摘要
构建杂合抗菌肽Attacin-Thanatin(简称mAT)融合表达重组菌株,对其在大肠杆菌系统中的表达特性及其融合表达产物的菌内抑菌活性进行了研究。将mAT重组表达质粒pET-mAT(pe)依次转化宿主菌E.coliDH5α、BL21(DE3)、Rosetta-gamiTM(DE3)pLysS,采用IPTG诱导表达;优化筛选该抗菌肽的敏感菌株、IPTG浓度、宿主菌诱导起始浓度等,初步建立了抗菌肽菌内抑菌活性检测方法,用于研究该杂合抗菌肽的体内抑菌活性。SDS-PAGE分析结果显示,宿主菌Rosetta-gamiTM(DE3)pLysS可获得表达的融合产物,而E.coliDH5α、BL21(DE3)经IPTG诱导后未检测到目的条带;优化结果显示,以E.coliDH5α宿主菌、IPTG工作浓度0.1 mmol/L、起始诱导菌浓度D600 nm值0.3为检测抗菌肽菌内抑菌活性的最佳条件;mAT融合表达产物抑菌活性的检测结果显示,宿主菌被本身所表达的杂合抗菌肽融合蛋白所抑制,增殖速度放缓。利用Rosetta-gamiTM(DE3)pLysS有效融合表达mAT并初步建立的抗菌肽菌内抑菌活性快速检测方法为进一步研究mAT奠定了基础。
To construct the fusion expression strains of the hybrid peptide Attacin-Thanatin（mAT）, and to detect it＇s expression property in Escherichia coli system as well as the bacteriostatic activity of the fusion expression product,the recombinant expression plasmids pET mAT（pe） was transformed into E. coli DHSa, BL2 1 （DE3）, Rosetta-gamiTM （DE3） pLysS respectively and induced with IPTG. The method for detection of bacteriostatic activity was established by selecting the susceptible strain,the concentration of IPTG and the starting D600nm of the host strain,and was applied to detect the bacteriostatic activity of mAT in vivo. SDS-PAGE analysis indicated that this mAT could be expressed in Rosetta-gami^TM （DE3） pLysS but not expressed in E. coli DH5α and BL21 （DE3）. Result of optimization analysis demonstrated that it was a feasible method when E. coli DH5α was taken as the host strain, meanwhile, the concentration of IPTG was 0. 1 mmol/L and the starting D600 of the host strain was 0.3. Detection of bacteriostatic activity showed that the host strain was inhibited by the fusion protein of mAT, and its propagation rate was slowed down. The successful expression of mAT in Rosetta-gamiTM （DE3） pLysS and the development of method for detection of its bacteriostatic activity in vivo established the foundation for further researches on mAT.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2009年第6期531-537,共7页
Chinese Veterinary Science
基金
四川省生物饲料关键技术项目(2007Z06-050)
教育部"长江学者和创新团队发展计划"项目(IRTO555-5)
