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.
Chen Bo; Guo Wei; Gui Jian-Fang (firstname.lastname@example.org).Expression characterization of ZP3 gene and isolation of chorion ZP3 protein in Carassius auratus gibelio,Acta Hydrobiologica Sinica,2005,29(3):233-238