酵母双杂交技术筛查与vasostatin相互作用的蛋白质

Yeast two-hybrid technique in screening for interacting proteins with vasostatin

目的:通过酵母双杂交技术筛查与血管生长抑制因子vasostatin相互作用的蛋白质,为进一步阐明其抑制血管生成作用的分子机制奠定基础。方法:应用第3代酵母双杂交系统,构建vasostatin诱饵质粒,经过表达和自激活验证后筛选预转化的人内皮细胞cDNA方库。将在四缺培养基中筛选得到的阳性克隆提取酵母质粒转化大肠杆菌,进行限制性酶切和测序,寻找可能与vastotatin具有相互作用的蛋白序列。结果:经过酵母双杂交共获得21个克隆,经过验证,有3个阳性克隆;经过NCBI的BIAST进行同源性分析,它们的核酸和蛋白序列与已知基因高度同源,它们分别为层黏连蛋白α5(7547～8112)和整合素αV(1257～1820、316～878)片段,均为重要的配体结合部位。Vasostatin蛋白通过与该区域结合,影响FAK、VEGF和bFGF等的信号通路,抑制血管生成。结论:酵母双杂交筛选获得的层黏连蛋白和整合素蛋白的3个蛋白片段,它们与肿瘤内血管内皮细胞增殖和迁移等密切相关。

Objective： To screen for proteins interacting with angiogenesis inhibitor vasostatin by yeast-two hybrid technique, so as to further elucidate its molecular mechanism of inhibiting angiogenesis. Methods： The 3rd generation yeast- two-hybrid system was applied to construct a bait plasmld of vasostatin gene. After validation and self-activating, the bait plasmid was used to screen human endothelial cells cDNA library to be transformed. The yeast plasmids extracted from positive clones, which were obtained in SD/-Trp-Leu-Ade-His medium, were transformed into E. coll. By restriction digestion and sequencing, the potential proteins interacting with vasostatin were identified. Results： Twenty one clones were obtained with the system and 3 of them were positive; and their nucleic acid sequences and protein sequences were highly homologous to the known genes by BLAST homology analysis of NCBI： they were both important ligand-binding sites, including Laminin protein ct5（7 547-8 112） and Integrin ctV （1 257-1 820, 316-878） fragments. By binding to these sites, vasostatin affected the signal pathway of FAK, VEGF and bFGF and subsequently inhibited angiogenesis. Conclusion： The laminin protein and integrin obtained by the yeast two-hybrid system are closely related to the proliferation and migration of intratumoral vascular endothelial cells.