银鲫ZP3的表达特征和卵壳ZP3蛋白的分离

EXPRESSION CHARACTERIZATION OF ZP3 GENE AND ISOLATION OFCHORION ZP3 PROTEIN IN CARASSIUS AURATUS GIBELIO

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。

Full length ZP3 cDNA had been cloned from Carassius auratus gibelio. In this study,the ZP3 and GST recombinant protein was produced by constructing the pGEX-KG expression vector and transforming the bacterium DE3 strain,and the antibody against the ZP3 recombinant protein was prepared from the immunized rabbit.Furthermore,expression characterization and status changes of ZP3 protein during oocyte maturation and early embryogenesis were analyzed by RT-PCR and Western blot.The data indicated that the ZP3 transcription was initiated before previtellogenic stage,while the ZP3 protein was translated from the stage of vitellogenesis,and the amount reached to its peak in the fully-grown oocytes.Significant changes of ZP3 status occurred after egg fertilization and early embryogenesis.After 30 minutes of post-fertilization,the original ZP3 protein disappeared from the extract,instead of that,the ZP3 antibody recognized bigger protein bands,and all of them could not be detected from the extract after 8—16 cell stage of embryogenesis. It implicated that the ZP3 dimeric complex and polymerized complexes were formed after egg fertilization and early embryogenesis.Moreover,the chorion proteins were separated by 12%SDS-PAGE and stained by Coomassie brilliant blue.Eight protein bands were respectively excised and recovered from the gel.The recovered proteins were again separated by 12%SDS-PAGE,and transferred to nitrocellulose membrane.Then they were detected by Western blot with the anti-ZP3 serum.The result indicated that the fifth protein band was positive ZP3 protein with about 50 KD,and the second protein band is also positive,but the molecular weight is higher than that of ZP3,which may be the complex of ZP3 and other protein.Therefore,we purified ZP3 protein of Carassius auratus gibelio from the isolated chorion by SDS-PAGE separation and Western blot detection.