论文标题:睾丸特异蛋白GGN1与GGNBP2的相互作用研究
Studies on the Anti-HIV Principles of Ilex Pubescens Hook.et Arn
论文作者 曹志国
论文导师 黄培堂；卢柏松,论文学位 硕士,论文专业 遗传学
论文单位 中国人民解放军军事医学科学院,点击次数 92,论文页数 68页File Size4913k
2005-05-18论文网 http://www.lw23.com/lunwen_123885692/ GGN1/GGNBP2;蛋白相互作用;精子发生
GGN1;GGNBP2;protein interaction;spermatogenesis
生殖细胞缺陷症(germ cell deficient,gcd)突变体小鼠,成年不育,它是男性缺精症和女性卵巢早衰的重要疾病模型。FancL(也叫Pog,proliferation of germ cells)的缺失是引起gcd突变小鼠的原因。FANCL是一种含有PHD结构域的泛素E3连接酶,参与DNA的损伤修复。睾丸生殖细胞特异的基因Ggn的表达产物GGN1和GGN3与FANCL相互作用。Ggn通过不同的剪接,产生三种不同的蛋白GGN1、GGN2、GGN3,其中GGN3与GGN1 C端完全同源。Ggn mRNA在睾丸中的表达时相表明Ggn在精子生成中可能起重要作用:尽管GGN1与FANCL相互作用,但GGN1参与的细胞过程还不清楚。揭示更多的参与该过程的蛋白质,特别是功能已知的蛋白质,为GGN1的功能研究以及了解精子生成过程具有重要价值。 本研究利用酵母双杂交技术,以GGN1蛋白的两个不同区段为诱饵蛋白,筛选成年小鼠睾丸cDNA文库,获得三个与GGN1有潜在相互作用的蛋白:GGNBP1、GGNBP2、OAZ3。我们制备了GGNBP2、GGN1高特异性多克隆抗体并进行了纯化、鉴定;通过细胞共定位和免疫共沉淀实验确证了GGN1与GGNBP2在哺乳动物细胞中的相互作用,通过构建突变体共转染酵母细胞确定了相互作用的区域即GGNBP2 N端400个氨基酸残基与GGN1 C端82个氨基酸残基足以介导两者的相互作用;两蛋白经翻译后修饰分子量变大。 GGNBP2原名DIF-3,在成年睾丸中高表达;脊椎动物的GGNBP2高度保守,如鸡与哺乳动物的GGNBP2有90%以上的同源性;但GGNBP2是一个功能未知的蛋白质,为此,本文对Ggnbp2的表达进行了研究,Northern blot实验结果表明:在成年小鼠的不同组织中,Ggnbp2基因mRNA在睾丸中有高表达,而在非睾丸组织中表达量很低;Ggnbp2基因mRNA的表达随小鼠年龄增大而升高。另外RT-PCR实验显示:小鼠睾丸组织的pre-mRNA的剪接方式与非睾丸组织的剪接方式不同。本研究为GGN1的功能研究、生殖细胞的发育调控、男性不育的治疗和节育措施的研究奠定了基础。
Germ-cell deficient (gcd) mouse is a mutant manifesting infertility at adulthood, which served as a mouse model for the sertoli-cell-only syndrome in male and premature ovarian failure (POF) disease in female[1]. Recently it was found that FancL (originally called Pog) was the gene underlying gcd mutation[2,3]. FANCL was then found to be an ubiquitin E3 ligase, which is involved in DNA damage repair[4-6]. It was recently found that a novel germ cell-specific gene Ggn (Gametogenetin) encoding products, GGNl and GGN3 interacting with FANCL[7]. Ggn encoded 3 putative proteins, gametogenetin protein 1 (GGNl), gametogenetin protein 2 (GGN2) and gametogenetin protein 3 (GGN3) by alternative splicing. GGN3 and GGNl C-termini are completely identical.The spatial and temporal expression of Ggn mRNA in the testis indicated that this gene might serve important role in spermatogenesis. Despite the finding that GGNl interacted with FANCL, the cellular pathway GGNl is involved in remained unknown. Finding more interacting partners of GGNl, especially the interacting partners with known function, will be helpful for the study of the function of GGNl and will also benefit our understanding of spermatogenesis[7].We used the yeast two-hybrid system to screen adult testis libraries to look for GGNl interacting proteins. Different regions of GGNl were used as the bait. After several screens we identified three proteins, GGNBP1, GGNBP2 and OAZ3 as the potential interaction partners of GGNl. We prepared the high-specificity polyclonal antibody of GGNBP2 and GGN3. The N-terminal 400 amino acid residues of GGNBP2 were enough to mediate the interaction with the C-terminal 82 amino acid residues of GGNl. The interaction between the two proteins was further confirmed by co-localization and coimmunoprecipitation experiments. Both proteins were post-translationally modified resulting in much more larger molecular weight than expected[8].
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