草鱼脂肪酸结合蛋白基因克隆及其在大肠杆菌中的表达

Gene cloning and expression of fatty acid-binding protein from grass carps(Ctenopharyngodon idellus) in Escherichia coli.

根据GenBank上已发表的其他物种的脂肪酸结合蛋白(fatty acid-binding protein,FABP)基因序列设计1对引物,从草鱼(Ctenopharyngodon idellus)cDNA文库中扩增出脂肪酸结合蛋白基因,定向克隆于表达质粒载体pET32A中构建成pET32A-FABP重组体,将重组体转化至大肠杆菌BL21(DE3)中,以终浓度1 mmol·L-1的IPTG对其进行诱导表达.表达产物经SDS-PAGE电泳分析表明,与阴性对照相比,阳性克隆在分子量33 kDa处有1条明显的蛋白带,大小与预期结果一致.经光密度分析可知,目的蛋白约占总可溶性蛋白的49.5%.这为进一步研究脂肪酸结合蛋白生物学特性以及FABP基因对脂肪代谢调控规律奠定了基础.

A pair of primers were designed based on the conservative sequences of FABP gene of other species published in the GenBank, and the FABP gene was amplified using grass carp （Ctenopharyngodon idellus） cDNA library as templates. Then the FABP gene was directionally cloned into expression vector pET32A to construct pET32A-FABP recombinant. This recombinant was transformed into Escherichia coli BE21 （DE3）. After induction by IPTG, an expected 33 kDa protein was found by SDS PAGE analysis, accounting for 49.5% of total soluble protein. The result lays helpful foundation for understanding the biological function of fatty acid-binding protein and effects of FABP gene on fatty acid metabolism.