应用酵母双杂交系统筛选与新基因nelin相互作用的蛋白质

Identification of Proteins Interacting with Novel Gene Nelin via Yeast Two-hybrid System

目的 通过筛选能够与新基因nelin的心肌特异表达产物相互作用的蛋白质从而揭示nelin的生物功能。方法 构建表达nelin阅读框全长的诱饵质粒并利用酵母双杂交系统筛选人心脏cDNA文库, 以寻找与nelin蛋白发生相互作用的蛋白质。并利用生物信息学方法对筛选得到的蛋白质进行功能结构域分析。结果 筛选得到3 个蛋白质分别为肌联蛋白 (Titin ) N2B亚型, 细丝蛋白 (Filamin ) C亚型即γ Filamin和α胰蛋白酶抑制物重链前体 (inter alphatrypsin inhibitor heavy chain precursor, ITIH)。对这3种蛋白的C末端结构域进行分析发现Titin和Filamin均含有免疫球蛋白样结构域 (Ig- like domain)。结论 实验结果揭示了新基因nelin很可能表达一个细胞骨架相关蛋白, 它可通过其蛋白质的C末端同Titin和Filamin的免疫球蛋白样结构域发生相互作用而参与细胞骨架网络的形成。

Objective To reveal the biological function of nelin,a novel cardiac-specific human gene by screening the proteins interacting with nelin.Methods Yeast two-hybrid method was used to screen human heart cDNA library with the bait plasmid that was constructed containing the whole ORF sequence of gene nelin.The protein functional domains of screened proteins were analyzed by the biological informatics databases,thus to detect the potential structure interacting with nelin. Results Three positive clones were retrieved.They were Titin N2B isoform,Filamin C isoform and inter-alpha trypsin inhibitor heavy chain precursor (ITIH).Titin and Filamin both had Ig-like domains in their C-terminus.Conclusion The novel gene nelin expresses a cytoskeleton-associated protein,which perhaps participates in the formation of cytoskeleton network by its C-terminus interacting with the Ig-like domains of Titin and Filamin.