节杆菌A·pascens DMDC12锰超氧化物歧化酶基因的克隆和表达

Cloning and expression of manganese-superoxide dismutase gene from Arthrobacter pascens DMDC12

利用PCR技术对自行筛选的高度耐盐节杆菌Arthrobacter pascensDMDC12中的超氧化物歧化酶基因进行克隆,获得了节杆菌A.pascensDMDC12的SOD新基因(GenBank收录号为DQ779150)。将该基因与原核表达质粒载体pET-22b(+)连接,构建重组质粒pET-sodAP,将质粒转化至表达宿主菌E.coliBL21(DE3),构建基因工程菌BL21/pET-sodAP。用异丙基硫代--βD-半乳糖苷(IPTG)诱导重组SOD蛋白表达,用酶活和SDS-PAGE分析表达产物,表达产物的相对分子质量为2.4×104,与sodAP推测的长度相同。表达目的蛋白占菌体可溶性蛋白的23%,比酶活达915.07 IU.mg-1,是对照菌株的8.73倍,具有较高的表达。本研究为进一步进行重组Mn-SOD的研究和应用奠定了基础。

The aim of the present study was to clone and express the superoxide dismutase gene （sodAP） of halophilic Athrobacter pascens DMDC12. The new sodAP （Accession NO. DQ779150） was amplified from A .pascens DMDC12. The sod AP was cloned in the vector plasmid pET-22b（ ＋ ） to yield the recombinant plasmid pET-sodAP. The pET-sod AP was then transformed into E. coli BL21（DE3） and recombinant E. coli BL21/pET-sod AP was obtained. The SOD was expressed under the induction of IPTG. The SDS-PAGE analysis showed that the molecular weight of expression product was about 2.4×10^4, which corresponded to prediction of sodAP. The expressed recombinant protein accounted for 23% of total cell protein. The relative SOD activity of crude cell free supematant was 915.07 IU·mg^-1 ,and was 8.73 times higher than the host with pET-22b（ ＋ ）. The cloning and efficient expression of sodAP will provide the foundation for the further research and development of Mn-SOD.