大黄鱼肝表达抗菌肽2基因的克隆和原核表达

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.

Cloning and prokaryotic expression of liver-expressed antimicrobial peptide-2 gene from Larimichthys crocea

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 cro- cea was cloned and sequenced. The cloned sequence was 2, 236 bp in length ranging from start eodon to stop codon, and included 78 bp exon I , 880 bp intron I , 179 bp exon II , 1044 bp intron II , and 55 bp exonIII. 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 punetatus and Larimichthys crocea was o- ver 95%. The recombinant expression plasmid pET-32a-LEAP-2 was constructed by linking Larimiehthys croeea 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.