Molecular cloning and expression analysis of IgMH and MHCⅡβ genes in the spotted sea bass (Lateolabrax maculatus)

To gain a deeper comprehension of the adaptive immune mechanism involved in disease prevention and control of Lateolabrax maculatus, this study employed RT-PCR and RACE techniques to clone the immunoglobulin M heavy chain (IgMH) and Major histocompatibility compatibility complex β (MHCII β). Additionally, real-time fluorescence quantitative PCR (qPCR) was utilized to examine the expression distribution in different tissues of spotted sea bass, along with the alterations in mRNA levels following LPS, Poly (I: C) stimulation, and Edwardia tarda infection. Finally, the expression of recombinant proteins IgMH, IgMCH_1-2, and MHCII β was achieved by constructing prokaryotic recombinant expression plasmids IgMH-pET21d, IgMCH_1-2-pET21d, and MHCII β-PET21d. The proteins were obtained through molecular sieve chromatography, and anti IgMH Polyclonal antibodies of L. maculatus were successfully prepared. The results indicated that the full length cDNA of IgMH and MHCⅡ β in L. maculatus were 1 977 bp and 1 242 bp, respectively. The genes IgMH and MHCII β exhibit high expression in immune-related tissues, including gills, spleen, and head kidney. LPS and Poly (I:C) stimulation and delayed Edwardsiella artificially infected L. maculatus led to significant changes in the expression levels of these two genes in gill, spleen and head and kidney, indicating that both IgMH and MHCII β participated in the anti-infection immune response of L. maculatus. Additionally, immunoblotting analysis demonstrated that anti-IgM Polyclonal antibodies display strong reactivity with the entire serum of L. maculatus, weak reactivity with Siniperca chuatsi, and no reactivity with Micropterus salmoides and Cienoyharyngodoni della. It is speculated that the reaction intensity reflects the genetic relationship between species. In summary, this study successfully accomplished the cloning of the complete sequences of IgMH and MHCII β genes, followed by the expression of recombinant IgMH and MHCII β proteins. Additionally, the production of Anti-IgM polyclonal antibody was achieved. The findings of this study provided evidence supporting the involvement of IgMH and MHCⅡ β in the immune response of L. maculatus. Furthermore, these results established a solid groundwork for future investigations on immune regulation and disease prevention and control strategies for L. maculatus.