Molecular Cloning, Expression And Functional Studies Of Creatine Kinase And Hepcidin From Roughskin Sculpin Trachidermus Fasciatus

Roughskin sculp in (Trachidermus fasciatus) is a catadromous fish. In view of the decrease of the population, it was listed as critically endangered species in Category II of the National Key Protected Wildlife List in 1988 in China. Aquaculture has become a crucial way to recover its population in the wild, however, the greatest challenge of the aquaculture seems to be the disease. In order to seek the effective measure to prevent the disease, our team dedicate to the research of the immune related genes in roughskin sculpin.Creatine kinase (CK) is a member of the phosphate kinase family, whose primary function is catalytic the reversible transfer of high-energy phosphate bond between creatine and ATP, consequently providing energy source for muscle contraction and the other life activities. As a peptide hormone, hepcidin is a member of antimicrobial peptides secreted by the liver, which plays a very important role in antibacterial. In this study we identified two genes termed as TfM-CK and Tf-Hepcidin respectively. The mRNA expression of TfM-CK and Tf-Hepcidin in fishes of healthy and challenged with Vibrio anguillarum was detected by quantitative real-time PCR. Meanwhile, eukaryotic and prokaryotic expression vectors were built to express the proteins for further studying their biological function in innate immune system.1. The in vivo and in vitro experiments show that creatine kinase (CK) might play a role in the innate immune of roughskin sculpin as a kind of immune factorTfM-CK was cloned from roughskin sculpin. The cDNA of TfM-CK is 1474 bp with an open reading frame (ORF) of 1146 bp that encodes a protein of 381 amino acids. It includes an ATP-gua_PtransN domain and an ATP-gua_Ptrans domain. Neighbor-joining tree shows that TfM-CK clusters to M-CKs from the other fishes in accordance with their traditional taxonomy. qRT-PCR analysis reveals that TfM-CK is constitutively expressed in all detected tissues, with a relatively high amount of mRNA in the muscle followed by the blood. Its expression is up-regulated in the four detected tissues (skin, spleen, muscle and head kidney) challenged with Vibrio anguillarum. In vivo, recombinant protein TfM-CK could significantly reduce mortality of Cyprinus carpio infected with Vibrio anguillarum.The research of binding activity assay, agglutination assay and antimicrobial activities also show that the reconmbinant protein might play a significantly role in the innate immune response.2. Tf-Hepcidin might act as pattern recognition receptors (PRR) as well as effectors in roughskin sculpin innate immune response.Tf-Hepcidin was cloned from roughskin sculpin. The cDNA of TfM-CK is 988 bp with an ORF of 273 bp that encodes a protein of 90 amino acids. It contains a signal peptide (from 1 to 24 amino acids), a propeptide (from 25 to 64 amino acids) and a mature peptide (from 65 to 90 amino acids). The mature peptide includes eight conserved disulfide-bonded cysteine residues. Neighbor-joining tree shows that Tf-Hepcidin clusters to Hepcidins from the other fishes in accordance with their traditional taxonomy. qRT-PCR analysis reveals that Tf-Hepcidin is constitutively expressed in all detected tissues, with a relatively high amount of mRNA in the liver. Its expression is up-regulated in the six detected tissues (blood, skin, liver, brain and spleen) challenged with LPS and heavy metal. The antimicrobial activities of recombinant expressed that Tf-Hepcidin is against many bacteria. Binding activity assay showed that Tf-Hepcidin could binding with LPS-LTA and PGN. Agglutination assay also indicated that Tf-Hepcidin could agglutinate many bacteria. In vivo, recombinant protein Tf-Hepcidin also could significantly reduce mortality of carp infected with Vibrio anguillarum.Therefore, we speculated that TfM-CK and Tf-Hepcidin might act as a pattern recognition receptors (PRR) and effectors which played a significantly role in the innate immune response of roughskin sculpin.