麻鸭磷脂氢谷胱甘肽过氧化物酶高效可溶性表达及其部分酶学性质  

Highly efficient soluble expression and partial enzymatic properties of shelduck phospholipid hydroperoxide glutathione peroxidase

作　　者：王晶晶[1,2] 张新笑 卞欢 耿志明[1] 李鹏鹏[1] 王道营[1] 徐为民[1,3] WANG Jing-jing;ZHANG Xin-xiao;BIAN Huan;GENG Zhi-ming;LI Peng-peng;WANG Dao-ying;XUWei-min(Institute of Agro-product Processing,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Key Laboratory of Meat Processing and Quality Control of Ministry of Education,Nanjing Agricultural University, Nanjing 210095,China;Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control,Nanjing 210095,China)

机构地区：[1]江苏省农业科学院农产品加工研究所,南京210014 [2]南京农业大学肉品加工与质量控制教育部重点实验室,南京210095 [3]江苏省肉类生产与加工质量安全控制协同创新中心,南京210095

出　　处：《南方农业学报》2019年第5期1120-1126,共7页Journal of Southern Agriculture

基　　金：国家自然科学基金青年科学基金项目(31701532);江苏省自然科学基金项目(BK20171324);江苏省农业科学院农产品加工研究所基金项目(JG201702)

摘　　要：[目的]克隆麻鸭磷脂氢谷胱甘肽过氧化物酶基因(GPx4)并在大肠杆菌中表达,分析其酶学特性,为研究GPx4功能及其在羟基脂肪酸形成过程中的调节机制打下基础。[方法]采用RT-PCR克隆麻鸭GPx4(DGPx4)的编码基因,利用定点突变的方法构建半胱氨酸突变体DGPx4表达载体pMBP-DGPx4(48Sec→ Cys),并在大肠杆菌中通过添加His标签进行可溶性表达,经Ni-NTA亲和层析和离子交换层析纯化得到融合蛋白DGPx4;采用总谷胱甘肽过氧化物酶活性检测试剂盒检测DGPx4活力以研究其酶学性质。[结果]克隆获得的DGPx4基因编码序列全长516 bp,编码172个氨基酸,编码蛋白分子量约20 kD,理论等电点(pI)为8.9。构建的突变体基因原核表达载体pMBP-DGPx4(48Sec→Cys)可在大肠杆菌中成功诱导表达获得融合蛋白DGPx4,经Ni-NTA亲和层析和离子交换层析即获得高纯度的DG-Px4。以烟酰胺腺嘌呤二核苷酸磷酸(NADPH)为底物时,DGPx4的最适反应温度为32 ℃,最适pH为8.0,对NaCl浓度较敏感;Cu^2+和Ni^2+对DGPx4活力有较高的促进作用,Mg^2+和Mn^2+对DGPx4活力有一定的抑制作用,Ca^2+和Zn^2+对DG-Px4活力则无明显的促进或抑制作用。[结论]从鸭肝细胞中克隆获得的DGPx4基因序列经定点突变后可在原核细胞中高效表达,且纯化后的高纯度融合蛋白DGPx4具有GPx4活力,能在抗脂质氧化过程中发挥作用。【Objective】The shelduck phospholipid hydroperoxide glutathione peroxidase gene(GPx4)was cloned and expressed in Escherichia coli,and its enzymatic properties were analyzed,which laid a foundation for preliminary study of GPx4 function and its regulation mechanism in the formation of hydroxy fatty acids.【Method】Shelduck GPx4 (DGPx4)encoded gene was cloned by RT-PCR,and site-directed mutation was used to established cysteine mutant DGPx4 expression vector pMBP-DGPx4(48Sec→Cys). His tag was added into E. coli to achieve soluble expression. Fusion protein DGPx4 was obtained by Ni-NTA affinity chromatography and ion exchange chromatography purification. DGPx4 activity and the enzymatic properties were measured by total glutathione peroxidase activity assay kit.【Result】The results showed that the obtained DGPx4 gene coding sequence was 516 bp in length and encoded 172 amino acids. The encoded protein had a molecular weight of approximately 20 kD and a theoretical isoelectric point(pI)of 8.9. The constructed mutant gene prokaryotic expression vector pMBP-DGPx4(48Sec→Cys)was successfully induced to express the fusion protein DGPx4 in Escherichia coli,and high purity DGPx4 was obtained by Ni-NTA affinity chromatography and ion exchange chromatography. When nicotinamide adenine dinucleotide phosphate(NADPH)was used as the substrate,the optimum reaction temperature for DGPx4 was 32 ℃,and the optimum pH was 8.0,and sensitive to NaCl concentration. Cu^2+ and Ni^2+ could activate the activity of DGPx4,Mg^2+ and Mn^2+ inhibited its activity,while Ca^2+ and Zn^2+ had no obvious effect on DGPx4 activity.【Conclusion】The DGPx4 gene sequence cloned from duck liver cells can be highly expressed in prokaryotic cells after site-directed mutagenesis,and the purified high-purity fusion protein DGPx4 has GPx4 activity and can play a role in anti-lipid oxidation.

关 键 词：麻鸭 磷脂氢谷胱甘肽过氧化物酶(GPx4) 原核表达 纯化 酶学性质 

分 类 号：S843.83[农业科学—畜牧兽医]