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

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 pGEX2KGexpression vector and transforming the bacteriumDE3 strain,and the antibody against the ZP3 recombinant protein was prepared from the immunized rabbit1Furthermore,expression characterization and status changes of ZP3 protein during oocyte maturation and early embryogenesis were analyzed by RT2PCR and Western blot1The 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 fully2grown oocytes1Significant changes of ZP3 status occurred after egg fertilization and early embryogenesis1After 30 minutes of post2fertilization,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 detectedfromthe 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 embryogenesis1Moreover,the chorion proteins were separated by 12 % SDS2PAGE and stained by Coomassie brilliant blue1Eight protein bands were respectively excised and recovered from the gel1The recovered proteins were again sepa-rated by 12 % SDS2PAGE,and transferred to nitrocellulose membrane1Then they were detected by Western blot with the anti ZP3 serum1The result indicated that the fifth protein band was positive ZP3 protein with about 50 K D,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 protein1Therefore,we purified ZP3 protein of Carassius auratus gibelio from the isolated chorion by SDS2PAGE separation and Western blot detection