本文選題：GGN2　切入點：GGNBP1　出處：《中國人民解放軍軍事醫(yī)學科學院》2005年博士論文　論文類型：學位論文

【摘要】：原始生殖細胞(PGC)是成年性腺生殖細胞的始祖細胞。一種胚胎期生殖腺內(nèi)PGC嚴重缺乏的生殖細胞缺失(germ cell-deficient,gcd)突變是外源基因插入失活FancL所致的隱性遺傳性變異,導致成年雌雄鼠不育。胚胎期FANCL與原始生殖細胞增殖密切相關,成年睪丸中FANCL與幾個睪丸生殖細胞特異性蛋白質(zhì)相互作用可能影響精子的生成。以FANCL蛋白為誘餌蛋白,通過酵母雙雜交實驗發(fā)現(xiàn)了配子生成素基因Ggn。Ggn基因至少有10多種不同的拼接方式,預期編碼GGN1、GGN2和GGN3三種蛋白。同樣,通過酵母雙雜交系統(tǒng)發(fā)現(xiàn)了與GGN1相互作用的配子生成素結合蛋白1(GGNBP1)。本課題旨在通過制備GGN2、GGNBP1和FANCL抗體,利用特異性抗體作為蛋白質(zhì)研究的有效工具進行體內(nèi)表達和相互作用研究,以探索它們在精子生成中所發(fā)揮的功能。 首先,通過克隆表達FANCL和GGNBP1蛋白以及人工合成GGN2短肽獲得抗原,皮下注射免疫家兔制備多抗血清。制備抗體親和層析柱親和純化抗體。 其次,利用純化抗體通過Western blotting方法分析小鼠多種組織勻漿蛋白。首次證實小鼠體內(nèi)確實存在FANCL、GGNBP1和GGN2蛋白。FANCL在小鼠組織細胞中廣泛表達,而GGN2和GGNBP1則是小鼠睪丸特異性表達的蛋白質(zhì)。 第三,提取小鼠多種組織細胞總RNA,Northern blotting分析表明Ggn和Ggnbp1也都是小鼠睪丸特異性轉錄基因。 第四,在NIH 3T3細胞中將帶綠色熒光蛋白標簽的EGFP-GGN1和EGFP-GGN2分別與帶Myc標簽的Myc-GGNBP1共表達,進行細胞免疫熒光共定位實驗表明GGNBP1與GGN1和GGN2之間相互作用。采用酵母雙雜交體系證實了它們之間的相互作用關系。 第五,Superdex 200分子篩凝膠層析小鼠睪丸勻漿蛋白,分段收集后經(jīng)親和純化抗體的Western blotting分析顯示FANCL、GGN2和GGNBP1蛋白共同分布在大小約230KDa的收集管內(nèi),表明小鼠睪丸細胞中FANCL、GGN2和GGNBP1共同存在于一個大約230KDa的復合物內(nèi)。 最后,細胞共定位研究發(fā)現(xiàn)EGFP-GGN2集中分布于細胞泡狀結構小體上。進
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2005
【分類號】：Q78

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1 趙慶國;GGNBP1和GGN2在小鼠睪丸中的表達及相互作用研究[D];中國人民解放軍軍事醫(yī)學科學院;2005年

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本文編號：1579765