哈维氏弧菌胞外蛋白酶基因的表型克隆及序列分析.pdfVIP

生物技术通报 BIO TE CHN OLOG Y B ULLETIN 2010 10 潘晓艺 沈锦玉 曹铮 尹文林 郝贵杰 徐洋 姚嘉赟 (, 313001) : 哈维氏弧菌是引起大黄鱼疾病 主要致病菌之一, 胞外蛋白酶是哈维氏弧菌 GYC11081 主要致病因子利 用表型克隆技术, 用 Sau3A 将提取 哈维氏弧菌基因组 DNA 酶切成短片段, 纯化回收 1- 5 kb 片段与BamH I酶切 pUC118在 T 连接酶 作用下进行连接,并转化感受态细胞 TG1,在含 0. 5%脱脂奶和 100 g/mL氨苄青霉素 L 平板上筛 4 选胞外蛋白酶阳性株, 命名为 YZ测定阳性株 YZ 胞外蛋白酶活性,并对阳性株 重组质粒 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 ORF编码 531 个氨基酸,在起始密码子 ATG 上游存在 一个 70 核糖体结合位点 ( SD) , 其上游 启动区与大肠杆菌 启动区和枯草芽孢杆菌 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 2 个转录终止子哈维氏弧菌胞外蛋白酶基因 克隆与诠释为哈维氏弧菌致病机理和疫苗 开发奠定了基础 : 哈维氏弧菌 大黄鱼 胞外蛋白酶 表型克隆 Phenotypic Cloning and Sequence Analysis of Extracellular Protease Gene of Vibrio harvey i PanX iaoyi Shen Jinyu Cao Zheng Y inW enlin HaoGuijie XuYang Yao Jiayun (K ey Laboratory of FishHealth and Immunology, ChineseA cademy of F ishery Science, Zhejiang Institute of Fresh ater Fisheries,Huzhou 313001) Abstrac:t A phenotypic cloning approach was used to isolate a Vibrio harveyi cDNA encoding extracellular protease. The genomic DNA of Vibrio harveyi GYC11081 digested by Sau3A I and the 1- 5 kb fragmentw as purified by agarose gel electrophoresis. Then the purifiedGYC11081DNA samplesw ere ligated intoBamH Idigested vector pUC118 and then electorporated into Escherichia coli strain TG1. Itw asmaintained on Luria ertani( L ) medium containing 0. 5% skmi medmilk and 100 g /mL Amp at 37!C. The positive strain of extracellular protease was selected which named YZ. The extracellular protease activity of the strain YZ w as determ inated. DNA se quence determ inationw as performed by Shanghai Invitrogen iotechnology Company w ith anA I PRISM 377 DNA Sequencer. Open read