留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码
周励, 王玉凤, 桂建芳. 银鲫HIRA多克隆抗体的制备及组织特异性表达分析[J]. 水生生物学报, 2008, 32(3): 354-359.
引用本文: 周励, 王玉凤, 桂建芳. 银鲫HIRA多克隆抗体的制备及组织特异性表达分析[J]. 水生生物学报, 2008, 32(3): 354-359.
ZHOU Li, WANG Yu-Feng, GUI Jian-Fang. PREPARATION OF POLYCLONAL ANTIBODY AND ANALYSIS OF SPATIAL EXPRESSION OF HIRA PROTEIN IN GIBEL CARP[J]. ACTA HYDROBIOLOGICA SINICA, 2008, 32(3): 354-359.
Citation: ZHOU Li, WANG Yu-Feng, GUI Jian-Fang. PREPARATION OF POLYCLONAL ANTIBODY AND ANALYSIS OF SPATIAL EXPRESSION OF HIRA PROTEIN IN GIBEL CARP[J]. ACTA HYDROBIOLOGICA SINICA, 2008, 32(3): 354-359.

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

  • 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。

     

    Abstract: 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.

     

/

返回文章
返回