重组人SOD2/3杂合酶的制备及其生物活性初探  

Preparation of Recombinant Hybrid Human SOD2/3 and Its Biological Activity Assay

作　　者：程安阳[1] 范立强[1] 赵健[1] 李小灵[1] 王刚[1] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

出　　处：《华东理工大学学报（自然科学版）》2011年第6期715-721,共7页Journal of East China University of Science and Technology

基　　金：国家自然科学基金资助项目(81071252)

摘　　要：为获得重组SOD2/3杂合酶,以及观察SOD2/3能否转导入宫颈癌细胞,抑制肿瘤细胞的增殖,构建了原核表达载体pET-SOD2/3,转化大肠杆菌BL21(DE3),IPTG诱导表达获得N端带有6个组氨酸标签的SOD2/3杂合酶;用Ni柱金属螯合亲和层析纯化重组SOD2/3;利用肝素柱层析验证SOD2/3的肝素靶向性;将纯化后的融合蛋白加入体外培养的宫颈癌细胞,通过FITC和MTT实验观察杂合酶SOD2/3的转导能力及其在细胞内的活性。成功构建了SOD2/3杂合酶的表达载体,重组SOD2/3表达量占菌体总蛋白30%左右;金属螯合亲和层析一步纯化,回收率在70%以上,纯度达90%左右;重组酶能与肝素亲和层析结合,说明杂合酶SOD2/3有肝素靶向性;细胞实验证实SOD2/3较SOD2能更好地抑制Hela细胞生长。获得了较纯的重组SOD2/3杂合酶,该酶能直接以天然有活性的形式转导人宫颈癌细胞内并抑制其细胞增殖,具有很好的作为肿瘤治疗、抗氧化损伤药物的潜力。To obtain recombinant hybrid human superoxide dismutase （rhSOD2/3） and investigate whether SOD2/3 fusion protein could be transduced into and inhibit the growth of human cervical carcinoma cell, the recombinant plasmid pET-SOD2/3 was constructed and transformed into E. coli BL21 （DE3） to express rhSOD2/3, rhSOD2/3 was purified by Ni-NTA affinity chromatography with the property of its N-terminal （His）6 affinity tag. Heparin affinity chromatography was used to test the heparin-binding ability of rhSOD2/3. The purified SOD2 and SOD2/3 were incubated with Hela cells. FITC and MTT assay was used to test their anti-tumor growth activities. The hybrid SOD2/3 was successfully expressed in E. coli, accounted for about 30% of the total bacterial protein and had normal SOD activity. After one-step Ni-NTA affinity chromatography, SOD2/3 of 90% pure was obtained. rhSOD2/3 could bind on a heparin affinity column, indicating that it was successfully targeted to heparin. Cell experiment showed that rhSOD2/3 inhibited the growth of Hela cells, while SOD2 did not. Relative pure hybrid rhSOD2/3 was obtained. Compared to SOD2, rhSOD2/3 fusion protein with natural and active form could be transduced more effectively into Hela ceils and inhibit more strongly cell growth. The hybrid rhSOD2/3 can bind to cell surfaces and may aid in anti-tumor activity of SOD2 and may function as a rational therapeutic agent for treating free-radical-mediated diseases.

关 键 词：SOD2/3杂合酶 表达 纯化 抗肿瘤 

分 类 号：Q812[生物学—生物工程]