牙鲆肝脏抗菌肽-2基因序列和表达分析

Sequence and Expression Analysis of LEAP-2 Gene from Paralichthys olivaceus

抗菌肽是鱼类先天免疫的组成部分,发现新的抗菌肽对于动物抗病机理的研究和寻找新药物有重要意义。经过对GenBank数据库中牙鲆EST文库进行同源比对后,筛选得到一个具有完整编码框的抗菌肽cDNA序列,结果再经过RT-PCR进行验证,命名为LEAP-2,对该序列进行注释,获得序列登录号EU586111。该抗菌肽能编码102个氨基酸的多肽,前28个氨基酸组成信号肽。推测编码蛋白质分子量为11 276.9 u,理论等电点(PI)为9.33。其蛋白质序列与另外9种鱼类的同源抗菌肽序列比较发现,同源性介于38.2%～61.7%。其中57至92位氨基酸为高度保守区,是维持其空间结构的主要区域,其中包含有4个保守的半胱氨酸,可能组成2个二硫键。荧光定量PCR分析显示,LEAP-2基因在肝脏中高表达,在牙鲆仔鱼不同发育时期中也有表达,但是表达水平存在明显差异,特别是在变态中期表达量比较高。

Antimicrobial peptides are found in several kinds of tissues of many vertebrates and invertebrates.These peptides are predicted to operate as a first-line host defense mechanism exerting broad-spectrum activity against pathogenic bacteria,fungi,parasites,and enveloped viruses.Followed basic local alignment search to the flounder EST library of GenBank database by using LEAP-2 from Oncorhynchus as probe sequence,an cDNA sequence owned a complete open reading frame of antimicrobial peptides was achieved.The sequence was verified by RT-PCR and named LEAP-2,accession number of EU586111 was acquired after sequence annotation and submitting to the GenBank database.Flounder LEAP-2 gene encode a peptide of 102 amino acids with a 28 amino acids signal peptide and a mature peptide of 74 amino acids,with a predicted mulecular weight of 11 276.9 u and pI at 9.33.The amino acid and nucleotide sequences were conserved between flounder and other organisms and the homology ranged 38.2%-61.7%,the 57-92 amino acids are highly conserved region is to maintain the spatial structure,including four conserved cysteine,may be composed of two disulfide bonds.The flounder LEAP-2 gene is expressed in a wide range of tissues and predominantly expressed in the liver.Its expression is developmentally regulated with no detection in early stages of metamorphosis but highly in the mid-metamorphosis.