| Southern catfish, Silurus meridionalis chen, is a valuable freshwater fish with important economic and food value in China. In past several years, extensive studies have been performed on the cloning of IgM in zebrafish, channel catfish, Mandarin fish, orange-spotted grouper, Atlantic salmon, European eel, rainbow trout and so on, especially the distribution of IgM-producing cells in the lymphoid tissues. However, there are no reports about the clonging of Ig heavy chain in any species of Siluridae. Knowledge of the Southern catfish Ig genetic structural organnizatin is of potential importance for the immunological control of disease in the catfish farms. In this study, the full-length cDNA of the Southern catfish immunoglobulin M heavy chain was cloned and the gene characterization was analyzed. The Southern catfish secreted IgM heavy chain was cloned using RT-PCR and RACE-PCR. The full-length cDNA sequence was 1980 bp-1990 bp, including 37-56 bp of 5’untranslated region,1716-1728 bp of the Southern catfish IgM heavy chain open reading frame,218 bp of 3’UTR (including the poly (A)+tail).A putative polyadenylation signal (AATAA) was identified 13 bp upstream of the polyA tail.In fact, We obtained 7 results of the full cDNA sequence of immunoglobulin M heavy chain. They were Sm-IgH-17, Sm-IgH-5, Sm-IgH-13, Sm-IgH-26, Sm-IgH-29, Sm-IgH-2-17, Sm-IgH-2-19, and Sm-IgH-2-32, respectively. Interestingly, Sm-IgH-5 and Sm-IgH-17 shareg an identical sequence in their full-length cDNA sequence, whereas Sm-IgH-13, Sm-IgH-26, Sm-IgH-29, Sm-IgH-2-17, Sm-IgH-2-19, and Sm-IgH-2-32 have distincactive sequences, sharing 93.5%and 99.6%nucleotides identity within the sequence corresponding to full-length cDNA.Sm-IgH-17 (1986 bp) was found to encode a 575 amino acid peptide, including open reading frame includes 1728 bp,5’UTR of 40 bp,3’UTR of 218 bp,144 amino acid of V region, and 431 amino acid of the four C domain. Sm-IgH-13 (1989 bp) was found to encode a 571 amino acid peptide, including open reading frame includes 1716 bp,5’UTR of 55 bp,3’UTR of 218 bp, including 140 amino acid of V region, and 431 amino acid of the four C domain. Sm-IgH-26 (1980 bp) was found to encode a 574 amino acid peptide, including open reading frame 1725 bp,5’UTR of 37 bp,3’UTR of 218 bp,143 amino acid of V region, and 431 amino acid of the four C domain. Sm-IgH-29 (1986 bp) was found to encode a 575 amino acid peptide, including open reading frame 1728 bp,5’UTR of 40 bp,3’UTR of 218 bp,144 amino acid of V region, and 431 amino acid of the four C domain. Sm-IgH-2-17 (1990 bp) was found to encode a 571 amino acid peptide, including open reading frame 1716 bp,5’UTR of 56 bp,3’UTR of 218 bp,140 amino acid of V region, and 431 amino acid of the four C domain. Sm-IgH-2-19 (1987 bp) was found to encode a 575 amino acid peptide, including open reading frame includes 1728bp,5’UTR of 41bp,3’UTR of 218 bp,144 amino acid of V region, and 431 amino acid of the four C domain. Sm-IgH-2-32 (1984 bp) was found to encode a 575 amino acid peptide, including open reading frame includes 1728 bp, 5’UTR of 38 bp,3’UTR of 218 bp,144 amino acid of V region, and 431 amino acid of the four C domain.The sequence was divided into leader peptide (L), CDRs, FRs, and CH domains on the basis of sequence comparisons with the H chain of other teleosts. Generally, the variable region of immunoglobulin M heavy chain of Southern catfish varied from 140 to 144 amino acid, and the constant region of that was 431 amino acid.The VH domain was shown to contain all of the canonical residues consereved in other teleosts and in most vertebrates.Comparisons of complementarity determing regions (CDRs) and frameworks (FRs) showed that FR2 and the last part of FR3 are the most conserved regions. The CDR3 was very heterogeneous and gave a major contribution to VH diversity. The CH domain was shown to contain CHI, CH2, CH3, CH4, and C-terminus.The FR1 domain was contained L—S—V—PG-S—L-C box. The CDR1 domain was contained SG-S box. The FR2 domain was contained WIRQ—GKGLEW box. The FR3 domain was contained S——F——D——Y——ED-AVYYCAR box. The FR4 domain was contained long block FDYWGKGT-VTV-S box.As can be seen in the alignment shown in residues were found to be conserved in the C region: ten cysteines and six tryptophan residues. The CH domain was shown to contain four continuous putative N-glycosylation sites. The CHI domain was not contained N-glycosylation sites. The CH2 domain was contained one N-glycosylation sites. The CH3 domain was contained one N-glycosylation sites. The CH4 domain was contained two N-glycosylation sites. The four N-glycosylation sites were NAS, NGT, NTT, and NLS respectively.The deduced amino acid sequences of the seven clones contained invariant cysteine and tryptophane residues characteristic for Ig domains, indicating that the seven Southern catfish sequences represent IgH chains. Tryptophane residues within the Ig domain loops were also found in the seven Southern catfish sequences at exactly the same positions as in the other teleost species.The primary structure, secondary structure and tertiary structure of IgM heavy chain was analysed and predicted. The relative molecular mass calculated from the amino acid composition is 63.8 KDa. Moreover, alignments of the deduced amino acid sequence of Southern catfish VH-CH domain of the secreted form was comparisoned with those of channel catfish, zebrafish, grass carp, orange-spotted grouper, and ladyfish. On the basis of the alignment, the Southern catfish VH-domain and CH-domain showed 53.7%-54.4%,39.8%-41.2%,40.5%-41.1%,32.2%-34.4%, and 35.2%～35.9%amino acid sequence identity with channel catfish, zebrafish, grass carp, orange-spotted grouper, and ladyfish.The entire amino acid sequence of the secreted form of Southern catfish VH-CH domain was assembled from Sm-IgH-17 and Sm-IgH-2-32, and aligned with VH-CH domain of other species using Clustal X software. The deduced amino acid sequence of the Southern catfish IgM heavy chain full-length cDNA shared similarities to that of the channel catfish with the identity of 53.7%～54.4%. A phylogenetic analysis was performed using IgH sequences of 29 species isolated from genomes and downloaded from GenBank. Subsequently, another phylogenetic analysis was performed using IgH sequences of 15 species in fish.Tissue distribution analysis by RT-PCR revealed that in adult Southern catfish, the abundance of IgM transcipts was found in the head-kidney (HK), kidney (K), spleen (S), blood cell (BL), stomach (ST), intestines (I), heart (H),skin (SK) and gill (G), whereas not detected in the liver (L), pancreas (P),gonads (O), brain (B) and mucle (M).RT-PCR analysis on ontogeny of IgH in Southern catfish indicated that mRNA didn’t expresse from unfertilization to 212hph after fertilization. In the present study, Ichthyophthirius multifilils treatment on tiny fish of Southern catfish (24 cm), IgH mRNA didn’t expresse from 0 hour to 12 day after infection. Ichthyophthirius multifilils treatment on tiny fish of Southern catfish(7-9 cm), IgH mRNA expresse from 0 hour to 218 hour in kidney, spleen, and intestine after infection. Interestingly, IgM mRNA expresse from 0 hour to 218 hour in control in kidney, spleen, and intestine. |
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