Molecular Cloning And Functional Expression Of TLR8Gene From Grass Carp, Ctenopharyngodon Idella

Grass carp (Ctenopharyngodon idella) belongs to osteichthyes, cypriniformes,cyprinidae, leuciscinae, ctenopharyngodon, as one of the important freshwater fish breedingin China. With the development of high density and intensive in breeding, the frequentoutbreaks of disease seriously affected aquaculture economic benefits. Hemorragic diseaseresulted from grass carp reovirus (GCRV) was one of the main disease of grass carp.Hemorragic disease of grass carp onset rapid, and lead to high mortality and strong resistanceto drug. Disinfectants and Chinese herbal medicine play barely roles in virus inhibition. Atpresent, the immune prevention is an effective method.TLR8(toll-like receptor8) is one of the members of the family of TLR. TLR8belongs totype Ⅰ transmembrane protein receptor, which contain extracellular region (LRR domain),transmembrane region and intracellular region (TIR domain). The role of LRR domain is toidentify pathogens on the surface of PAMPs, while intracellular TIR is mainly responsible forthe corresponding response signal transduction in vivo. TLR8is typically a member ofintracellular members, which can identify ssRNA virus, small dsRNA viruses,oligoribonucleotide and a series of chemical synthetic ligands. As one of TLR antiviralreceptors, the exploration on TLR8genes and functional mechanisms will help to furtherunderstand the innate immune mechanism of grass carp and apply to grass carp breeding anddisease control. The main results and conclusion are summarized as below:1. By homology-based cloning, rapid amplification cDNA end and gradualamplification, we cloned the grass carp TLR8gene (CiTLR8). CiTLR8gene had4605nucleotides. The full-length cDNA of CiTLR8was of3766bp. The open reading frame was of3072bp and encoded a polypeptide of1023amino acids. A single intron with the size of839bp was found.2. By quantitative RT-PCR, CiTLR8mRNA was tested in the15tissues of healthy grasscarp. CiTLR8mRNA was ubiquitously expressed and expression level in gas bladder, spleen,brain tissues was high. Skin, hindgut, trunk kidney, gill, head kidney, foregut, eyes, heart andintestine expressed less TLR8mRNA. The lowest expression was in the blood, muscle and hepatopancreas.3. By quantitative RT-PCR, the CiTLR8expression in spleen and head kidney wassignificantly up-regulated and reached peak at24h post-injection of GCRV. After that, thedetection of CiTLR8mRNA expression was carried on the cellular level. CiTLR8transcriptionreached peak at8h and then declined below the normal level post-GCRV infection in the C.idella kidney (CIK) cell line; and it was rapidly and significantly down-regulated by thestimulation of the synthetic double-stranded RNA polyriboinosinic-polyribocytidylic acidsodium salt (poly I:C) in CIK at5μg/ml and25μg/ml.4. With the aid of RNA interfernce, quantitative RT-PCR and antiviral activity detection,we simply explored the expression and the roles of CiTLR8playing in the innate immuneresponse to GCRV. The inhibitor expression vectors were constructed and transfected intoCIK cell line to obtain stably expressing shRNA targeting TLR8. In CiTLR8-knockdown cells,CiTLR7transcript weakly increased, CiIFN-I mRNA was significantly down-regulated, andthe expression of CiMyD88, CiIRF7and CiMx1scarcely changed. Post poly I:C stimulation,CiTLR8, CiTLR7and CiMyD88transcripts significantly increased, CiIRF7wasdown-regulated after an initial phase of increase, and CiIFN-I and CiMx1transcripts wereup-regulated. After GCRV infection, the transcripts of CiTLR8, CiTLR7, CiMyD88andCiIRF7were up-regulated, but CiIFN-I and CiMx1transcripts were inhibited. Nevertheless,cells transfected with pshTLR8vectors had strong resistance against GCRV injection,suggesting CiTLR8might play a negative role in antiviral immune response. Therefore, onehypothesis is to silence CiTLR8gene in the resistance GCRV exploration.The above results laid a solid foundation for further analyzing the roles which TLR8genes involved in antiviral immune response signal pathway and mining CiTLR8geneticmarkers, and provided ideas for exploring effect mechanism and prevention of grass carphemorrhagic disease as well as resistance genetic breeding.