多头带绦虫铜锌超氧化物歧化酶基因的原核表达及其表达产物的酶学活性分析  

Prokaryotic expression of Taenia multiceps copper-zinc superoxide dismutase gene and analysis of enzymatic activity of the expressed Cu/Zn-SOD protein

作　　者：黄兴[1,2] 陈林[1] 王宇[1] 徐璟[1] 郭承[1] 古小彬[1] 杨光友[1] HUANG Xing CHEN Lin WANG Yu XU Jing GUO Cheng GU Xiao-bin YANG Guang-you(College of Veterinary Medicine ,Sichuan A gricultural University , Chengdu 611130,China Chengdu A gricultural College, Che ngdu 611130, China)

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]成都农业科技职业学院,四川成都611130

出　　处：《中国兽医科学》2016年第11期1406-1411,共6页Chinese Veterinary Science

基　　金：四川省科技支撑计划项目(2015NZ1504)

摘　　要：为探讨多头带绦虫铜锌超氧化物歧化酶(C u/Zn-SO D)基因的特征及该酶在多头带绦虫上的抗氧化作用,采用RT-PCR技术扩增脑多头蚴C u/Zn-SO D基因,构建原核表达载体p ET-32a-C u/Zn-SO D;然后将其转化到大肠杆菌BL21(DE3)中进行诱导表达;对表达产物进行W estern-blot检测,并进行酶比活性分析。结果显示,多头带绦虫C u/Zn-SO D基因编码蛋白的氨基酸序列与猪带绦虫和肥头绦虫C u/Zn-SO D基因编码蛋白的氨基酸序列的相似性分别为98%和92%;纯化后的多头带绦虫重组Cu/Zn-SOD蛋白的浓度为1.587 m g/m L;用羟胺法测得该重组蛋白的酶比活性为3 125.64 U/mg±76.82 U/mg;H2O2、SDS和EDTA对重组的多头带绦虫Cu/Zn-SOD的酶活性具有较强的抑制作用,而氯仿-乙醇和脲素则对多头带绦虫Cu/Zn-SOD的酶活性并无影响。上述结果为进一步研究多头带绦虫C u/Zn-SO D基因的生物学功能奠定了基础。The aim of this study was to characterize the Cu/Zn superoxide dismutase of Taenia mul- ticeps（T. multiceps Cu/Zn-SOD）,and analyze its roles in the parasite antioxidant system. The T. multiceps Cu/Zn-SODgene was cloned by RT-PCR and analyzed by bioinformatic approaches. A prokaryotic expression vector pET32a-Cu/Zn-SODwas reconstructed and then induced with IPTG. The expression product was puri- fied by Ni-NTA affinity chromatography and analysed by Western-blot and enzymatic activity. Sequence alignment showed that the amino acid sequence of Cu/Zn-SOD gene was highly homologous to those from Taenia solium（98%identity）andTaenia crassiceps（92%）. The enzyme activity of the purified recombi- nant Cu/Zn-SOD protein was 3 125.64 IU/mg±76.82 IU/mg hydroxylamine method,and the activity can be inhibited by H2O2,SDS and EDTA,but C2H5OH-CHCl3 and urea had no effect on the activity of recombinant Cu/Zn-SOD protein. In conclusion,these results provide fundaments for study on biofunctions of T. mul- ticeps Cu/Zn-SOD.

关 键 词：多头带绦虫 Cu/Zn-SOD基因 原核表达 酶学活性分析 

分 类 号：S852.734[农业科学—基础兽医学]