豚鼠自身免疫性梅尼埃病相关抗原相对分子质量68000蛋白的二维电泳和质谱分析

Identification of relative molecular mass 68000 antigens related to autoimmune Meniere disease with two-dimensional electrophoresis and mass spectrometric analysis in Guinea pigs

目的:探寻导致豚鼠自身免疫性梅尼埃病模型的内耳组织抗原成分中相对分子质量为68 000的蛋白质成分。方法:双向凝胶电泳方法分离豚鼠内耳组织抗原成分,用ImageMaster图像分析软件选择相对分子质量为68 000的蛋白质点,对这些蛋白质点进行质谱分析,通过Mascot软件查询Swiss-prot数据库,利用PathwayStud io软件对查询到的蛋白进行生物信息学分析并选取蛋白进行免疫印迹法验证。结果:获得重复性和分辨率都较好的双向电泳图谱,软件分析相对分子质量为68 000蛋白共获得22个蛋白质点;质谱分析后查询数据库鉴定得到13个蛋白质。这些蛋白分别与免疫、凋亡和分化等功能相关,其中波形蛋白从相对分子质量、Mascot评分和蛋白功能等方面综合评估可能为相对分子质量68 000蛋白中引起豚鼠自身免疫性梅尼埃病的主要蛋白。结论:成功获得豚鼠自身免疫性梅尼埃病模型主要内耳抗原成分中相对分子质量68 000蛋白的双向凝胶电泳图谱,并应用质谱技术鉴定蛋白质点。波形蛋白可能为相对分子质量68 000蛋白中引起豚鼠自身免疫性梅尼埃病的主要蛋白。

Objective： To investigate the components of relative molecular mass 68 000 inner ear antigens inducing autoimmune Meniere disease（AIMD） in Guinea pigs.Methods： Inner ear antigens of Guinea pigs were separated by two-dimensional polyacrylamide gel electrophoresis（2-DE）.The relative molecular mass 68 000 protein spots were chosen by ImageMaster software and mass spectrometric analysis was used to test these protein spots.The Swiss-prot database was searched with Mascot software and then the PathwayStudio software was used to carry out the bioinformatics analysis for proteins searched.Western blotting was performed to prove selective proteins.Results： 2-DE pattern with higher resolution and reproducibility was obtained.It was found that a total of 22 kinds of protein spots contained in relative molecular mass 68 000 antigens were acquired by Image Master software analysis,and 13 kinds of protein were identified with mass spectrometric analysis and Swiss-prot database searching.These proteins were related to immunity,apoptosis,differentiation and other functions respectively,in which vimentin may be the main proteins in relative molecular mass 68 000 inner ear antigens for inducing AIMD in Guinea pigs according to assessment from relative molecular mass,Mascot score and protein functions.Conclusion： 2-DE patterns of relative molecular mass 68 000 proteins of Guinea pigs inner ear antigens are successfully obtained,and the total of protein spots are identified with mass-spectrometric technique.Vimentin may be the main proteins in relative molecular mass 68 000 antigens for inducing AIMD in Guinea pigs.