天蚕素A-蜂毒素杂合肽用pBV220载体的表达、纯化和活性测定  

Expression of Cecropin A-Melittin Hybrid Peptide by pBV220 Vector and Its Purification and Activity Assay

作　　者：王云起[1] 刘飞鹏[2] 李月琴[2] 张欣[2] 蔡继业[3] 

机构地区：[1]暨南大学化学系 [2]暨南大学生物工程系 [3]暨南大学化学系广州510632

出　　处：《中国生物工程杂志》2005年第10期29-33,共5页China Biotechnology

基　　金：国家"973"重点基础研究发展规划项目(2001CB510101);国家自然科学基金重点项目(30230350);广东省自然科学基金资助项目(970636)

摘　　要：为提高抗菌肽的表达,在抗菌肽的N端融合了1段酸性小肽以中和表达产物对宿主的毒性;并将融合肽基因同向串连成多拷贝,在大肠杆菌中获得了较高的表达。用化学合成法分别合成了编码天蚕素A(1-8)-蜂毒素(1-10)杂合肽和酸性小肽的DNA片段,首先将其拼接成融合肽的完整基因,然后通过前后接头将融合肽基因连接成两侧具有EcoRI和SalI酶切位点的同向串连的多拷贝基因。将5份拷贝的基因克隆至pBV220表达载体,转化E.coliDH5α,温度诱导得到表达量为35%的融合蛋白。表达产物主要以包涵体形式存在,将包涵体溶解,经Ni2+-NTA琼脂糖亲和层析获得纯化的融合蛋白。融合蛋白再经CNBr切割和阳离子交换层析,得到纯化的抗菌肽,经蛋白质N端测序确认序列正确。琼脂糖扩散法和液相测定法证明了纯化的抗菌肽具有抗菌活性。To improve antimicrobial peptide＇ s yield, an acidic peptide was fused to nitrogen end of antimicrobial peptide to neutralize the virulence of antimicrobial peptide on the host cells. Multiple copies of the fusion peptide gene were linked in tandem and were expressed in E. coli expression system at high levels. Two DNA fragments encoding cecropin A （1-8）-melittin （1-10） hybrid and an acidic peptide were chemically synthesized, respectively. First, the two fragments were linked to a whole fusion peptide gene, then the whole gene was ligated to tandem repeats which are flanked with EcoRI and SalI cohesive end. Five-copy gene was cloned into pBV220 vector and transformed into E. coli DH5α. The resulting expression level of multi-copy fusion peptide reached up to 35% of total cell proteins. The fusion protein mainly existed as inclusion bodies. The inclusion bodies were solved and purified by Ni^2＋ -NTA-Agarose affinity chromatography. The target product, CAME peptide, was purified after CNBr treatment and cation-exchange chromatography and was confirmed with Nterminal amino acid sequencing. Results of agarose diffusion assay and liquid turbidity analysis indicated that CAME peptide exhibited the antibacterial activity.

关 键 词：天蚕素A-蜂毒素杂合肽 pBV220载体 基因表达 基因纯化 基因活性 抗菌肽 免疫系统 

分 类 号：Q516[生物学—生物化学] R978[医药卫生—药品]