Research On TLRs And Immune-Related Genes Expression Profile Of Grouper Infected With Cryptocaryon Irritans

Cryptocaryon irritans is a pathogenic ecto-parasitic ciliate that can infect almostall marine teleosts, and cause serious economic losses for aquaculture with itswidespread distribution, indiscriminate host specificity and rapid proliferation.Recently, grouper (Epinephelus coioids) become the main aquaculture species rearedin coastal areas of China for its delicious quality and rich nutrition, etc. However, theannual outbreak of Ichthyophthiriasis has posed very serious harm to the grouperaquaculture industry. Therefore, the immunity studies of grouper not only give us aninsight into the mechanisms of host defense against parasite, but also help us todevelop immune prophylaxis to prevent this disease. In the present study, we firstlycloned a cDNA sequence of grouper TLR2EST, TLR3, TLR9EST, TLR21, TLR22andan adaptor MyD88, and analyzed the structure characteristics and tissue-expressionpattern of each gene. Secondly, post C. irritans infection, we examined the expressionprofile of some immune-related genes including TLRs in local infection sites andsystemic immune organs at different time points. Thirdly, the recombinant TLR9,TLR21and MyD88was expressed in E. coli, and polyclonal antibody anti-theseprotein was produced using mice. Lastly, we identified the signal transduction role of TLR9, TLR21and MyD88in HEK293T cell. The main results of this thesis were asfollows:To begin the understanding of the potential role of grouper TLRs, We firstlycloned the full-length of grouper TLR21, TLR3and MyD88cDNA. TLR21ORF was2937bp encoding a putative polypeptide of979Aa, which contains a N-terminalCys cap (LRR-NT),27LRR motifs, a C-terminal Cys cap (LRR-CT), atrans-membrane domain and a cytoplasm TIR domain. The LRR-NT and LRR-CT ofTLR21contains two and four Cys, respectively. TLR3ORF was2730bp encoding aputative polypeptide of909Aa, which contains a LRR-NT,25LRR motifs, a LRR-CT,a trans-membrane domain and a cytoplasm TIR domain. The LRR-NT and LRR-CTof TLR21contains three and four Cys, respectively. MyD88ORF was873bpencoding291amino acid residues. Similar to other species, the EcMyD88genecontains of five conserved exons and four diverse introns. SMART program analysisshowed that MyD88consisted of conserved DD domain and TIR domain. TheMyD88-TIR domain consisted of a5-strand parallel β-sheet surrounded by sixα-helices that formed a global fold, which was similar to the structure of the humanMyD88-TIR domain. Five highly conserved amino acid residues (Arg¹⁸⁹, Asp¹⁹⁰,Lys²¹⁰, Gln²⁷⁵and Arg²⁸¹) were also found in this domain. Homology alignmentindicated that the TIR domain of these three proteins was highly conserved than otherdomain such as LRR domain and DD domain. Phylogenetic analysis implied thatthese proteins showed a highly correlated evolutionary relationship with other fishortholog and branched into the same cluster. In this part, we also isolated the EST ofgrouper TLR2, TLR9and TLR22. Tissues expression pattern analysis suggested thatTLR21, TLR3, TLR22and MyD88were broadly expressed in the tested tissues.To identify the potential role of immune-related genes in anti-parasitic immuneresponses in fish, we monitored the expression change of some genes in local andsystemic immune organs of grouper post-C. irritans infection.1) TLR9and TLR21expression was up-regulated in the skin and gill, while TLR2expression wasincreased in the spleen and head kidney. The expression of TLR3and TLR22was only increased at some time points. MyD88expression was up-regulated in the skin andspleen post infection.2) other six immune-related genes' expression were alsoregulated post C. irritans infection, especially the acute phase protein C-type lectinand pro-inflammatory IL-1β, which expression were significantly increased at almostall time points, while cox-2expression was significantly down-regulated in the spleen(during the first round infection) and head kidney.3) some specific immune cellMarker genes' expression was increased such as CD8, and IgM expression wassignificantly up-regulated in the skin, gill and head kidney at the later phase postchallenge. Besides, a large number of leucocytes, including neutrophil-like cells,eosinophilic granule cells, lymphocytes and monocytes, infiltrated into the gills invicinity of the parasite infection sites. In conclusion, the immune-related genes'expression could be regulated at local and systemic immune organs post parasiteinfection, and expression pattern was different in both two immune systems, alsothese changes may be due to the migration of immune cell as showed above.Recombinant grouper TLR21, TLR9and MyD88was expressed in E. coli, andpurified directly by cutting and exatracting the protein from SDS-PAGE gel.Anti-serum was then collected from the mice immunized with purified protein, whichdisplayed immunological reactivity and specificity when analyzed in Western blot.The antibody can be used next to study the location of TLR21, TLR9and MyD88ingrouper tissues.pFLAG-CMV2-TLR21-TIR/TLR9-TIR and pcDNA3.1-MyD88expressionvector was constructed by inserting target fragments into vector, respectively, andrespectively transfected into HEK293T cell with pGL-NF-κB/pGL-IFN-β fireflyreporter plasmid and pRL-TK renilla luciferase plasmid.24h post transfection,luciferase activity was measured. HEK293T cell transfected with plasmid encodingTLR21-TIR and TLR9-TIR inhibited NF-κB activation, while the later plasmidincreased IFN-β activation. Cell transfected with plasmid encoding MyD88increasedNF-κB and IFN-β activation, which was dose-dependent. Interestingly,co-transfection with the plasmid encoding MyD88, TLR9-TIR could enhance the activation of NF-κB. MyD88located in the special sites in293T cell.