ1-b in crucian carp (Carassius carassius)

Transforming growth factor β1 (TGFβ1) is a polyfunctional cytokine with important roles in growth, differentiation and immune function in various animals. In this study, PCR, bioinformatics, real-time quantitative PCR, prokaryotic expression, protein purification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF-MS) were applied to investigate the structural features and function of TGFβ1-b in crucian carp. The complete coding sequence (CDS) of TGFβ1-b was 1134 bp in length and was submitted to GenBank (ID: MH473141). TGFβ1-b encoded a putative protein of 377 amino acids and included a signal peptide consisting of 22 amino acids. TGFβ1-b was relatively conservative in fish and distant from mammals in terms of evolutionary relationship. TGFβ1-b was found to be expressed in various tissues, with the highest expression in the kidney. The expressions of TGFβ1-b in muscle, heart and liver were increased with the addition of Rhodopseudomonas palustris, Bacillus subtilis and Enterococcus faecium at 30 days (p < 0.01). While, the expressions of SMAD2, SMAD3 and SMAD7 were also up-regulated with the addition of R. palustris at 20 days (p < 0.01). The expression of TGFβ1-b could be affected by time and group factors (p < 0.05). Moreover, the expression vector TGFβ1-b-pDE2 was successfully constructed. Prokaryotic expression indicated that a 43 kDa target protein was obtained after induction with 1.5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for 3.5 h at 37 °C for 200 r/h. The activities of alkaline phosphatase and lysozyme in injection TGFβ1-b protein group (ITg) and feeding broken bacterial liquid group (BTg) were significantly increased at 24 h (p < 0.01). And the activities of superoxide dismutase in ITg were significantly increased at 36 h (p < 0.01). Besides, the expressions of heat shock protein 30 and heat shock protein 47 in ITg and BTg were significantly increased (p < 0.01). Whereas, the expression of interleukin-11 was significantly reduced (p < 0.01). These results indicated that TGFβ1-b protein might play a role in immunity of crucian carp.