短短芽胞杆菌FJAT-0809-GLX超氧化物歧化酶基因的克隆及原核表达

摘要

The gene (GenBank No. KM255665) encoding superoxide dismutase (SOD) in the strain of Brevibacillus brevis FJAT-0809-GLX was cloned by PCR with total DNA extracted from this strain as a template. The PCR product was purified and ligated with pMD18 and then transformed into Escherichia coli DH5α. The recombinant plasmid DNA was used for nucleotide sequencing. The results indicated that the gene was 609 bp and it encoded 202 amino acid residues. The DNA product was purified and then inserted into the expression vector pET-28a to generate the recombinant expression vector pET-28a-SOD. Both the inserted fragment and its reading frame were confirmed by BamHⅠ/XhoⅠdigestion. Vector pET-28a-SOD was transformed into E. coli BL-21 and induced with Isopropyl-β-D- thiogalactopyranoside (IPTG). SDS-PAGE showed that the relative molecular weight of the expressed protein was about 25 ku in cells, which was close to the ORF of SOD. The data laid a foundation for the further study on the biochemical and physiological characteristics of this enzyme.%短短芽胞杆菌FJAT-0809-GLX具有抑菌、 抗褐变和保鲜功能.以短短芽胞杆菌菌株FJAT-0809-GLX总DNA为模板,采用PCR扩增超氧化物歧化酶(Supseroxide dismutase,SOD)基因,将该片段纯化回收后与pMD18-T连接并转入大肠杆菌(Escherichia coli)DH5α,进行序列测定,结果显示SOD基因序列长度为609 bp(GenBank登录号:KM255665),编码202个氨基酸残基.将SOD基因片段与同样酶切的表达载体pET-28a连接,构建重组表达载体pET-28a-SOD,转入大肠杆菌BL-21,采用异丙基-β-D-硫代吡喃半乳糖苷(Isopropyl-β-D-thiogalactopyranoside,IPTG)进行诱导表达.SDS-PAGE结果表明,在菌体中存在约25 ku的蛋白表达产物,与该基因ORF预期大小接近.以上结果为进一步研究该酶的生理生化特性奠定了基础.