论文部分内容阅读
克隆得到银鲫(Carassius auratus gibelio)ZP3基因全长cDNA,在体外表达出银鲫的ZP3融合蛋白,并制备出多抗血清;通过免疫印迹和RT-PCR分析,研究了银鲫ZP3在卵子发生过程中的表达特征和在早期发育胚胎中的状态变化。研究结果表明,银鲫ZP3的转录发生在卵黄发生以前,而ZP3蛋白的翻译起始于卵黄形成阶段,且随着卵母细胞进一步成熟,其含量不断增加。ZP3蛋白的存在状态在受精后和早期胚胎发育过程中发生了明显变化。在受精后5—30min期间,抽提液中原始的ZP3蛋白带迅速消失,取而代之的是分子量大约为90KD以及更高分子量的蛋白带;而在受精后80min和8—16胞期的胚胎抽提液中,二聚体和多聚体复合体蛋白带也相继消失。这种状态变化意味着ZP3蛋白在卵子受精后有可能与自身或其他蛋白共价结合转变成了二聚体和多聚体。接着,用获得的ZP3抗体作为检测指标,通过卵壳蛋白的凝胶分离,从卵壳中分离纯化出银鲫ZP3蛋白。
The full-length cDNA of ZP3 gene of Carassius auratus gibelio was cloned and the ZP3 fusion protein of silver carp was expressed in vitro and multiple antisera were prepared. By immunoblotting and RT-PCR analysis, The process of expression characteristics and changes in the early development of embryos in the state. The results showed that the transcription of ZP3 in silver carp occurred before yolk and the translation of ZP3 protein began in the yolk formation stage, and its content increased with the further maturation of oocytes. The presence of ZP3 protein changed significantly after fertilization and early embryonic development. During 5-30min after fertilization, the original ZP3 protein band in the extract quickly disappeared, replaced by a protein band with a molecular weight of about 90KD and higher molecular weight; however, embryos extracted 80min and 8-16 after fertilization Liquid, dimer and polymer complex protein band also disappeared. This state change means that the ZP3 protein may be covalently bound to itself or other proteins to become dimers and multimers upon fertilization of the egg. Next, ZP3 antibody was used as a detection index to isolate and purify silver carp ZP3 protein from egg shell by gel separation of egg shell protein.