从大肠杆菌中表达、纯化宁乡猪N-乙酰谷氨酸合成酶并制备其多克隆抗体

Expression and Purification of N-acetylglutamate Synthase from Ningxiang Pig in Escherichia Coli and Preparation of Its Polyclonal Antibody

N-乙酰谷氨酸合成酶催化生成的N-乙酰谷氨酸(NAGS)对于哺乳动物尿素循环第一个酶—氨基甲酰磷酸合成酶I变象异构激活是必需的。N-乙酰谷氨酸合成酶定位于肝脏和小肠线粒体基质中,通过提供N-乙酰谷氨酸调节氨基甲酰磷酸合成酶I的活性来调节尿素合成。我们用RT-PCR方法从宁乡猪肝脏中扩增了N-乙酰谷氨酸合成酶的开发阅读框,并将此基因连接到原核表达载体上,构建了pET-NAGS质粒。将重组质粒转化到Ec.oliBL21(DE3),在IPTG诱导下表达His-NAGS融合蛋白。通过SDS-PAGE,得出NAGS分子量约为40kDa。一步亲和层析纯化后,我们将纯化后的NAGS蛋白注射到新西兰大白兔中制备多克隆抗体。通过免疫组化和免疫印迹测试抗体,结果表明此抗体有较好的抗原性和特异性。据我们所知,这是第一次在大肠杆菌中表达来源于宁乡猪的NAGS。

N-acetylglutamate synthase（NAGS） catalyzes the formation of N-acetylglutamate（ NAG）, which is an obligatory allosterie activator of carbamylphosphate synthetase Ⅰ （ CPSI）, the first enzyme of the urea cycle in mammals. NAGS is localized in the mitochondrial matrix of the liver and intestinal cells and has a potential regulatory role in urea genesis by supplying NAG for modulating CPSI activity. In this study, the coding region of pNAGS was amplified from Ningxiang pig liver tissue by RT-PCR and the recombinant prokaryotic expression vector pET-NAGS was constructed. The vector was transformed into E. coli BI21 （ DE3 ） to express the his-NAGS fusion protein in the bacteria under induction of IPTG. The molecular mass of NAGS was 40 kDa by SDS-PAGE, and appeared to be an oligomer. After purification by one-step affinity chromatography, the fusion protein was injected into New Zealand rabbits to prepare polyclonal antibody. Then the antibody was tested by immunobistochemisty and western blot, and the results showed that NAGS had fine antigenicity and specificity. As we know, this is the first report of expression in E. coli of the NAGS protein from Ningxiang pig.