Cloning and expression analysis of immunoglobulin M (IgM) from humphead snapper Lutjanus sanguineus

• 1、School of life sciences, Nantong university,Nantong 226019
• 2、Zhongkai University of Agriculture and Engineering
• 3、Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals

Abstract：In the present study, full-length cDNA sequences of immunoglobulin M (IgM) was cloned by rapid amplification of cDNA ends technique (RACE) from humphead snapper (Lutjanus sanguineus). Full-length cDNA sequence of IgM is 2045 bp, encoding 595 amino acids. BLAST analysis revealed that the IgM amino acids sequence of humphead snapper shared high identity (80%) with that of others. Phylogenetic tree was constructed by the Neighbor-Joining method, and the results suggested that IgM of humphead sanpper shared the closest genetic relationship with the IgM of Larimichthys crocea. The results of fluorescent real-time quantitative RT-PCR showed that the expression of IgM mRNA could be detected in head kidney, and increased continuously as time goes on in 48 h post infection. In addition, IgM was subcloned into pET28a(+) to construct expression plasmids pET28a-IgM. SDS-PAGE and western blot analysis indicated that the recombinant proteins were successfully expressed in Escherichia coli BL21 (DE3). Then the recombinant proteins were purified and the antiserum was obtained by immuning rabbits with the purified recombinant proteins emulsified with adjuvant. ELISA analysis showed that the titer of the antiserum prepared in this study was 1 : 40000. The results of the western blot revealed that specific antigen-antibody reaction was occurred between the antiserum and the recombinant proteins.

Keywords： Humphead Snapper；IgM； RACE； Real-time RT-PCR；Prokaryotic Expression