斑点叉尾鮰(Ictalurus punctatus)LEAP2成熟肽在大肠杆菌中的融合表达  

Fusion Expression of Channel Catfish(Ictalurus punctatus)LEAP2 Mature Peptide in Escherichia coli

作　　者：高北[1] 陶妍[1] 

机构地区：[1]上海海洋大学食品学院,上海201306

出　　处：《生物技术通报》2014年第2期130-135,共6页Biotechnology Bulletin

基　　金：上海市教育委员会重点创新基金项目(11ZZ148)

摘　　要：以斑点叉尾鮰(Ictalurus punctatus)肝脏为基因克隆的材料,通过RT-PCR对编码LEAP2成熟肽区域41个氨基酸的cDNA片段(mLEAP2)进行克隆。根据斑点叉尾鮰与其他鱼类、哺乳动物及两栖动物等其他物种LEAP2成熟肽区域氨基酸序列的比对结果,发现存在14个保守的氨基酸残基,其中4个高度保守的半胱氨酸残基位于成熟肽的羧基端区域,在空间上可形成两对二硫键,推测与LEAP2的抗菌活性有关。进一步构建重组表达质粒pET32a-mLEAP2,使mLEAP2基因与携带有6×His-tag标签和肠激酶识别位点的硫氧还蛋白trxA基因融合,转化至大肠杆菌BL21(DE3)后,在25℃下,经0.7 mmol/L IPTG诱导培养16 h后,成功表达了trxA-mLEAP2融合蛋白。Tricine-SDS-PAGE显示,细胞经超声破碎后,上清和沉淀中均含融合蛋白,采用固化金属离子亲和层析(IMAC)对其进行纯化,得到了纯化的融合蛋白。Antimicrobial peptides generally are some small molecule peptides with strong ability to fight against microbial organisms. They play an important role in the host＇s immune system and are considered to be good substitutes for traditional antibiotics. A cDNA fragment（mLEAP2）encoding the LEAP2（liver-expressed antimicrobial peptide 2）mature peptide consisted of 41-amino-acid was cloned from the liver of channel catfish（Ictalurus punctatus）by RT-PCR. When the amino acid sequence of channel catfish mLEAP2 was compared with those of poikilothermal fish and other species, fourteen residues were observed at conserved positions, especially four highly conserved cysteine residues occurred at C-terminal regions of these mLEAP2s, suggesting that two disulfide bridges resulted from these four cysteines possibly related to antimicrobial activity of LEAP2. Subsequently, the mLEAP2 gene was fused with a trxA partner gene with a 6×His-tag and an enterokinase site to construct the recombinant expression plasmid pET32a-mLEAP2. The fusion protein＂ trxA-mLEAP2＂ was successfully expressed in E.coli BL21（DE3）at 25 ℃ after a 16 h induction with 0.7 mmol/L IPTG. Tricine-SDS-PAGE showed that the fusion protein existed in both the supernatant and inclusion body after sonication and centrifugation. The purified fusion protein was successfully obtained by immobilized metal affinity chromatography（IMAC）.

关 键 词：斑点叉尾鮰 LEAP2成熟肽 RT-PCR 融合表达 

分 类 号：S917.4[农业科学—水产科学]