The Cloning and Expression Characteristics of A Full-length cDNA of MHC I from Snakehead Channa argus

doi:10.13560/j.cnki.biotech.bull.1985.2015.12.024

Abstract

Abstract:

By the methods of suppression subtractive hybridization(SSH)and rapid amplification of cDNA ends(RACE), 3 full-length sequences, Char-Ia-1, Char-Ia-2 and Char-Ib of major histocompatibility complex(MHC)I from snakehead(Channa argus)were cloned and characterized. The sequences of these clones were predicted to be in a high degree of homology with known teleost's MHC I. Char-Ia-1 and Char-Ia-2 contained an open reading frame(ORF)of 1 167 and 1 083 bp, and encoded a putative peptide of 388 and 360 amino acid respectively. However, the cDNA of Char-Ib contained an ORF of 978 bp encoding putative peptide of 325 amino acid, and a significantly shorter carboxyl terminal indicated that it was a molecule of secreting type I. Comparing Char-Ia and Char-Ib demonstrated significant difference in 3' untranslated region, and low homology of amino acid's sequences in extracellular domains, transmembrane and cytoplasmic regions. These suggested that Char-Ia and Char-Ib were encoded by two different loci. Alignment of amino acid sequence indicated that key antigenic peptide-anchoring residues binding with snakehead's MHC I were conserved, and there was amino acid deletion in α1 and α3 domains as the same characteristics of teleost. A phylogenetic analysis indicated that Char-Ia-1 and Char-Ia-2 branched together in the tree, and away from Char-Ib, this further demonstrated that they were from two different loci. RT-PCR analysis showed that snakehead MHC I gene was constitutively expressed in all detected tissues. Moreover, we designed specific primers to determine the expression level of Char-Ia and Char-Ib. The results illustrated that Char-Ia was ubiquitously expressed at low level, whereas the Char-Ib was predominantly expressed in spleen, intestine, gill and peripheral blood, suggesting the significant specificity of expression in tissue, which indicated that Char-Ia and Char-Ib played the different physiological functions in the immune responses of fish.

Key words: major histocompatibility complex, loci, expression characteristics, snakehead Channa argus

Wang Qiangqiang, Jia Weizhang, Huang Zebo. The Cloning and Expression Characteristics of A Full-length cDNA of MHC I from Snakehead Channa argus[J]. Biotechnology Bulletin, 2015, 31(12): 167-173.

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