Abstract
The complement component 8α and 8β are glycoproteins that mediate formation of the membrane attack complex (MAC) on the surface of target cells. Full-length complement C8α (Rb-C8α) and C8β (Rb-C8β) sequences were identified from a cDNA library of rock bream (Oplegnathus fasciatus), and their genomic sequences were obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The Rb-C8α gene contains 64bp of 5′-UTR, open reading frame (ORF) of 1794bp, which encodes a polypeptide of 598 amino acids, 212bp of 3′-UTR. The Rb-C8β gene contains 5′-UTR of 27bp, open reading frame (ORF) of 1761bp, which encodes a polypeptide of 587 amino acids, 3′-UTR of 164bp. Rb-C8α consists of 11 exons interrupted by 10 introns and Rb-C8β consists of 12 exons interrupted by 11 introns. Sequence analysis revealed that both Rb-C8α and Rb-C8β contain thrombospondin type-1, a low-density lipoprotein receptor domain class A, membrane attack complex/perforin (MACPF) domain and epidermal growth factor like domain. The promoter regions of both genes contain important putative transcription factor binding sites including those for NF-κB, SP-1, C/EBP, AP-1, and OCT-1. Rb-C8α and Rb-C8β showed the highest amino acid identity of 62% and 83% to rainbow trout C8α and Japanese flounder C8β respectively. Quantitative real-time PCR analysis confirmed that Rb-C8α and Rb-C8β were constitutively expressed in all examined tissues, isolated from healthy rock bream, with highest expression occurring in liver. Pathogen challenge, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus led to up regulation of Rb-C8α and Rb-C8β in liver. Positive regulations upon bacterial and viral challenges, and high degree of evolutionary relationship to respective orthologues, confirmed that Rb-C8α and Rb-C8β important immune genes, likely involved in the complement system lytic pathway of rock bream.
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