抗菌肽cecropin P1在大肠杆菌中的融合表达及活性测定  

Fusion Expression of Cecropin P1 in Escherichia Coli and Its Antimicrobial Activity Analysis

作　　者：翟琼[1] 叶江[1] 莫剑[1] 张惠展[1] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

出　　处：《食品与药品》2011年第1期1-5,共5页Food and Drug

摘　　要：目的在大肠杆菌中融合表达抗菌肽cecropin P1。方法根据抗菌肽cecropin P1的氨基酸序列,依照大肠杆菌密码子的偏爱性,采用SOE技术合成cecropin P1的编码基因;将其克隆至表达载体pET-32a(+)上,经IPTG诱导表达,超声上清液用Ni-NTA亲和层析初步纯化,经肠激酶切割后,用薄层平皿琼脂糖孔穴扩散法初步测定重组cecropin P1的活性。结果构建得表达质粒pET32-P1,经IPTG诱导表达获得融合蛋白TrxA-cecropin P1,占总蛋白质25%以上;纯化产物经肠激酶切割后,Tricine-SDS-PAGE显示成功释放抗菌肽cecropin P1;初步测定结果显示其有抑菌活性。结论本研究为研发重组抗菌肽cecropin P1的生产工艺奠定了基础。Objective To achieve the fusion expression of cecropin P1 in E.coli. Methods According to the biased coden used in E.coli, the gene sequence encoding cecropin P1 was designed. The gene fragment of cecropin P1 was first obtained through gene SOEing; then the DNA sequence was inserted into pET-32a（＋） vector. After IPTG induction and Ni-NTA affinity chromatography, the fusion protein was cleaved by recombinant enterokinase. The activity of recombinant cecropin P 1 was determined by agarose diffusion assay on thin-layer plate. Results The fusion expression vector pET32-P 1 was constructed. After IPTG induction, TrxA-cecropin P 1 fusion protein was successfully expressed, exceeding 25% of the total soluble proteins. After cleavage by enterokinase, Tricine-SDS-PAGE showed that cecropin P1 fusion protein was released successfully. The recombinant cecropin P1 exhibited antibacterial activity, which suggested the functional cecropin P1 has been achieved by gene engineering method. Conclusion This study layed a foundation for the development of production technology of cecropin P 1.

关 键 词：CECROPIN P1 融合表达 Ni—NTA亲和层析 肠激酶 抑菌活性 

分 类 号：R978.16[医药卫生—药品]