利用E.coli-App穿梭载体构建温控双基因裂解载体pMC-WK  

Construction of E.coli-App temperature controlled double gene cracking vect or pMC-WK

作　　者：孙颖[1] 金天明[1] 冯立文[2] 袁洁[3] 

机构地区：[1]天津农学院动物科学系,天津300384 [2]吉林大学畜牧兽医学院,吉林长春130062 [3]广东省农业科学院兽医研究所,广东广州510640

出　　处：《中国兽医学报》2011年第9期1304-1308,共5页Chinese Journal of Veterinary Science

基　　金：天津市高等学校科技发展基金计划项目(20060720);国家自然科学基金资助项目(31072109)

摘　　要：采用PCR方法从噬菌体PhiX174基因组中扩增出裂解蛋白E基因,从金黄色葡萄球菌基因组中扩增出核酸酶A基因(SN),通过15个柔性氨基酸linker将双基因串联,插入PBV220表达载体,构建温控双基因裂解系统(DLS)。PCR扩增DLS,将其与E.coli-App穿梭载体pMC-Express相连,构建重组穿梭载体pMC-WK。裂解试验表明,E-15aalinker-SN裂解较E裂解迅速而彻底,菌体裂解率达到99.999 4%;经PCR扩增出的DLS具有裂解活性。本试验成功构建了温控双基因裂解载体pMC-WK,为App菌影疫苗的研究奠定了基础。Using the PCR method to clone lysis protein E gene from the genome of phage PhiX174 and nuclease A gene（SN） from the genome of Staphylococcus aureus. Link the two genes with 15 amino acid flexible linker to get double-gene tandem, then insert it into PBV220 plasmid.Amplifing DLS through PCR to construct pMC-W K on the basis of E.coli-App shuttle vector pMC-Express.Lysis experiments s how th at E-15aalinker-SN system has better cracking efficiency than E gene system.Th e lysis rate can be 99.999 4% and DLS gene amplified by PCR with the cleavage acti vity.The test successfully constructed temperature controlled double gene cracki ng vector pMC-WK,making foundation for the App bacterial ghost vaccine research.

关 键 词：裂解蛋白E 核酸酶A App菌影 穿梭载体 

分 类 号：S852.2[农业科学—基础兽医学] S852.61[农业科学—兽医学]