白眉蝮蛇去整合素Adinbitor的基因克隆、表达及其部分生物学活性

Cloning, Expression and Some Biological Functions of Adinbitor, a Disintegrin from Agkistrodon halys brevicaudus stejneger

为了寻找去整合素家族的新成员 ,研究其在抗血栓与抗肿瘤等方面的作用 ,从中国白眉蝮蛇毒腺中提取总RNA进行RT PCR扩增 ,获得了 2 19bp的白眉蝮蛇去整合素基因 .测序结果显示 ,其与韩国的同为蝮蛇去整合素的saxatilin的DNA序列同源性为 95 8% ,蛋白质序列同源性为 91 8% ,且蛋白质中含有去整合素的特征模体 RGD .将去整合素基因进行克隆、转化与诱导后 ,得到了该蛋白的可溶性高效表达 .经组氨酸亲和层析纯化 ,获得了分子量为 9kD的均质蛋白 ,并将其命名为adinbitor .活性测定结果显示 ,adinbitor能明显抑制由碱性成纤维细胞生长因子诱导的人脐静脉血管内皮细胞ECV30 4的增殖 ,诱导ECV30 4细胞发生凋亡 。

In order to find a novel disintegrin from Chinese snake (Agkistrodon halys brevicaudus stejneger) and its functions of antithrombosis and anti-tumor, total RNA was extracted from venom gland and was reversely transcribed into cDNA. By amplifying the cDNA, adinbitor gene from Agkistrodon halys brevicaudus stejneger with 219 bp was obtained. Sequencing analysis indicated that adinbitor's DNA was 95.8% homology with saxatilin and the protein was 91.8% homology with saxatilin. Adinbitor contained the motif- RGD of disintegrin. After cloning, transforming, and expression of adinbitor gene, the adinbitor was purified by His-Bind column chromatography. A 9 kD soluble homogeneous protein was purified, and the protein could induce a significantly apoptosis in the ECV304 cells.The protein also could inhibit proliferation of ECV304 cells induced by basic fibroblast growth factor (bFGF) and inhibit a dose-dependent platelet aggregation induced by ADP.