PREPARATION OF POLYCLONAL ANTIBODY AND ANALYSIS OF SPATIAL EXPRESSION OF HIRA PROTEIN IN GIBEL CARP

Hir/Hira (Histone regulation) genes were first identified in yeast as negative regulators of histone gene expression It has been confirmed that HIRA is a conserved family of proteins present in various animals and plants1The function of Hira gene in the development still remains unclear1It was shown that during Drosophila fertilization,the male pronucleus remain abnormally condensed in eggs laid by Hira mutated females,indicating that HIRA complex may have a role in decondensation of sperm nucleus and the formation of male pronucleus after fertilization1Gibel carp Carassius auratus gibelio is a unique triploid species that has two different reproduction modes : allogynogenetic reproduction and gonochoristic reproduction1It has a similar phenotype of abnormal male pronucleus to that in Hira mutated Drosophila1In order to analyse the function of Hira gene in fish development and in gynogenesis,we have previously cloned the full -length cDNA of Hira homologs from gynogenetic gibel carp (cagHira) and gonochoristic colored crucian carp (caHira), respectively, and analyzed the spatial and temporal transcriptional expression1An anti CAGHIRA polyclonal antibody was now prepared and subsequently used to investigate the expression of this protein in different tissues of gibel carp1The cDNA fragment encoding 184 amino acids (Aa 497 —680) of cagHira was cloned and inserted into a prokaryotic expression vector pET232a1Then the recombinant protein approximately similar to the expected size was induced to express and was purified1The anti2CAGHIRA polyclonal antibody was prepared by immunization of mice using this purified fusion protein1The specific recognition of anti2CAGHIRA polyclonal antibody was further verified by western blot analysis of the serumfrom pre and postimmunized mice The preparation of CAGHIRA antibody will greatly facilitate further study of Hira function in fish development and in gynogenesis1The CAGHIRA protein expression in various tissues was then analyzed using this new prepared antibody by western blot1The result showed that CAGHIRA was specifically expressed in ovary1No obvious band was observed in brain,heart,liver,spleen,kidney,muscle and testis tissues1The result is consistent with the detection by RT2PCR and suggests that HIRA may play an important role in oogenesis and/or early embryogenesis.