乙酰乙酰辅酶A还原酶基因phbB克隆及其表达初探

Molecular cloning and primary expressed exploration of acetoacetylCoA reductase gene (phbB)

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。

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.