摘要
Angioarrestin是一种具有潜在应用价值的肿瘤血管形成抑制因子.利用DNA重组法构建了angioarrestinC端hFDcDNA和麦芽糖结合蛋白(MBP)重组原核表达质粒pMAL-C2-hFD.将重组质粒转入大肠杆菌E.coliBL21(DE3),经0.3mmol/LIPTG在37℃条件下诱导表达4h,SDS-PAGE检测,融合蛋白表达量约占细菌总蛋白的20%.Western印迹证实,目的蛋白N端带有MBP标签.取表达上清纯化、透析、浓缩并冻干,以此为抗原免疫Balb/c小鼠制备多克隆抗体.此多抗可以与pET-22b(+)表达系统获得的hFD重组蛋白发生良好的抗原抗体反应,ELISA检测多抗效价达1∶10240.实验证明:通过基因重组可获得angioarrestinC端hFD在大肠杆菌中的高效表达蛋白,且该蛋白具有较高的免疫活性.以此为抗原制备的抗angioarrestin多克隆抗体为深入研究angioarrestin提供了材料.
Angioarrestin is one kind of potential antiangiogenesis factors. By DNA recombinant technique to construct E.coli BL21 (DE3) expressed plasmid pMAL-C2-hFD,and hFD cDNA fused to the 3'end of the gene encoding the MBP protein. The fusion protein was expressed in E. coli BL21 ( DE3 ) at 37℃ after 0.3 mmol/L IPTG induction for 4 hours. The fusion protein of high expression has a molecular weight 66 kD by SDS-PAGE. The yield of fusion protein was 20 percent in total proteins measured by SDS-PAGE. Supernatant of purified recombinant protein was used as immunogen to prepare the polyclonal antibody. The result of ELISA shows that antibody potency is 1 : 10240. The fusion protein highly expressed in E. coli BL21 (DE3)posses well antigen activity. And preparation of polyclonal antibody will provide material for further studies of angioarrestin.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2007年第2期136-140,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
上海市科学技术发展基金项目(No024319115)~~