单 位：
东北农业大学生命科学学院;北京大学蛋白质工程及植物基因工程国家重点实验室
关键词：
聚-β-羟基丁酸酯;重组表达;基因克隆;纯化;表达载体
摘 要：
从真养产碱杆菌(Alcaligenes eutrophus)中克隆得到PHB合成的关键酶基因,NADPH依赖性的乙酰乙酰辅酶A还原酶基因phbB(GenBank ID:KC191672),其DNA长度为741 bp,编码246个氨基酸(aa),与GenBank数据库中的entrophusPhbB(FJ897462.1)的同源性为70.99%。属于膜蛋白或分泌蛋白,推测该蛋白可能定位在细胞膜上。成功构建原核表达载体pET28a(+)-phbB-HE。经SDS-PAGE电泳检测获得phbB基因表达蛋白,其分子质量为31 ku,为研究乙酰乙酰辅酶A还原酶活性及后期蛋白功能鉴定奠定基础。
译 名：
Molecular cloning and primary expressed exploration of acetoacetylCoA reductase gene (phbB)
作 者：
CANG Jing;JIANG Xiaojuan;XU Zhichao;LIN Zhongping;DU Juan;School of Life Sciences, Northeast Agricultural University;National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University;
关键词：
poly-β-hydroxyl butyrate;;recombinant expression;;gene clone;;purification;;expression vector
摘 要：
The full-length cDNA sequence of NADPH-dependent acetyl-CoA enzyme reductase gene named phbB(GenBank ID: KC191672) as the key enzymes of PHB synthase gene were cloned from Alcaligenes faecalis(Alcaligenes eutrophus). According to entrophusPhbB(FJ897462.1) sequences that has been speculated. The gene homology was 70.99%, with the length of 740 bp encoded 246 amino acids( aa). The recombinant plasmid named pET28a(+)- phbB- HE were constructed successfully.The products were identified by SDS- PAGE analysis, it confirmed that high efficiency expression of the phbB protein fragments(ca.31 ku) were demonstrated. It laid a solid foundation for further study of phbB gene activity and late protein identification.
