猪肝羧酸酯酶1基因的克隆和在原核细胞中的功能性表达  

Cloning and Functional Expression of Pig Liver Esterase 1 Gene in Prokaryotic Cells

作　　者：肖启玲 李朋辉[1,2] 闫秉芳 杨路[1,2] 王喜亮[1,2] 肖运才[1,2] 李自力[1,2] 刘梅[1,2] 毕丁仁[1,2] 石德时[1,2] 

机构地区：[1]华中农业大学动物医学院农业微生物学国家重点实验室,武汉430070 [2]华中农业大学动物医学院农业部兽用诊断制剂创制重点实验室,武汉430070 [3]罗德岛大学生物医学科学系药物基因组学与分子治疗中心,美国金士顿02881

出　　处：《畜牧兽医学报》2015年第4期650-656,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31372484);湖北省自然科学基金(2012FFB02906);中央高校基本科研业务费专项资金(2011PY117)

摘　　要：为获得有活性的猪肝羧酸酯酶1(PLE1)并研究其功能,利用RT-PCR与常规PCR方法,从大白猪肝组织中扩增PLE1基因的ORF。将PLE1的ORF序列连接到pET15b原核表达载体,在Origami TM2(DE3)中与pGro7质粒共诱导表达,纯化目的蛋白质并检测其酶活性。测序结果表明克隆到PLE1基因编码区全长1 686bp的片段,该片段编码562个氨基酸。SDS-PAGE和Western blotting结果显示,PLE1融合蛋白质成功表达,在30℃的培养温度下,IPTG诱导6h后,PLE1重组蛋白质表达量最高,且大部分以可溶性形式存在。酶活性试验结果表明,PLE1融合蛋白质水解底物p-NPA的酶活力为1.24U。研究结果不仅为研究PLE1水解兽药特性提供高纯度的具有原有酶活性的重组蛋白质,也为PLE同工酶家族其他重要成员的活性功能研究提供理论依据和技术手段。In order to obtain active pig liver carboxylesterase 1(PLE1)and study its function,PLE1 were obtained by RT-PCR and normal PCR from large white pig liver.PLE1 ORF was linked into the prokaryotic expression vector pET15 b,and was functional expressed in E.coli Origami TM2(DE3)cells which were transformed ahead with pGro7.The target protein was purified and its enzyme activity was tested.The sequencing results showed that total length of PLE1 ORF is 1 686 bp,which coding 562 aa.The SDS-PAGE and Western Blotting results demonstrated that recombinant protein PLE1 was expressed successfully,and the highest amount was acquired after culture in 30 ℃ for 6h,and most of the protein is soluble,and the recombinant PLE1 hydrolysis activity for p-NPA is 1.24 U.The research results not only provide high purity activePLE1 for the study of PLE1 hydrolysis of veterinary medicine,but also provide the theory basis,technical methods for the function study of other important PLE isoenzymes.

关 键 词：猪肝羧酸酯酶1 功能性表达 活性检测 

分 类 号：S828[农业科学—畜牧学]