抗菌肽Dermadistinctin串联表达载体的构建及表达产物抗菌活性的鉴定  

Construction of tandem expression vector expressing antibacterial peptide Dermadistinctin and identification of antimicrobial activity of the expressed fusion protein

作　　者：卢帅[1] 黎满香[1] 刘珂[1] 颜运秋[1] 郑元敬[1] 林荣高[1] 

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128

出　　处：《中国兽医科学》2009年第12期1099-1103,共5页Chinese Veterinary Science

基　　金：湖南省教育厅科技科研项目(06C406);湖南省科技厅科技科研项目(06FJ4101)

摘　　要：根据GenBank中Dermadistinctin K(DDK)和Dermadistinctin L(DDL)的氨基酸序列,选择大肠杆菌偏嗜性密码子,采用SOE法合成了DDK和DDL基因。在DDK及DDL基因序列中引入肠激酶裂解位点,并将其依次克隆入pET-32a(+)质粒,构建了串联体融合表达载体P-DDK,并将其转入Rosetta(DE3)中进行融合表达;表达产物在终浓度为0.1 mmol/LIPTG诱导4 h后可达到最大表达量。对重组蛋白DDK进行镍离子亲和层析柱纯化以及肠激酶裂解之后,对酶切产物进行了抑菌活性分析。分析结果表明,抗菌肽DDK与DDL的混合物对金黄色葡萄球菌(CowanⅠ)、大肠杆菌DH5α、致病性大肠杆菌O1、猪霍乱沙门氏菌、多杀性巴氏杆菌均具有一定的抑菌活性。Based on the gene sequences encoding antibacterial peptide Dermadistinctin K（DDK） and Dermadistinctin L（DDL）registered in GenBank,DDK and DDL genes with enterokinase cleavage sites were designed and synthesized according to the preferential codon of Escherichia coli by SOE method. The modified antibacterial peptide genes of DDK and DDL were both cloned into the pET-32a（＋） vector to con- struct the recombinant expression vector P-DDK which was subsequently transformed into Rosetta（DE3） and induced with IPTG. The expressed proteins of DDK and DDL was obtained,and the maximum expression product was achieved after induction with 0. 1 mmol/L IPTG for 4 hours. Then the fusion protein named DDK was purified, and cleaved by enterokinase to generate antibacterial peptides DDK and DDL. The antibacterial activity of the hybird of DDK with DDL was analyzed by the microtitre dilution method. The results demonstrated that the hybird of DDK with DDL had broad-spectrum antibacterial abilities against Gram-negative bacteria and Gram-positive bacteria including Staphylococcus aureus （Cowan I ）, pathogenic E. coli 01 ,E. coli DHSa, Salmonella choleraesuis and Pasteurella multocida.

关 键 词：抗菌肽Dermadistinctin 串联体 抗菌活性 

分 类 号：S852.42[农业科学—基础兽医学]