海洋微生物信号降解酶基因aiiA克隆与表达载体的构建  

Clone and construction of recombinant expression vector of signal degrading-enzyme gene aiiA from marine bacteria

作　　者：丁贤[1,2] 殷波 张善夫 唐维可 孙威文[2] 周世宁[2] 

机构地区：[1]中国水产科学研究院南海水产研究所农业部南海渔业资源开发利用重点实验室,广东广州510300 [2]中山大学生命科学学院有害生物控制与资源利用国家重点实验室,广东广州510275 [3]中科院南海海洋研究所LTO国家重点实验室,广东广州510301 [4]淮安大江水产饲料有限公司,江苏淮安223003

出　　处：《中国微生态学杂志》2012年第6期485-489,共5页Chinese Journal of Microecology

基　　金：国家863项目(2008AA09Z402);广东省科技计划项目(2009B020311003);广东省教育部产学研结合项目(2011B090400151);江苏省科技项目(BN2010038);中国水产科学研究院基本科研业务费(2010TS01)

摘　　要：根据已知的微生物信号降解酶基因aiiA的序列设计、合成特异性引物探针,以从海洋分离的微生物ZD02的基因组为模板,PCR扩增编码蛋白aiiA信号降解酶的基因aiiA序列,产物经PCR验证后用于构建克隆载体pMD18-ZD02aiiA,并以此克隆载体为模板,以带酶切位点的引物扩增基因,经BamHI和EcoRI双酶切后将其插入表达载体pET-17b,构建原核表达质粒pET-ZD02aiiA。经酶切、PCR鉴定及序列测定等,结果表明:克隆基因已正确插入到载体的多克隆位点,序列和读码框正确,为海洋微生物ZD02信号降解酶基因的体外重组和诱导表达研究打下基础。A pair of proper primers were designed and synthesized for Polymerase Chain Reaction （PCR） of signal de- grading-enzyme gene aiiA which encodes protein aiiA from marine bacterial genome, according to the gene sequences of aiiA. The plasmid pMD18-ZDO2aiiA was constructed after PCR testing of aliA gene. The gene sequence of aliA was amplified with proper primers （with active locations by enzymes BamHland EcoRI） from moulding board of pMD18-ZDO2aiiA and linked into expression vector pET-17b after it was hydrolyzed by enzymes of BamHI and EcoRI. And then plasmids pET-ZDO2aiiA was constructed. The results of hydrolyzation of enzymes, PCR amplification and sequencing demonstrated that the target gene frag- ments was inserted into vector pET-17b correctly, the sequences of gene aliA and frame of reading codes were right. Thus, it may provide basis for the recombinant expression and induced expression of signal degrading-enzyme gene from marine bacteria.

关 键 词：细菌 信号降解酶因aiiA 克隆 

分 类 号：Q936[生物学—微生物学]