大黄鱼肝表达抗菌肽2基因的克隆和原核表达

摘要

抗菌肽是在多种细胞中表达具有抗菌活性的肽类物质的总称,在免疫反应中发挥着非常重要的作用.通过同源克隆法克隆到大黄鱼肝脏表达的抗菌肽2(liver-expressed antimicrobial peptide-2,LEAP-2)基因的完整开放阅读框(Opening Reading Frame,ORF).克隆到的大黄鱼LEAP-2全长2236 bp,包含外显子Ⅰ78 bp,内含子Ⅰ880 bp,外显子Ⅱ179 bp,内含子Ⅱ1044 bp,外显子Ⅲ55 bp,编码序列312 bp,编码103个氨基酸.推断的氨基酸序列羧基端区域存在高度保守的4 个半胱氨酸残基,符合LEAP-2超家族的结构特征.同源性对比后显示LEAP-2基因在进化上高度保守,大黄鱼LEAP-2推断的氨基酸序列与牙鲆、黄颡鱼、蓝色鲶鱼和斑点叉尾鮰等鱼类之间的同源性均在95%以上.将大黄鱼LEAP-2 cDNA连接到pET-32a(+),构建了重组表达质粒pET-32a-LEAP-2,将其转化到大肠杆菌BL21上并用1.0 mmol/L IPTG诱导表达,获得了大小约为27 kDa的重组蛋白,与预期的一致.%Antimicrobial peptide is generally referred to peptides which exhibit antimicrobial activity expressed in several cells, and has an important role in immune reaction. In this experiment, liver-expressed antimicrobial peptide-2 (LEAP-2) gene from Larimichthys crocea was cloned and sequenced. The cloned sequence was 2, 236 bp in length ranging from start codon to stop codon, and included 78 bp exon Ⅰ, 880 bp intronⅠ , 179 bp exon Ⅱ 1044 bp intron Ⅱ , and 55 bp exon Ⅲ. The coding sequence was 312 bp in length, and encoded 103 amino acids. C-terminal regions of the deduced amino acid sequence contained four conserved cysteines, in accordance with structure characteristic of the LEAP-2 superfamily. LEAP-2 amino acid sequence was highly conserved in the evolution, and the similarity among Paralichthys olivaceus, Pelteobagrus fulvidraco, Ictalurus furcatus, Ictalurus punctatus and Larimichthys crocea was o-ver 95% . The recombinant expression plasmid pET-32a-LEAP-2 was constructed by linking Larimichthys crocea LEAP-2 with pET-32a ( + ) , and transformed into BL21 cells. The expression of recombinant protein in the transformed cells was induced by by 1. 0mmol/L IPTG. The expressed recombinant protein was about 27kDa.