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

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.