Cloning and in vitro expression of antibacterial peptide gene Hepcidin in Pacific cod Gadus macrocephalus
WU Lingmei1, GU Jie1, WEN Zhiqing 2，3, Jia Peng 2，3* , LIU Ruiting1, JIANG Jielan1, LIU Hong 2，3, ZHENG Xiaocong 2，3, MAO Mingguang1*
1.Key Laboratory of Mariculture and Stock Enhancement in North China’s Sea, Ministry of Agriculture and Rural Affairs, Key Laboratory of Fish Applied Biology and Aquaculture in North China, Liaoning Province, Dalian Ocean University, Dalian 116023, China; 2. Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center, Shenzhen 518045, China；3.Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen 518045, China
Abstract: The expression patterns of GmHep were investigated in liver, kidney, spleen, brain, intestine, gill and muscle of Pacific cod Gadus macrocephalus with average body weight of 2.438 kg and in 5, 10, 30, 37 and 40 day old larvae using qPCR, and the prokaryotic (pET-32a- GmHep) and eukaryotic(pEGFP-GmHep) expression vectors were constructed in order to reveal the molecular structure and expression characteristics of the Hepcidin (GmHep), and to develop a new type of antimicrobial peptide. It was found that the GmHep gene had an open reading frame of 297 bp length, encoding 98 amino acids, and molecular weight of 10 833.56. There were 22 amino acid residues on the N-terminal of the peptide which mature peptide contained 8 cysteine residues and 4 disulfide bonds. The sequence alignments showed that the Hepcidin homology between Pacific cod and Atlantic cod G.morhua was the maximaum (93%), and the homology of GmHep and other fish was lower (less than 60%), indicating that the function of GmHep was unique. The expression level of GmHep was higher in liver than that in other tissues, and GmHep was expressed in 5, 10, 30, 37, 40-day old larvae and juveniles, indicating that GmHep can play roles in the early stage of development. The optimal inducing expression of GmHep in vitro was observed under the conditions of IPTG concentration of 0.01 mmol/L, 30 ℃, and induction for 4 hours. GmHep was found to be mainly located in the cytoplasm of carp epithelioma cell (EPC) line. In conclusion, two kinds of GmHep expression vectors were successfully constructed in vitro, and it was approved that GmHep is a liver secretion type, and it plays a certain role in the early development of Pacific cod. The findings provide a basis for the development of new antimicrobial peptides in fish.
2020, Vol. 35 
