抗菌肽DCD-1L在大肠杆菌中的表达及活性鉴定  

Fusion Expression of DCD-1L in Escherichia coli and Its Antimicrobial Activity Analysis

作　　者：王炯蓉[1] 李志忠[1] 王芳[2] 袁红霞[2] 田彩平[2] 廖世奇[2] 马瑾[2] 马宁[2] 黄小娟[1] 杨楠[3] 

机构地区：[1]兰州理工大学生命科学与工程学院,甘肃兰州730050 [2]甘肃省医学科学研究院,甘肃兰州730050 [3]西北师范大学生命科学学院,甘肃兰州730070

出　　处：《生物技术》2013年第6期37-41,共5页Biotechnology

摘　　要：目的:利用大肠杆菌表达系统表达抗菌肽DCD-1L并鉴定其抑菌活性。方法:根据DCD-1L氨基酸序列以及大肠杆菌密码子偏爱性设计出DCD-1L的基因序列,通过重叠延伸PCR扩增出完整的DCD-1L编码序列,接着用酶切,连接,转化等方法获得重组表达载体pET-32a-DCD-1L,并将其进行PCR,酶切及测序鉴定。将鉴定正确的重组体转化E.coli transetta菌株,经终浓度0.5mmol/L IPTG诱导表达后,用SDS-PAGE分析目的蛋白表达状况,并用抑菌圈实验检测其抑菌活性。结果:SDS-PAGE检测DCD-1L在28KDa处有目的条带,抑菌圈实验结果显示DCD-1L对地衣芽胞杆菌,金黄色葡萄球菌,绿脓杆菌以及肺炎链球菌均有明显的抑菌活性。结论:成功构建重组表达载体pET-32a-DCD-1L并在E.coli transetta菌株中成功表达,获得了对多种革兰氏阳性菌和革兰氏阴性菌都有抑菌活性的抗菌肽DCD-1L。Objective ： To achieve the DCD - 1L in E. coli expression system and identificate the anti - bacterial activity. Method ： The gene sequence of DCD - 1L was designed based on the amino acid sequence of DCD - 1L and the codon bias of E. coli. Then,through re- striction enzymes digestion, connection and transformation achieve the recombinant expression vector pET -32a - DCD - 1L , identifypET - 32a - DCD - 1L by having it undergo PCR, enzyme cleavage, and sequencing, transformed right pET - 32a - DCD - 1L into E. coli transetta cell to express DCD -1L. Have the induced by IPTG of final concentration is 0. 5mmol/L and use SDS- PAGE to analyze pro- tein expression. Use the inhibition zone test to test for inhibition of anti - bacterial activity. Result： SDS - PAGE analysis showed that the molecular weight of 28 kDa, and the inhibition zone test indicates that DCD - 1L has obvious inhibiting effects on B. licheniformis, S. aureus, P. aerugirtosa and S. pneumoniae. Conclusion ：The recombinant expression veetor pET - 32a - DCD - 1L was successfully con- structed and expressed in E. coli transetta, the expression product DCD - 1L has obvious inhibiting effects on Gram - positive and Gram - negative bacterium.

关 键 词：抗菌肽 DCD-1L 大肠杆菌 基因重组 融合表达 抑菌活性 

分 类 号：Q786[生物学—分子生物学]