牛乳铁蛋白肽基因(LfcinB)的合成及其在大肠杆菌中的表达  

Synthesis of Bovine Lactoferricin B Gene (LfcinB) and Its Expression in Escherichia coli

作　　者：郭东华[1] 孙东波[1] 孙斌[1] 范春玲[1] 武瑞[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319

出　　处：《农业生物技术学报》2009年第3期451-454,共4页Journal of Agricultural Biotechnology

基　　金：黑龙江八一农垦大学博士启动基金

摘　　要：根据抗菌肽数据库(APD)报道的牛乳铁蛋白肽(LfcinB)氨基酸序列,合成编码LfcinB基因的两条互补的寡核苷酸链,退火后在5'端和3'端分别形成BamHⅠ和XhoⅠ位点的粘性末端。合成的LfcinB基因定向克隆到pET-32a原核表达载体,阳性克隆菌用TB培养基进行扩大培养,然后用终浓度1.0mmol/L的IPTG进行诱导表达。SDS-PAGE结果显示,LfcinB基因在大肠杆菌(Escherichiacoli)中获得了大量表达,表达蛋白以包涵体形式存在,利用TGE透析液对纯化的LfcinB重组蛋白包涵体进行了复性。抑菌结果显示,复性的LfcinB重组蛋白对氨苄青霉素耐药性大肠杆菌具有良好的抑菌活性。Two complementary sense and antisense oligo-nucleotides, encoding bovine lactoferricin B （LfcinB） gene, were synthesized based on amino acids sequence of bovine lactoferricin B published in the Antimicrobial Peptide Database （APD）. After the sense and antisense oligo-nucleotides annealed, a cohesive BamI-I I site at 5＇ terminus and a cohesive Xho I site at 3＇ terminus were formed. The synthetic LfcinB gene was cloned into prokaryotic expression vector pET-32a. The positive bacteria clones were amplifled and cultured in terrific broth （TB） medium. Then the aimed protein was expressed by 0.1 mmol/L IPTG. The result of SDS-PAGE indicated that LfcinB gene was strongly expressed in Escherichia coil BL21, and the expressed recombinant protein was in form of inclusion body. Inclusion bodies of LfcinB recombinant protein were purified. The purified inclusion bodies were renatured by TGE dialysate. Result of antibacterial test indicated that the refolded recombinant protein showed good antibacterial activity against Escherichia coli BL21 with ampicillin-resistant.

关 键 词：牛乳铁蛋白肽 LfcinB基因 原核表达 

分 类 号：S188[农业科学—农业基础科学]