Molecular Cloning And Immunoresponse Of TLR1, TLR2 And MyD88 Genes In Epinephelus Coioides

Toll-like receptors (TLR) is type I transmembrane protein. After recognizing pathogen-associated molecular patterns (PAMP), TLR induces expression of transcription factors NF-κB and IRF through myeloid differentiation factor 88 (MyD88) dependent or TIR domain-containing adaptor-inducing interferon-P (TRIF) dependent signaling pathway, finally initiates innate and adaptive immunoresponse against pathogen invasion. At least 17 TLR types have been identified in teleosts. The molecular structure and functional characteristics of piscine TLR differs from these of mammalian. Epinephelus coioides is a species of important marine fish cultivating in coastal areas of Southeast China. Diseases often occur and cause severe economic loss in Epinephelus coioides culture industry with intensive farming. To understand more information about the immune system of Epinephelus coioides, in this study the full-length cDNAs of Epinephelus coioides TLR1 (EcTLR1), TLR2 (EcTLR2) and MyD88 (EcMyD88) genes were cloned using RT-PCR and RACE-PCR techniques. The structures and functions of the genes were analyzed by bio-informatic method. In addition, the expression profiles of the genes in tissues of normal Epinephelus coioides were detected, and the immunoresponses post polyI:C or bacterial LPS stimulation, or Vibrio alginolyticus infection were also determined by using qPCR.The full-length cDNA of the EcTLR1, EcTLR2 and EcMyD88 genes were confirmed as 3195 bp,3439 bp and 1795 bp respectively, and the putative length of ORF in the three genes were predicted as 2406 bp,2466 bp and 870 bp that encoded 801 aa,821 aa and 289 aa, respectively. The length of the 5'UTR of the three genes were verified as 240 bp,378 bp and 243 bp, and the lengh of the 3' UTR of the three genes were identified as 549 bp,595 bp and 682 bp, respectively. Each 3'UTR in the three genes possessed a polyadenylation signal sequene of AATAAA. Two mRNA unstable motifs (ATTTA) were found in the 3'-UTR of EcMyD88.To predict the functional domains, the SMART program was used to analyze the structures of the proteins. As the results, extracellular portions of EcTLRl and EcTLR2 proteins comprised 9 and 10 leucine-rich repeat (LRR) motifs respectively, while the cytoplasmic region of each protein contained a unique Toll/interleukin-1 receptor (TIR) domain. EcMyD88 protein contained a N-terminal death domain and a C-terminal TIR domain. Five kinds of potential functional sites on putative EcTLR1 protein were predicted, including N-glycosylation sites, protein kinase C phosphorylation sites, casein kinase II phosphorylation sites, N-acylation sites and tyrosine kinase phosphorylation sites. The EcTLR2 protein comprised cAMP/cGMP-dependent protein kinase II phosphorylation sites, cell-attachment sequence and leucine zipper, except for the N-glycosylation sites, protein kinase C phosphorylation sites, casein kinase II phosphorylation sites and N-acylation sites. However, EcMyD88 protein only contained casein kinase II phosphorylation site.According to amino acid sequence alignment analysis, the EcTLRl shared 47.5%-62.4% identity with that of other fish,36%-37.2% and 36.2%-38.2% identities with that of mammals and birds respectively. The EcTLR2 had 46.3%-74.8% identity with that of teleost, higher than that of mammalian 39.7%-40.7% and avian 39.1%-40.1%, respectively. The EcMyD88 shared high identity with other piscine MyD88 (67%-78.7%), whist the lower identities were showed with mammalian 60.9%-62.6% and chicken 57.1%. The TIR domain of EcTLR1, EcTLR2 and EcMyD88 showed 56.8%-89.8% identity with that of other vertebrates. Based on TLR1, TLR2 or MyD88 deduced amino acid sequences, the three phylogenetic trees were constructed and showed similar results. The animal species on the phylogenetic trees were divided into three branchs:fish, birds and mammals. By the identity and phylogenetic tree analysis to EcTLR1,EcTLR2 and EcMyD88, a close phylogenetic relationship was indicated between Epinephelus coioides and other fish.The EcTLR1,EcTLR2 and EcMyD88 mRNAs were examined in all organs/tissues. The EcTLRl and EcTLR2 mRNAs were mainly expressed in spleen, head kidney and thymus, while EcMyD88 was mainly found in liver, spleen, head kidney and thymus. After Epinephelus coioides were injected with LPS, the levels of EcTLRl, EcTLR2 MyD88 and IL-1βmRNAs increased 1.52-2.47 folds in spleen and thymus. The EcTLR1, EcTLR2 MyD88 and IL-1βmRNAs increased 1.52-3.64 folds in head kidney, spleen and thymus of Epinephelus coioides post injection with PolyI:C. The levels of EcTLRl and EcTLR2 mRNA in head kidney were significantly up-regulated 1.59 to 2.57 folds from 3 d to 7 d after Epinephelus coioides infected with V. alginolyticus. In spleen of Epinephelus coioides, the EcTLR1 and EcTLR2 mRNAs were up-regulated 1.47 and 1.9 folds at 1 d after V. alginolyticus infection. The MyD88 and IL-1βmRNAs in head kidney and spleen of Epinephelus coioides increased 2-39.75 folds from 1 d to 7 d post V. alginolyticus infection. The results suggested that TLRl, TLR2 and MyD88 strongly involved in immune responses during Vibrio alginolyticus infection.To prepare polyclonal antibody against EcMyD88, the coding region of EcMyD88 was cloned into pET-32a and pET32a-EcMyD88, a prokaryotic expression vector, was constructed. The pET32a-EcMyD88 was transformed into Escherichia coli BL21 and EcMyD88 was expressed under induction by IPTG. Then the recombinant EcMyD88 protein was successfully purified by His-Bind Resin column. The production of polyclonal antibodies against EcMyD88 using the recombinant protein is in progress.