Angioarrestin(hARP1)C端FD区的克隆、表达及其免疫活性鉴定

Cloning,Expression and Polyclonal Antibody Preparation of C-terminal FD Domain of Human Angioarrestin(hARP1)

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.