哈维氏弧菌胞外蛋白酶基因的表型克隆与序列分析

Phenotypic Cloning and Sequence Analysis of Extracellular Protease Gene of Vibrio harveyi

哈维氏弧菌是引起大黄鱼疾病的主要致病菌之一,胞外蛋白酶是哈维氏弧菌GYC1108-1的主要致病因子。利用表型克隆技术,用Sau3AⅠ将提取的哈维氏弧菌基因组DNA酶切成短片段,纯化回收1-5 kb的片段与BamH I酶切的pUC118在T4连接酶的作用下进行连接,并转化感受态细胞TG1,在含0.5%脱脂奶和100μg/mL氨苄青霉素的LB平板上筛选胞外蛋白酶阳性株,命名为YZ。测定阳性株YZ的胞外蛋白酶活性,并对阳性株的重组质粒的插入片段进行序列测定,分析开放阅读框,确定胞外蛋白酶基因编码区。发现蛋白酶基因的ORF编码531个氨基酸,在起始密码子ATG上游存在一个核糖体结合位点(SD),其上游的启动区与大肠杆菌的σ70启动区和枯草芽孢杆菌的启动区高度同源。在终止子(TAG)下游,存在两个反向重复序列,为推导的2个转录终止子。哈维氏弧菌胞外蛋白酶基因的克隆与诠释为哈维氏弧菌致病机理和疫苗的开发奠定了基础。

A phenotypic cloning approach was used to isolate a Vibrio harveyi eDNA encoding extracellular protease. The genomic DNA of Vibrio harveyi GYC1108-1 digested by Sau3A I and the 1 - 5 kb fragment was purified by agarose gel electrophoresis. Then the purified GYC1108-1 DNA samples were ligated into BamH l-digested vector pUCll8 and then electorporated into Escherichia coli strain TG1. It was maintained on Luria-Bertani （LB） medium containing O. 5 % skimmed milk and 100 μg,/mL Amp at 37℃. The positive strain of extracellular protease was selected which named YZ. The extracellular protease activity of the strain YZ was determinated. DNA se- quence determination was performed by Shanghai Invitrogen Biotechnology Company with an ABI PRISM 377 DNA Sequencer. Open read- ing frame （ORF） of extracellular protease gene was analysised. Subsequent sequence analysis revealed that the ORF of protease gene encoded 531 amino acids,a ribosome binding site （SD） in the upstream of initiation codon ATG,and the promoter region was homologous to motifs of the σ^70 promoter region of Escherichia coli and the promoter region of Bacillus subtilis. There are two inverted repeat sequenees in the downstream of termination codon （TAG）, which derive the two transcription terminators. Cloning and interpretation of Vibrio harveyi extracellular protease gene ,it laid the foundation of the research of pathogenesis of Vibrio harveyi and the development of vaccines.