SPECIES-SPECIFIC RECOGNITION PATTERNS OF MONOCLONAL-ANTIBODIES DIRECTED AGAINST VIMENTIN
SPECIES-SPECIFIC RECOGNITION PATTERNS OF MONOCLONAL-ANTIBODIES DIRECTED AGAINST VIMENTIN
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DOI：
10.1016/0014-4827(92)90341-5
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发表时间：
1992-07-01
影响因子：
3.7
通讯作者：
TRAUB, P
中科院分区：
文献类型：
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作者：
BOHN, W;WIEGERS, W;TRAUB, P
两个商业上可获得的单克隆抗体提出的中间丝蛋白波形蛋白的特征在于其物种特异性的反应模式对脊椎动物细胞。抗体V9表现出与所有测试的哺乳动物物种的波形蛋白的广泛反应性，但在小鼠细胞和鸡成纤维细胞中没有特异性地检测到波形蛋白。抗体Vim 3B4识别鸡和大多数哺乳动物物种（啮齿动物物种除外）细胞中的波形蛋白。通过有限的蛋白水解和免疫印迹以及序列比较对人波形蛋白上Vim 3B4的结合位点进行表征，强烈表明该表位位于波形蛋白杆状结构域的螺旋2部分。小鼠波形蛋白cDNA克隆的定点突变，随后在体内表达表明，Vim 3B4可以检测啮齿动物波形蛋白含有一个单一的氨基酸取代（缬氨酸为亮氨酸）的位置353的小鼠波形蛋白序列。这一发现的实际应用证明了明确的鉴定修饰的小鼠波形蛋白，不同于内源性波形蛋白，在细胞质中间丝网络中的小鼠皮肤成纤维细胞转染重组质粒表达载体。
Two commercially available monoclonal antibodies raised against the intermediate filament protein vimentin were characterized concerning their species-specific reaction pattern on vertebrate cells. The antibody V9 exhibited extensive reactivity with vimentin of all mammalian species tested, but specifically did not detect vimentin in mouse cells and chicken fibroblasts. The antibody VIM 3B4 recognized vimentin in cells of chicken and most mammalian species, except for rodent species. Characterization of the binding site of VIM 3B4 on human vimentin by limited proteolysis and immunoblotting as well as by sequence comparison strongly suggested that the epitope is located in the coil 2 part of the vimentin rod domain. Site-directed mutagenesis of a mouse vimentin cDNA clone followed byin vivoexpression showed that VIM 3B4 could detect rodent vimentin containing a single amino acid substitution (valine for leucine) at position 353 of the mouse vimentin sequence. Practical application for this finding was demonstrated by the unequivocal identification of a modified murine vimentin protein, distinct from the endogenous vimentin, in a cytoplasmic intermediate filament network in mouse skin fibroblasts transfected with a recombinant plasmid expression vector.