Molecular Cloning And Characterization Of MyD88 In Common Carp, Cyprinus Carpio L.

The innate inunune system is the first line of the defensive mechanisms that protect hosts from invading microbial pathogens.Host cells express various patterm recognition receptors(PRRs) that sense diverse pathogen-associated molecular patterns(PAMPs), ranging from lipids, lipoproteins, proteins and nucleic acids.Recognition of PAMPs by PRRs activates intracellular signaling pathways that culminate in the induction of inflammatory cytokines, chemokines,interferons(IFNs). Toll-like receptors (TLRs) are one of the impertant PRRs families which recognit PAMPs.TLRs have a central role in innate immunity and are also required for the development of an adaptive immune response, bridging innate and adaptive immunity. As one of the interleukin-1 receptor/toll-like receptor (IL-1R/TLR) superfamily, myeloid differentiation factor 88 (MyD88) ) is an important adapter protein in the signal transduction pathway mediated by Toll-like receptors except TLR3. Infection ultimately increases the abundance of the mRNA molecules encoding acute-phase proteins(APP).The APPs represent a group of serum proteins produced and released predominantly by hepatocytes,primarily protecting the host from an overwhelming inflammatory response.Serum amyloid A is a major APP reported to exert a number of immune functions.Common carp (Cyprinus carpio L.) was bought from the mart. We have isolated, sequenced and characterized the full-length cDNA and gene of MyD88 from common carp by the method of molecular cloning and rapid-amplification of cDNA ends (RACE). In the present study, we report the full-length sequence of MyD88 gene in Common carp (Cyprinus carpio L.).In the 3748 bp genomic sequence,five exons and four introns were identified.The cloned cDNA exhibited 870 bp of 3'UTR and 858 bp of the entire open reading frame encoding a polypeptide of 284 amino acids.Sequence analysis indicated that MyD88 with 284 amino acids has a calculated molecular mass of 32.86kDa and a theoretical pI of 5.79. To determine the struetural domains in MyD88, the amino acid sequence was analyzed using the SMART program. The dedueed amino acid sequence of MyD88 Possesses a typical MyD88 domain ineluding an N-terminal death domain as well as inierm ediate and C-terminal TIR domains. Protein organization, multiple alignment and signal peptide prediction showed that the MyD88 sequence shares highly identity with known MyD88 members from other species. The amino acid sequence of the carp MyD88 is 86.0%,66.9%,60.4% and 59.6% identical to that of Danio rerio, Paralichthys olivaceus, human, and mouse.We studied their expression pattern in tissues in relatively health common carp by RT-PCR. MyD88 constitutively detectable in various tissues and organs selected, both are highly expressed in the foregut, spleen, kidney, gill and gonad; moderately expressed in the hindgut and musle. Expression of MyD88 and SAA were measured in previously non-exposed fish, 6h, 1, 3, and 5 days after experimental Vibrio anguillarum challenge to determine if they are associated with host response to V. anguillarum infection by real-time quantitative PCR. The mRNA copy numbers of carp MyD88 and SAA were increased in some organs including spleen, kidney, foregutl and hindgut. These results imply that MyD88 and SAA have an important role in the immunity in common carp.