Molecular cloning,characterization and expression analysis of a C-type lectin (Ec-CTL) in orange-spotted grouper,Epinephelus coioides |
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| Authors: | Jingguang Wei Dan Xu Jinggeng Zhou Huachun Cui Yang Yan Zhengliang Ouyang Jie Gong Youhua Huang Xiaohong Huang Qiwei Qin |
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| Affiliation: | 1. State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, 135 West Xingang Road, Guangzhou 510275, PR China;2. Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, PR China;1. Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China;2. Hubei Collaborative Innovation Center for Freshwater Aquaculture, Wuhan 430070, People’s Republic of China;3. Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Wuhan 430070, People’s Republic of China;4. State Key Laboratory of Agricultural Microbiology, Wuhan 430070, People’s Republic of China;1. Fish Health Management Division, ICAR-Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751 002, India;2. Institute of Marine Research, Nordnesgaten 50, 5817 Bergen, Norway;3. Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110 067, India;4. Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India;1. Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China;2. Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China;3. University of Chinese Academy of Sciences, Beijing, China;4. College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China |
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| Abstract: | C-type lectins play crucial roles in pathogen recognition, innate immunity, and cell–cell interactions. In this study, a new C-type lectin (Ec-CTL) gene was cloned from grouper, Epinephelus coioides by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-CTL was composed of 840 bp with a 651 bp open reading frame (ORF) that encodes a 216-residue protein. The deduced amino acid sequence of Ec-CTL possessed all conserved features crucial for the fundamental structure, such as the four cysteine residues (Cys71, Cys152, Cys167, Cys175) involved in the formation of disulphide bridges and the potential Ca2+/carbohydrate-binding sites. Ec-CTL contains a signal peptide and a single carbohydrate recognition domain (CRD). The genomic DNA of the gene consists of three exons and two introns. Ec-CTL showed high similarity of 54% to the C-type lectin of killifish Fundulus heteroclitus. Ec-CTL mRNA is predominately expressed in liver and skin, and lower expressed in kidney, intestine, heart, brain and spleen. The expression of Ec-CTL was differentially up-regulated in orange-spotted grouper challenged with Saccharomyces cerevisiae, Vibrio vulnificus, Staphyloccocus aureus and Singapore grouper iridovirus (SGIV). Recombinant mature Ec-CTL (rEc-CTL) was expressed in E. coli BL21, purified and characterized as a typical Ca2+-dependent carbohydrate-binding protein possessing hemagglutinating activity. It bound to all examined bacterial and yeast strains, and aggregated with S. cerevisiae, V. vulnificus and S. aureus in a Ca2+-dependent manner. |
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