人铜锌超氧化物歧化酶原核表达载体的构建、表达及蛋白纯化  

Cloning,expression and purification of human Cu,Zn superoxide dismutase of in E.coli

作　　者：刘磊[1] 许淑芬[1] 方艳秋[1] 谭岩[1] 

机构地区：[1]吉林大学第一医院中心实验室,吉林长春130021

出　　处：《中国老年学杂志》2008年第5期434-436,共3页Chinese Journal of Gerontology

基　　金：吉林省杰出青年资助基金(No.20050113);吉林省科技厅国际合作项目(No.20060722)

摘　　要：目的构建人铜锌超氧化物歧化酶(hCu,Zn-SOD)原核表达载体并诱导其表达,纯化及鉴定目的蛋白,为其临床应用奠定基础。方法根据已报道的hCu,Zn-SOD基因序列,采用RT-PCR技术从外周血单个核细胞(PBMC)中获得SODcDNA序列。将所得的PCR产物插入克隆载体pGEM-Teasy中,重组质粒经酶切,PCR及测序鉴定正确后,将目的片段插入原核表达载体PET20b(+)中并转化大肠杆菌DE3,通过IPTG诱导表达出目的蛋白,经镍固定金属亲和层析纯化。采用邻苯三酚自身氧化法测定SOD生物学活性。结果序列分析表明SOD基因成熟肽编码区含有465bp与GenBank(X02317)中已报道序列一致的SOD核苷酸。经IPTG诱导表达,SDS-PAGE电泳和免疫印迹分析显示表达的目的蛋白分子质量约为19kD并与商品化的人SOD单抗呈特异性反应。经Ni2+-NTA琼脂糖纯化获得SDS-PAGE电泳下单一条带。可溶蛋白酶活力达1300U/ml。结论在大肠杆菌中获得了人SOD的高效表达,为研究其生物学功能和广泛应用奠定了基础。Objective To construct prokaryotic expression plasmid of human Cu,Zn superoxide dismutase （hCu,Zn-SOD）, then to obtain recombinant SOD for clinical use. Methods The encoding sequence for mature peptide of human SOD was amplified with RT-PCR and inserted into pGEM-Teasy vector to choose the target gene by double digestion, PCR and sequencing, then to establish the prokaryotic expressing system. The competent cells of host strain of DE3 were transformed by the recombinant plasmid. Expression of the target protein was induced with IPTG and purified by Ni^2＋ -NTA agarose column. The method of pyrogallol self oxidation was used to test the activity of purified protein. Results The cloned fragment of human SOD was 100% consistent with that in GenBank （X02317）. The expressed fusion-protein was 19 kD in SDS-PAGE as expected,was recognized by a commercial McAb. After preliminary purification through Ni^2＋ -NTA agarose affinity chromatography,the activity of the product was 1 300 U/ml. Conclusions The homologous recombinant is highly obtained in prokaryotic expressing system which may benefit for some related clinical uses.

关 键 词：人超氧化物歧化酶 表达纯化 大肠杆菌 

分 类 号：Q78[生物学—分子生物学]