人血管能抑素基因N端片段的表达、纯化及其生物活性测定

以中国人胎盘脐带组织为材料 ,提取组织总RNA ,用RT PCR方法合成人血管能抑素cDNA ,将该cD NA克隆进 pSP72载体获得重组质粒 pSP72C。以pSP72C为模板 ,PCR方法合成编码血管能抑素N端 189aa的基因片段 ,将其克隆进pET 3c载体获得重组表达质粒 pET CN ,转化E .coliBL2 1(DE3) ,SDS PAGE分析显示 ,在IPTG诱导下 ,人血管能抑素N端基因片段获得了有效表达 ,表达量约占菌体总蛋白质的 35 .3% ,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白质复性以及SephadexG 10 0凝胶过滤层析等步骤纯化后 ,获得了纯度约92 .6 %的人血管能抑素N端片段 ,CAM实验证明具有显著抑制鸡胚新生血管生成活性。

Expression and Purification of the N-domain of Human Canstatin and Its Bioactivity

Total RNA was extracted from placenta umbilical tissue. The canstatin cDNA was amplified from total RNA by net-RT-PCR technique and cloned into pSP72, and the resulted plasmid pSP72C was used as template to amplify its N-domain. The amplified 1-89 aa N-domain was then cloned into pET-3c. The resulted plasmid pET-CN was transformed into E. coli BL21(DE3) . The N-domain was efficiently expressed after IPTG induction as a 10 kD band on SDS-PAGE. The expressed product accounted for approximately 35.3 % of the total bacterial proteins, as estimated by densitometry and existed mainly as inclusion body. The inclusion bodies were washed, lysed and the reactivated proteins were purified by the Sephadex G-100 gel filtration to a purity of 92.6 %. CAM assay showed that N-domain effectively inhibited the angiogenesis of chichen embryo microcapillary vessel.