斑马鱼胸腺苷酸合成酶基因的克隆与表达  

Cloning of thymidylate synthase from zebrafish and its fusion expression

作　　者：杜长青[1,2] 牛荣丽[1] 张培军[1] 林秀坤[1] 

机构地区：[1]中国科学院海洋研究所实验海洋生物学重点实验室,青岛266071 [2]中国科学院研究生院,北京100039

出　　处：《高技术通讯》2006年第4期420-424,共5页Chinese High Technology Letters

基　　金：国家自然科学基金（30472043）资助项目.

摘　　要：用RT-PCR和RACE方法，从斑马鱼胚胎中克隆出胸腺苷酸合成酶（TS）的cDNA全长序列，分析表明，它的编码框序列含有954个核苷酸,编码一个分子量为36kD的318个氨基酸的蛋白序列。将斑马鱼TS的cDNA序列连接到pET-28表达载体上，构建了一个表达载体pET-28/Z-TS,并在大肠杆菌BL21（DE3）中进行了IPTG诱导表达,纯化的TS蛋白在28℃表现出比较高的生物活性，与人的TS蛋白的Km值比较接近。Using total RNA as template, the complete TS eDNA encoding thymidylate synthase was amplified by RT-PCR and RACE. The open reading frame （ORF） of zebrafish TS eDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular weight of 36 kD. The complete ORF encoding the zebrafish TS gene was inserted into pET-28a and an expression vector pET-28/Z-TS was constructed. The zebrafish TS protein was expressed in E. coli BL21（DE3） and purified to homogeneity. The purified zebrafish TS showed the highest activity at 28℃ with similar Km value as human TS.

关 键 词：斑马鱼 胸腺苷酸合成酶 克隆表达 催化活性 

分 类 号：Q78[生物学—分子生物学] Q959.4