Characterization of the fertilization antigen 1 for the development of a contraceptive vaccine.
Characterization of the fertilization antigen 1 for the development of a contraceptive vaccine.
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用于开发避孕疫苗的受精抗原 1 的表征。
DOI：
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发表时间：
1986
影响因子：
11.1
通讯作者：
B. Rosenblum
中科院分区：
文献类型：
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作者：
R. Naz;T. Phillips;B. Rosenblum
使用可抑制体外受精的单克隆抗体 MA-24，通过免疫亲和层析从溶解脱氧胆酸盐或二碘水杨酸锂的小鼠睾丸中纯化出受精抗原 FA-1。分别使用脱氧胆酸盐或二碘水杨酸锂提取物的逐步洗脱程序，在高 pH (11.4) 或低 pH (2.8) 下回收 FA-1。通过 NaDodSO4/PAGE 和银染色分析时，这两个级分均显示 47 kDa 的单条带。去除去垢剂并在 pH 5.8 下进行广泛透析或用 0.15 M NaCl 处理后，即使存在去垢剂，也可检测到 23 kDa 的单体。 FA-1 的二维 PAGE 显示有 4 或 5 个多肽在 47 kDa 或 23 kDa 范围内。当在蔗糖和氯化铯梯度上分离时，透析的 FA-1 包含主要 23 kDa 和次要 48 kDa 带。高效尺寸排阻色谱显示 23 kDa 处有一个主峰，50 kDa 处有一个次峰。通过反相色谱法对 23-kDa 峰的进一步分析将抗原解析为三个峰，这给出了与天然 FA-1 相似的二维凝胶图案。透镜状凝集素柱上的凝集素亲和层析证明，部分抗原与凝集素结合，而其余部分则没有。 FA-1与周期性希夫试剂呈阳性反应，并含有葡萄糖和甘露糖，二者合计占抗原总质量的18.8%。氨基酸分析显示天冬氨酸、谷氨酸、丝氨酸和甘氨酸的比例很高。由于单次注射 MA-24 显着降低体内受精率，因此纯化的 FA-1 是开发避孕疫苗的有吸引力的候选者。
A fertilization antigen, FA-1, was purified from either deoxycholate- or lithium diiodosalicylate-solubilized murine testes by immunoaffinity chromatography using a monoclonal antibody, MA-24, which inhibited fertilization in vitro. The FA-1 was recovered at high (11.4) or low (2.8) pH using stepwise elution procedures of the deoxycholate or lithium diiodosalicylate extracts, respectively. Both of these fractions showed a single band of 47 kDa when analyzed by NaDodSO4/PAGE and silver staining. Following removal of the detergent and extensive dialysis at pH 5.8 or treatment with 0.15 M NaCl, even in the presence of detergent, a monomer of 23 kDa was detected. Two-dimensional PAGE of FA-1 showed, four or five polypeptides in the 47-kDa or 23-kDa range. The dialyzed FA-1 contained a major 23-kDa and a minor 48-kDa band when separated on both sucrose and cesium chloride gradients. High performance size-exclusion chromatography showed a major peak at 23 kDa and a minor peak at 50 kDa. Further analysis of the 23-kDa peak by reverse-phase chromatography resolved the antigen into three peaks, which gave similar two-dimensional gel patterns as the native FA-1. Lectin affinity chromatography on a lens culinaris column demonstrated that a part of the antigen was bound to the lectin while the rest was not. The FA-1 revealed a positive reaction with periodic-Schiff reagent and contained glucose and mannose, which together constituted 18.8% of the total antigen mass. Amino acid analysis showed a high percentage of aspartic acid, glutamic acid, serine, and glycine. As a single injection of MA-24 significantly reduced fertilization rates in vivo, the purified FA-1 is an attractive candidate for the development of contraceptive vaccine.