血管抑制素和内皮抑制素在大肠杆菌中的融合表达与鉴定

Fusion Expression and Identification of Angiostatin and Endostatin in E.coli BL21(DE3)

血管抑制素(A S)和内皮抑制素(ES)是两种具有较强活性的内源性血管抑制剂,联合应用具有协同作用。本研究通过大肠杆菌表达A S-ES融合蛋白,观察其对血管生成的抑制作用。首先用RT-PCR方法分别获得A S和ES基因,通过基因拼接获得融合基因,构建了含有该融合基因的原核表达质粒——pET-42(b)/A S-ES。表达菌株经IPTG诱导后目的蛋白以包涵体形式表达,表达量为14%,分子量约65 KD。W estern b lotting检测表明表达产物可分别与A S和ES抗体产生特异的免疫反应。经复性、肝素亲和层析柱纯化的表达产物对鸡胚尿囊膜血管有明显抑制作用。本研究获得了A S、ES在大肠杆菌中的融合表达,目的蛋白具有特异的免疫反应性和血管抑制活性。

Angiostatin（AS） and endostatin（ES） are both potent endogenous angiogenesis inhibitors, and the combination of AS and ES has been shown to have synergistic antiangiogenic effects. Here we report the fusion protein AS-ES expressed in E. coli which has antiangiogenic effects. At first, AS and ES genes were cloned respectively through RT-PCR,then fusion gene was made through gene splicing , finally pET-42 （b）/AS-ES expression plasmid was contrusted and transduced in E. coli BL21 （DE3）. Target protein was in form of inclusion body, the rate of expression was about 14%, and MW about 65KD. Western blotting assay showed expressed protein had specific immune reaction to both the antibodies of AS and ES. The expressed protein which was refolded and purified through heparin affinity chromatography had antiangiogenic effect to vessels on chicken embryo chorioallantoic membrane. The results show that fusion protein AS-ES was expressed successfully in E. coli ,and the expressed protein ,which was renatured and purified, had imuno-reactivhy to anti-AS and anti-ES in Western blotting and angiogenesis inhibition activity.