水产学报2017，Vol.41Issue(10)：1500-1511,12.DOI:10.11964/jfc.20160910553
草鱼NK-lysin基因的克隆、原核表达与活性分析
The cDNA cloning, expression and antibacterial activity of NK-lysin from grass carp (Ctenopharyngodon idella)
摘要
Abstract
NK-lysin is a cationic antimicrobial peptide mainly produced by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells and stored in cytolytic granules together with perforin and granzymes. NK-lysin is a member of saposin-like protein family and is orthologous with human granulysin. Mammalian NK-lysin is known to possess antibacterial and antitumor property. Homologues ofNK-lysin have been identified in diverse organisms including human, pig, cattle, chicken, horse, water buffalo and teleost species. In fish,NK-lysin genes have been reported in five species, i.e. Japanese flounder (Paralichthys olivaceus), channel catfish (Ictalurus punctatus), zebrafish (Danio rerio), half-smooth tongue sole (Cynoglossus semilaevis) and large yellow croaker (Larimichthys crocea). In this study, we aimed to explore the structure and expression difference in tissues, and theNK-lysin (Cinkl) was identified from grass carpCtenopharyngodon idellausing reverse transcriptase polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE), and primers were designed according to the conserved sequence of known fishNK-lysin genes. The expression of theCinkl gene in different tissues was analyzed by real-time quantitative PCR. In order to explore the antibacterial activity ofCiNkl, the recombinant expression vector was constructed by cloning its mature peptide into the prokaryotic expression plasmid pET-28b-MBP. The antibacterial analysis of recombinant protein was evaluated using agar disc diffusion. The full-length cDNA sequence ofCinkl was 768 bp, encoding 121 amino acids. Genomic DNA was 3361 bp including 4 exons and 3 introns that was similar to zebrafishnklc andnkld genome. The multiple sequence alignment analysis showed that theCiNkl shared 57.98% and 63.03% identity with zebrafish Nklc and Nkld respectively.CiNkl possesses a Saposin B domain of members of saposin-like protein (SAPLIP) family and six conserved cysteins (Cys) residues that in mammals are known to form three intramolecular disulfide bonds essential to antimicrobial activity. Phylogenetic tree suggested thatCiNkl is clustered closely withD. rerio Nklc and Nkld. Quantitative RT-PCR results showed thatCinkl mRNA was expressed in all tissues examined and highly in the spleen, with small amount in heart, gill, kidney, head kidney and weakly in skin, brain, liver and intestine. In addition, SDS-PAGE showed that the molecular mass of recombinantCiNkl expressed in host bacteria Rosetta (DE3) was approximately 57 ku. The recombinant protein purified by Ni-NTA His·Bind resin showed the antibacterial activity against Escherichia coli M15,Aeromonas hydrophila andStaphylococcus aureus. The results indicate that grass carp NK-lysin has potential immunomodulatory effects in fish innate immunity.关键词
草鱼/NK-lysin/基因克隆/组织表达/抑菌活性Key words
Ctenopharyngodon idella/NK-lysin/gene cloning/organization expression/antibacterial activity分类
生物科学引用本文复制引用
王改玲,王明成,李传凤,潘磊,刘盼婷..草鱼NK-lysin基因的克隆、原核表达与活性分析[J].水产学报,2017,41(10):1500-1511,12.基金项目
国家自然科学基金(31402334) （31402334）
河南省基础与前沿技术研究计划(142300410111)National Natural Science Foundation of China (31402334) （142300410111）
Basic and Frontier Technology Research Program of Henan Province (142300410111) （142300410111）
