以原核表达的具有明胶水解活性的人基质金属蛋白酶 2的催化区 (MCD)为靶标 ,筛选噬菌体随机环七肽库和十二肽库 .找到 6种与MCD特异结合的小肽 ,将 6种小肽基因分别与GST表达质粒重组 ,进行GST融合表达 ,制备融合蛋白 .采用Glutathione Sepharose 4B亲和层析法纯化融合蛋白 ,通过酶抑制实验、体外侵袭实验检测融合蛋白的活性 .结果表明 ,GST C71能够抑制MCD水解 β酪蛋白的活性 ,并且对人纤维肉瘤细胞HT10 80的体外侵袭有明显的抑制作用
Abstract
Matrix metalloproteinase 2 (MMP 2) is an important matrix metalloproteinase in the degradation of basement membranes and denatured collagens,and is believed to play a key role in cancer invasion and metastasis. The isolation of an inhibitor of MMP 2 from phage display random peptide libraries was described. The catalytic domain of MMP 2 (MCD) was used as the target to screen the binding peptides from phage display random peptide library. After three rounds of biopanning, six phage clones were isolated. The results of ELISA showed that all the selected phages could specifically bind MCD. The six peptides genes were expressed in E.coli as GST peptide fusion proteins, and the fusion proteins were purified by Glutathione\|Sepharose 4B column. The MMP 2 inhibitory activities of the purified GST peptide fusion proteins were analyzed by enzyme inhibition assays. The results showed that GST C71 could inhibit the degradation of casein by MCD. GST C71 also exhibited the activity of inhibiting HT1080 fibrosarcoma cell invasion through a layer of Matrigel.
关键词
基质金属蛋白酶2(MMP-2) /
小肽 /
侵袭
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