Moleculer Cloning And Characteristic Analysis Of CTLP And Txndc12from Orange-spotted Grouper:Epinephelus Coioides

Epinephelus coioides is a high economy value marine culture fish. With the rapid development of marine farming activities and the limit of culture environment, outbreaks of viral and bacteria diseases have affected grouper aquaculture industry causing heavy economic losses in recent years. Therefore, it is necessary to find some fuctional gene from E. coioides to resist disease and bad environment.1. Lectin is a very significant factor of innate immunity. It plays an important role in pathogen recognition and cell-cell interaction. However, its homologue may have the function of antifreeze activity. In this study, a C-type lectin-like protein from Epinephelus coioides (Ec-CTLP) was successfully cloned and expressed. The full-length cDNA of Ec-CTLP was composed of905bp with a522bp open reading frame that encodes a174-residue protein. The putative amino acid sequence of Ec-CTLP contains a signal peptide of19residues at the N-terminal and a CLECT domain from Cys43to Arg169and a conserved imperfect WND (Trp-Asn-Asp) motif. The homologous identity of deduced amino acid sequences is from38%to70%with other fishes. Quantitative real-time PCR analysis revealed that Ec-CTLP transcript was most abundant in liver. Intracellular localization revealed that Ec-CTLP was only distributed in cytoplasm. The expression of Ec-CTLP was differently upregulated in grouper spleen (GS) cells after challenging with16℃and4℃. Recombinant Ec-CTLP (rEc-CTLP) was expressed in Escherichia BL21(DE3) and purified for mouse anti-Ec-CTLP serum preparation. The rEc-CTLP fusion protein was demonstrated to enhance the cold resistance of bacterial, and the survival of bacteria (including Gram-negative E.coli and Gram-positive S.aureus) was positive correlation with the concentration of the rEc-CTLP. These results could help in further investigations of the properties and function of C-type lectin, and may improve the cold-tolerance ability of organism in genetic thremmatology.2. Thioredoxin domain-containing protein12(Txndc12) belongs to the thioredoxin superfamily, and has roles in redox regulation, defense against oxidative stress, refolding of disulfide-containing proteins, and regulation of transcription factors. In this study, a thioredoxin domain-containing protein12was cloned from the marine fish grouper, Epinephelus coioides by RACE PCR, named as Ec-Txndcl2. The Ec-Txndc12encodes173amino acid residues with signal peptide in its N-terminal and a thioredoxin (Trx) domain that is homologous with some genes in Mus musculus, Xenopus laveis, etc. Ec-Txndc12mRNA is predominately expressed in liver, brain and muscle. The expression of Ec-Txndc12was up-regulated in the liver of grouper challenged with SGIV. In order to elucidate its biological functions, Ec-Txndcl2was recombined and expressed in Escherichia coli BL21(DE3). The rEc-Txndc12fusion protein was demonstrated to possess the antioxidant activity. The grouper spleen (GS) cells were treated with a high concentration of rEc-Txndcl2(30mg/mL), which significantly enhanced cells viability under oxidative damage caused by viral infection. These results together indicated that Ec-Txndc12could function as an important antioxidant in a physiological context, and might be involved in the responses to viral challenge...