家蝇抗菌肽Defensin基因同向串联表达载体的构建和鉴定  

Construction and Identification of Cloned Vector with Multi-copy Musca domestica Antimicrobial Peptides-Defensin in the Same Direction

作　　者：王婷婷[1] 金小宝[2] 朱家勇[2] 刘雷山[2] 

机构地区：[1]广东医学院,广东湛江520423 [2]广东药学院病原生物学教研室,广东广州510224

出　　处：《生物技术》2006年第2期3-5,共3页Biotechnology

基　　金：广州市科技局科技攻关项目(No.2005Z3-E0211)

摘　　要：目的:构建家蝇抗菌肽Defensin基因多拷贝串联体,并克隆到甲醇酵母分泌表达载体pPIC9K上。方法:PCR法扩增家蝇抗菌肽Defensin基因成熟肽片断,目的片断的上游5′端带有EcoRⅠ和NheⅠ位点,下游5′端带有NotⅠ和XbaⅠ位点,目的片断首先克隆入pMD18-T载体,利用pMD18-T载体的NdeⅠ位点和目的片断上的一对同尾酶(NheⅠ和XbaⅠ),多次酶切连接,串联成多拷贝的Defensin成熟肽基因,再用EcoRⅠ和NotⅠ双酶切,最后克隆入甲醇酵母分泌表达载体pPIC9K。结果:PCR鉴定、酶切鉴定和DNA测序证明多拷贝基因重组质粒构建成功。结论:该方法能方便高效地获得所需的多拷贝基因,为进一步进行高效表达打下基础。Objectives： To construct a tandem repeated gene recombinant plasmid pPIC9K Defensin-8. Methods： Musca-domstica anfimicrobial peptides-Defensin without singnal pepfide was cloned using PCR.The DNA containe EcoR Ⅰ and Nhe Ⅰ in up 5′, Not Ⅰ and Xba Ⅰ in down 5′. Defensin was constructed into was cloned in pMD18 - T. Using Nde Ⅰ in pMD18 - T , Xba Ⅰ and Nhe Ⅰwho have the same position. Attacin was constructed into pPIC9K following restriction endonuclease and link again and again. Results： PCR amplification and DNA sequencing analys is confirmed that 8 - copy gene was correctly in serted into the vector. Conclusion：With this method we can conveniently and efficiently obtain the desired multi- copy gene , which lays the basis for the cost effective expression.

关 键 词：家蝇 抗菌肽 Defensin基因 同向串联 多拷贝 

分 类 号：Q963[生物学—昆虫学]