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

 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angioarrestin C端 hFD cDNA 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.coli BL21（DE3）,经0.3 mmol/LIPTG 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angioarrestin C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.

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

Angioarrestin is one kind of potential antiangiogenesis factors.By DNA recombinant technique to construct E.coli BL21（DE3） expressed plasmid pMAL-C2-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.