草鱼NK-lysin基因的克隆、原核表达与活性分析

The c DNA cloning, expression and antibacterial activity of NK-lysin from grass carp(Ctenopharyngodon idella)

为研究草鱼抗菌肽NK-lysin基因的组成结构、在组织中的表达差异及其抑菌活性,实验根据已知鱼类NK-lysin基因保守区序列设计上下游引物,采用RT-PCR和RACEPCR技术克隆草鱼NK-lysin基因(Cinkl),RT-PCR进行各组织间的表达分析,并构建Cinkl成熟肽的原核表达载体,采用琼脂糖孔穴扩散法检测重组蛋白的抑菌活性。结果显示,Cinkl c DNA全长768 bp,编码121个氨基酸;基因组DNA全长3 361 bp,包含4个外显子和3个内含子。经多序列比对分析和结构域预测表明,Ci Nkl氨基酸序列具有SAPLIP家族的特征:含有6个保守的半胱氨酸和saposin B蛋白结构域,与斑马鱼Nklc、Nkld的相似性分别为57.98%和63.03%。构建NK-lysin氨基酸序列系统进化树,发现Ci Nkl与斑马鱼Nklc、Nkld聚为一支。RT-PCR检测结果显示,Cinkl m RNA在所有检测的草鱼组织中均有表达,在脾脏中表达量最高,心脏、鳃、中肾和头肾的表达量次之,皮肤、脑、肝脏和肠有微量表达。p ET-28b-MBP与Ci Nkl成熟肽序列构建的原核表达质粒在宿主菌Rosetta(DE3)表达出约57 ku的可溶性融合蛋白,利用Ni-NTA His·Bind树脂进行纯化,Ci Nkl重组蛋白对G–细菌如大肠杆菌M15株、嗜水气单胞菌,以及G+细菌如金黄色葡萄球菌均具有抑菌活性。基于草鱼NK-lysin的组织表达特征和重组蛋白的抑菌活性,推测NK-lysin在草鱼的先天性免疫防御中发挥调节作用。

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 of NK-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 the NK-lysin（Cinkl） was identified from grass carp Ctenopharyngodon idella using reverse transcriptase polymerase chain reaction（RT-PCR） and rapid-amplification of c DNA ends（RACE）, and primers were designed according to the conserved sequence of known fish NK-lysin genes. The expression of the Cinkl gene in different tissues was analyzed by real-time quantitative PCR. In order to explore the antibacterial activity of Ci Nkl, the recombinant expression vector was constructed by cloning its mature peptide into the prokaryotic expression plasmid p ET-28 bMBP. The antibacterial analysis of recombinant protein was evaluated using agar disc diffusion. The full-length c DNA sequence of Cinkl was 768 bp, encoding 121 amino acids. Genomic DNA was 3 361 bp including 4 exons and 3 introns that was similar to zebrafish nklc and nkld genome. The multiple sequence alignment analysis showed that the Ci Nkl shared 57.98% and 63.03% identity with zebrafish Nklc and Nkld respectively. Ci Nkl 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 that Ci Nkl is clustered closely with D. rerio Nklc and Nkld. Quantitative RTPCR results showed that Cinkl m RNA 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 recombinant Ci Nkl expressed in host bacteria Rosetta（DE3） was approximately57 ku. The recombinant protein purified by Ni-NTA His·Bind resin showed the antibacterial activity against Escherichia coli M15, Aeromonas hydrophila and Staphylococcus aureus. The results indicate that grass carp NKlysin has potential immunomodulatory effects in fish innate immunity.