银鲫HIRA多克隆抗体的制备及组织特异性表达分析  

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

作　　者：周励[1] 王玉凤[1] 桂建芳[2] 

机构地区：[1]华中师范大学生命科学学院,遗传调控与整合生物学湖北省重点实验室,武汉430079 [2]中国科学院水生生物研究所,淡水生态与生物技术国家重点实验室,武汉430072

出　　处：《水生生物学报》2008年第3期354-359,共6页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金(30671609);淡水生态与生物技术国家重点实验室开放基金(2005FB20)资助

摘　　要：Hir/Hira基因家族的成员广泛存在于多种生物体中,但有关其在生物体发育过程中的具体功能还不甚清楚。对果蝇的研究表明,dHira基因产物可能在受精时精核的解凝过程和雄性原核的正常形成过程中起重要作用。本研究组前期已经分别克隆出雌核发育银鲫和两性生殖彩鲫的Hira基因(cagHira和caHira),本实验在银鲫cagHira基因的特异区域,设计一对引物,以银鲫成熟卵母细胞总RNA逆转录出的cDNA为模板,扩增出cagHira的特异片段。再将该片段克隆到原核表达载体pET-32a上,转化BL21(DE3)菌株,经诱导后表达出融合蛋白。分析表明,该融合蛋白主要以包涵体形式表达。以纯化的融合蛋白作为抗原去免疫小鼠,制备多克隆抗血清,经蛋白质印迹分析检测,该抗血清(稀释到1∶2000)与包涵体蛋白识别反应良好,确定获得了具有高效价的特异性银鲫CAGHIRA多克隆抗体,为进一步研究HIRA在鱼类发育和雌核生殖过程中的作用奠定了基础。对银鲫HIRA蛋白的组织特异性表达分析发现,该蛋白仅在成熟卵巢组织中特异表达,故表明HIRA可能对鱼类卵子发生和/或早期胚胎发育具有重要作用。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 plants. The function of Hira gene in the development still remains unclear. It 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 fertilization. Gibel carp Carassius auratus gibelio is a unique triploid species that has two different reproduction modes： allogynogenetic reproduction and gonochoristic reproduction. It has a similar phenotype of abnormal male pronucleus to that in Hira mutated Drosophila. In 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 expression. An anti-CAGHIRA polyclonal antibody was now prepared and subsequently used to investigate the expression of this protein in different tissues of gibel carp. The cDNA fragment encoding 184 amino acids （Aa 497-680） of cagHira was cloned and inserted into a prokaryotic expression vector pET-32a. Then the recombinant protein approximately similar to the expected size was induced to express and was purified. The anti-CAGHIRA polyclonal antibody was prepared by immunization of mice using this pu- rified fusion protein. The specific recognition of anti-CAGHIRA polyclonal antibody was further verified by western blot analysis of the serum from pre- and post-immunized mice. The preparation of CAGHIRA antibody will greatly facilitate further study of Hira function in fish development and in gynogenesis. The CAGHIRA protein expression in various tissues was then analyzed using this

关 键 词：银鲫 HIRA 抗体制备 组织表达 

分 类 号：Q344.13[生物学—遗传学]