[人铜/锌超氧化物歧化酶基因在乳酸乳球菌中的克隆与表达]

[Cloning and expression of human Cu/Zn superoxide dismutase gene in Lactococcus lactis].

作者信息

Xiang H, Wei W Z, Tan H R, Guo S X

机构信息

Institute of Microbiology, Chinese Academy of Sciences, Beijing.

出版信息

Sheng Wu Gong Cheng Xue Bao. 2000 Jan;16(1):6-9.

PMID:10883266

Abstract

The cDNA encoding human Cu/Zn SOD was amplified by RT-PCR using the total RNA of human liver as the template, and was cloned into an E. coli expression vector pET23b. After the DNA sequence was determined, the recombinant plasmid pET23bsod was introduced into E. coli BL21(DE3)/pLysS. SDS-PAGE analysis revealed that the recombinant E. coli expressed the predicted 19 kDa human Cu/Zn SOD, and its amount was over 50% of total proteins. The Cu/Zn SOD cDNA was then subcloned into a lactococcal expression vector pMG36e, and resulting pMG36esod was introduced into L. lactis MG1363 by electroporation. The human Cu/Zn-SOD was expressed up to 5% of the soluble proteins, and the enzymatic activity was also observed by SOD activity dying.

摘要

以人肝脏的总RNA为模板，通过RT-PCR扩增编码人铜锌超氧化物歧化酶（Cu/Zn SOD）的cDNA，并将其克隆到大肠杆菌表达载体pET23b中。测定DNA序列后，将重组质粒pET23bsod导入大肠杆菌BL21(DE3)/pLysS。SDS-PAGE分析表明，重组大肠杆菌表达了预测的19 kDa人铜锌超氧化物歧化酶，其含量超过总蛋白的50%。然后将铜锌超氧化物歧化酶cDNA亚克隆到乳酸乳球菌表达载体pMG36e中，并通过电穿孔将所得的pMG36esod导入乳酸乳球菌MG1363。人铜锌超氧化物歧化酶的表达量高达可溶性蛋白的5%，并且通过超氧化物歧化酶活性染色也观察到了酶活性。