Molecular Cloning And Study The Immune Function Of RLR In Common Carp (Cyprinus Carpio L.)

Common carp Cyprinus carpio L. is an important economic freshwater fish because of itshigh content of nutrients and its medicinal value. However, with the increased production density,and the water contains more complex microbiota, there has appeared so many carp diseasescaused by the virus or bacterial, it has became one of the main problems faced in production. Asa kind of lower vertebrates, the acquired immune system in common carp is not developedenough compared with mammals, and therefore it relies mainly on innate immune system toresist the invading pathogens.The innate immune system is the first line of defense against pathogenic microorganisms.The organisms identify pathogen-associated molecular patterns (PAMPs) through patternrecognition receptors (PRRs), including TLRs, NLRs and RLRs, inducing the expression ofinterferon and pro-inflammatory cytokines. RLR family is a new PRR discovered in2004, it hasthree members including RIG-I, MDA5and LGP2. The CARDs of RIG-I and MDA5bindingwith the IPS-1CARD domain, which activates downstream signaling molecules, then promoteIRF3/7and NF-κB signaling pathway. LGP is a positive regulatory factor involved in RLRantiviral signaling pathways.In this study, with the primers which were designed based on sequence alignement, usingsemi-quantitative RT-PCR and RACE (Rapid amplification of cDNA end), the complete cDNAof common carp MDA5and LGP2genes were coloned. Bioinformatics was applied for theanalysis of the protein structures and the phylogeny of RLRs. We detected and analyzed theMDA5and LGP2mRNA expression levels in various tissues of common carps and the carpsafter poly I:C injections. This study provided the role of MDA5and LGP2in common carpantiviral innate immune system.The full length of common carp MDA5cDNA is3851bp, encoding993amino acids. Thededuced amino acids contained six functional domains: a CARD (Capase activation andrecruitment domain), a DEXDc (DExD/H box-containing domain), two HELICc (helicase superfamily C-terminal domain), a ResIII (Conserved restriction domain of bacterial type IIIrestriction enzyme) and an RD (C-terminal regulatory domain). MDA5mRNA expressed in alltested tissues, with a high level in gills and spleen, follow by skin, brain, muscle and hindgut,while lower expressed in gonads and blood. Compared with the controls which common carpswere injected with PBS, MDA5mRNA expession level was significantly up-regulated by polyI:C injection. The MDA5transcripts in liver were significantly up-regulated at3h,6h afterinjection, increased86.3folds in a maximum level, then recovered to normal level at1d. Inspleen, MDA5showed a highest level at6h (18.8folds, P﹤0.05), and recovered to normal levelat2d post-injection. For headkidney tissues, the expression was significantly up-regulated at6hwith the highest level (24.1folds, P﹤0.05), and then recoverd to the original level at3dpost-injection. The expression of MDA5reached the highest level at6h post-injection both inskin and gills (26.8and139.3folds, P﹤0.05), and then recoverd to the original level at2dpost-injection. For forgut and hindgut, the expression was significantly up-regulated at3h (500.2and15.0folds, P﹤0.05), and then reduced significantly at6h post-injection.The full length of common carp LGP2cDNA is2978bp, encoding679amino acids. Thededuced amino acids contained five functional domains: two DEXDc, a ResIII, a HELICc and anRD. LGP2mRNA expressed in all tested tissues, with a high level in gills, brain and spleen,while lower expressed in oral epithelial, gonads and blood. Compared with the controls whichcommon carps were injected with PBS, LGP2mRNA expession level was significantlyup-regulated by poly I:C injection. The LGP2transcripts in liver were significantly up-regulatedat3h post-injection increased in a maximum level (175.3folds, P﹤0.05), then recovered tonormal level at2d post-injection. In spleen, LGP2showed a highest level at12h (55.5folds, P﹤0.05), and then recovered to normal level at3d post-injection. For headkidney tissues, theexpression was significantly up-regulated at6h with the highest level (77.2folds, P﹤0.05), andthen recoverd to the original level at1d post-injection. In skin and gills, the expression of LGP2reached the highest level respectively at6h and12h post-injection (54.6and57.5folds, P﹤0.05), and then recoverd to the original level respectively at3d and1d post-injection. For forgutand hindgut, the expression was significantly up-regulated at3h (224.6and11.5folds, P﹤0.05),and then reduced at6h post-injection.These results implied that MDA5and LGP2have participated in the carp antiviral innate immune process, and provided an important theoretical basis for the study of the mechanism ofinnate immune system in bony fish.