Recombinant protein and synthetic THY domain peptide of Macrobrachium rosenbergii thymosin β4 exhibit protective effect against Aeromonas hydrophila infection

Abstract

Thymosin β4 (Tβ4) is involved in cytokine secretion, nerve development, stimulation of wound healing, cancer progression, growth rate and immune response. The functions of Tβ4 in vertebrate have been widely studied, but the activities of each THY domain in invertebrate multirepeat β-thymosins remain unclear. In the present study, a new four THY domains Tβ4 gene was identified and characterized. The four THY domains have certain similarities in amino acid sequence, but MrThy4-D1 and MrThy4-D4 are predicted with α-helical structure, while MrThy4-D2 and MrThy4-D3 are random coils. The CD spectra results have confirmed the α-helical structure of synthetic MrThy4-D1 and MrThy4-D4 peptides. MrTβ4 was mostly expressed in hemocyte and hepatopancreas. The expression of MrTβ4 could be significantly up-regulated after Aeromonas hydrophila infections. The recombinant MrTβ4 protein was successfully produced and purified as the inclusion body. After refolding, both the recombinant MrTβ4 protein and the synthetic D1, D4 peptides exhibit certain antimicrobial activities to the selected Gram-negative and Gram-positive bacteria. The rMrTβ4 protein, synthetic MrThy4-D1 and MrThy4-D4 peptides all could increase the survival rate of M. rosenbergii against A. hydrophila infections, by improving the circulating hemocytes count, enhancing the antioxidant enzyme activities in hemolymph, or directly inhibition effect on bacteria. The present study not only enrich the understanding of the immune system of shrimp, but also lay an important foundation for the further development of MrTβ4 and antimicrobial peptide drugs.