Generation of an antibody specific to Xenopus fertilized eggs by subtractive immunization
Generation of an antibody specific to Xenopus fertilized eggs by subtractive immunization
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DOI：
10.1111/j.1365-2443.2005.00838.x
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发表时间：
2005-04-01
期刊：
影响因子：
2.1
通讯作者：
Fukami, Y
中科院分区：
文献类型：
--
作者：
Sakakibara, K;Sato, K;Fukami, Y
在此，我们报告了单克隆抗体mAb 5H7 - G1的制备及其特性。该抗体能够识别受精后的非洲爪蟾卵动物皮层中的卵抗原，但对未受精的卵则无此识别能力。mAb 5H7 - G1是通过对小鼠进行消减免疫制备而成：先用未受精卵提取物进行初次免疫，随后用环磷酰胺进行免疫抑制处理，再反复用受精卵提取物进行免疫。在免疫印迹分析中，mAb 5H7 - G1能够识别受精后的非洲爪蟾卵（而非未受精或经离子载体激活的卵）中的多条蛋白条带。N - 连接多糖很可能是mAb 5H7 - G1的靶标，因为当用N - 糖苷酶F处理蛋白质样品时，mAb 5H7 - G1的免疫反应性会显著降低。此外，mAb 5H7 - G1能够识别经过氧化氢（卵酪氨酸激酶Src的人工激活剂）处理的卵中的部分（而非全部）酪氨酸磷酸化蛋白，这表明这些蛋白也含有N - 连接糖。当将mAb 5H7 - G1显微注射到受精的非洲爪蟾胚胎中时，会导致第一次细胞分裂延迟或完全抑制，这表明mAb 5H7 - G1所识别的抗原在这一过程中发挥着重要作用。这些结果表明，mAb 5H7 - G1有助于分析受精及早期发育过程中的差异蛋白质组展示。更广泛地说，消减免疫可能作为一种策略，用于揭示在不同细胞条件下发生的细胞事件。
Here we report the generation and characterization of a monoclonal antibody, mAb 5H7-G1, which recognizes egg antigens in the animal cortex of fertilized, but not unfertilized, Xenopus eggs. The mAb 5H7-G1 was generated by subtractive immunization of mice: primary immunization with unfertilized egg extract followed by immunosuppression treatment with cyclophosphamide and repeated immunization with fertilized egg extract. In immunoblotting analysis, mAb 5H7-G1 recognizes multiple protein bands of fertilized (but not unfertilized or the ionophore-activated) Xenopus eggs. N-linked polysaccharide is most likely the target of mAb 5H7-G1 because immunoreactivity of mAb 5H7-G1 is effectively diminished when protein samples are treated with N-glycosidase F. Moreover, mAb 5H7-G1 recognizes some, but not all, tyrosine-phosphorylated proteins in eggs treated with H2O2, an artificial activator of the egg tyrosine kinase Src, suggesting that these proteins also contain N-linked sugars. When microinjected into fertilized Xenopus embryos, mAb 5H7-G1 causes a retardation or complete inhibition of first cell cleavage, suggesting that the mAb 5H7-G1-reactive antigens play an important role in this event. These results demonstrate that mAb 5H7-G1 is useful to analyze differential proteome display during fertilization and early development. More generally, subtractive immunization may work as a strategy to uncover cellular events that operate during different cellular conditions of interest.