GGNBP1和GGN2在小鼠睾丸中的表达及相互作用研究

发布时间：2018-03-07 14:55

本文选题：GGN2　切入点：GGNBP1　出处：《中国人民解放军军事医学科学院》2005年博士论文　论文类型：学位论文

【摘要】：原始生殖细胞(PGC)是成年性腺生殖细胞的始祖细胞。一种胚胎期生殖腺内PGC严重缺乏的生殖细胞缺失(germ cell-deficient,gcd)突变是外源基因插入失活FancL所致的隐性遗传性变异,导致成年雌雄鼠不育。胚胎期FANCL与原始生殖细胞增殖密切相关,成年睾丸中FANCL与几个睾丸生殖细胞特异性蛋白质相互作用可能影响精子的生成。以FANCL蛋白为诱饵蛋白,通过酵母双杂交实验发现了配子生成素基因Ggn。Ggn基因至少有10多种不同的拼接方式,预期编码GGN1、GGN2和GGN3三种蛋白。同样,通过酵母双杂交系统发现了与GGN1相互作用的配子生成素结合蛋白1(GGNBP1)。本课题旨在通过制备GGN2、GGNBP1和FANCL抗体,利用特异性抗体作为蛋白质研究的有效工具进行体内表达和相互作用研究,以探索它们在精子生成中所发挥的功能。首先,通过克隆表达FANCL和GGNBP1蛋白以及人工合成GGN2短肽获得抗原,皮下注射免疫家兔制备多抗血清。制备抗体亲和层析柱亲和纯化抗体。其次,利用纯化抗体通过Western blotting方法分析小鼠多种组织匀浆蛋白。首次证实小鼠体内确实存在FANCL、GGNBP1和GGN2蛋白。FANCL在小鼠组织细胞中广泛表达,而GGN2和GGNBP1则是小鼠睾丸特异性表达的蛋白质。第三,提取小鼠多种组织细胞总RNA,Northern blotting分析表明Ggn和Ggnbp1也都是小鼠睾丸特异性转录基因。第四,在NIH 3T3细胞中将带绿色荧光蛋白标签的EGFP-GGN1和EGFP-GGN2分别与带Myc标签的Myc-GGNBP1共表达,进行细胞免疫荧光共定位实验表明GGNBP1与GGN1和GGN2之间相互作用。采用酵母双杂交体系证实了它们之间的相互作用关系。第五,Superdex 200分子筛凝胶层析小鼠睾丸匀浆蛋白,分段收集后经亲和纯化抗体的Western blotting分析显示FANCL、GGN2和GGNBP1蛋白共同分布在大小约230KDa的收集管内,表明小鼠睾丸细胞中FANCL、GGN2和GGNBP1共同存在于一个大约230KDa的复合物内。最后,细胞共定位研究发现EGFP-GGN2集中分布于细胞泡状结构小体上。进
[Abstract]:Primordial germ cell (PGC) is the progenitor cell of adult gonadal germ cell. A germ-cell deletion mutation caused by the insertion of foreign gene into inactivated FancL is a recessive genetic variation caused by the serious lack of PGC in the gonad of embryo. FANCL is closely related to the proliferation of primordial germ cells. The interaction between FANCL and several testicular germ cell-specific proteins in adult testis may affect the formation of spermatozoa. FANCL protein is used as bait protein. Yeast two-hybrid experiments have found that there are at least 10 different splicing modes of the gametopoietin gene Ggn.Ggn gene, which is expected to encode three proteins, GGN1, GGN2 and GGN3. Gametin-binding protein (GGNBP1) interacting with GGN1 was found by yeast two-hybrid system. The purpose of this study was to study the expression and interaction of GGNBP1 and FANCL by using specific antibodies as an effective tool for protein research. To explore their role in spermatogenesis. Firstly, the antigens were obtained by cloning and expressing FANCL and GGNBP1 proteins and synthesizing GGN2 short peptides. Rabbits were immunized subcutaneously to prepare multi-antiserum, and the antibodies were purified by affinity chromatography. Secondly, the purified antibody was used to analyze the homogenate proteins of various tissues of mice by Western blotting method. It was first confirmed that FANCLN GGNBP1 and GGN2 proteins were widely expressed in mouse tissue cells. GGN2 and GGNBP1 are specific proteins expressed in mouse testis. Third, Northern blotting analysis showed that Ggn and Ggnbp1 were also mouse testis specific transcription genes. 4th, EGFP-GGN1 and EGFP-GGN2 with green fluorescent protein tag and Myc-GGNBP1 with Myc tag were co-expressed in NIH 3T3 cells, respectively. The interaction between GGNBP1 and GGN1 and GGN2 was confirmed by using yeast two-hybrid system. Mouse testicular homogenate protein was separated by 5th and Superdex 200 molecular sieve gel chromatography. Western blotting analysis of affinity purified antibody showed that FANCLN GGN2 and GGNBP1 protein were distributed in a collection tube about 230 KDa in size. The results showed that FANCLN GGN2 and GGNBP1 co-existed in a complex of about 230 KDa in mouse testicular cells. Finally, the co-localization of EGFP-GGN2 was found to be concentrated on the vesicular structure of the cell.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：Q78

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