本文選題：日本血吸蟲 + Tsunagi　； 參考：《安徽醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 血吸蟲病是一種分布廣泛、危害嚴(yán)重的人獸共患寄生蟲病。血吸蟲致病主要是由于性成熟雌蟲所產(chǎn)蟲卵在人畜肝臟大量沉積形成的蟲卵肉芽腫和纖維化,而沉積在腸壁的蟲卵排出體外是該病流行傳播的重要途徑。另一方面,血吸蟲雌雄合抱是雌蟲生殖系統(tǒng)正常發(fā)育和產(chǎn)卵的前提條件,雌蟲的發(fā)育成熟又是雄蟲依賴性的。因此,控制血吸蟲性別分化、性成熟及雌蟲產(chǎn)卵成為防治血吸蟲病的重要策略。 Mago nashi基因在模式生物如果蠅和秀麗隱桿線蟲中是調(diào)節(jié)雌雄同體性別決定基因即決定生殖細(xì)胞雄性化作用的基因,該基因的蛋白產(chǎn)物與Tsunagi(或Y14)蛋白結(jié)合成復(fù)合物共同行使功能。已有實(shí)驗(yàn)報(bào)道這兩種蛋白在進(jìn)化過程中均高度保守表達(dá)。本課題組在前期工作中已經(jīng)用RNAi方法研究了日本血吸蟲Mago nashi基因(登錄號BM735619)干擾后血吸蟲的表型變化,發(fā)現(xiàn)部分實(shí)驗(yàn)組雄蟲睪丸小葉中的精母細(xì)胞表現(xiàn)出泛雄性化表型。但是日本血吸蟲Tsunagi基因至今未有報(bào)導(dǎo),本文旨在獲得日本血吸蟲Tsunagi基因序列并對該基因的功能進(jìn)行初步鑒定。 根據(jù)遺傳密碼的簡并性,本文利用黑腹果蠅和秀麗隱桿線蟲Tsunagi蛋白高度保守的60個(gè)氨基酸序列,使用blastp,blockmarker,CodeHop等網(wǎng)絡(luò)工具及數(shù)據(jù)庫設(shè)計(jì)一對簡并引物,從日本血吸蟲成蟲cDNA中擴(kuò)增出了一段長180bp的PCR產(chǎn)物,測序后經(jīng)blastx檢索GenBank,發(fā)現(xiàn)這段基因的氨基酸序列與黑腹果蠅和秀麗隱桿線蟲Tsunagi蛋白的氨基酸序列高度同源。然后利用5’端和3’端錨定PCR方法分別從日本血吸蟲童蟲cDNA文庫中擴(kuò)增出日本血吸蟲Tsunagi蛋白基因上、下游的未知序列。再根據(jù)錨定PCR產(chǎn)物序列設(shè)計(jì)一對特異性引物,并在引物5’端分別引入BamⅠ和XhoⅠ酶切位點(diǎn),從日本血吸蟲童蟲cDNA文庫和日本血吸蟲成蟲cDNA中均擴(kuò)增出了日本血吸蟲Tsunagi基因的開放閱讀框(Open reading frame, ORF)。經(jīng)基因測序分析后該cDNA長度為531bp,編碼177個(gè)氨基酸,蛋白的理論分子量為20.078 KD,等電點(diǎn)為6.195。SjTsunagi基因同源性比對后發(fā)現(xiàn)其編碼的氨基酸序列與其它物種的Tsunagi蛋白有很高的相似性:與黑腹果蠅Tsunagi蛋白的氨基酸序列有55.9%的同源性和67.2%的相似性;與秀麗隱桿線蟲Tsunagi蛋白的氨基酸序列有40.6%的同源性和51.4%的相似性。蛋白功能分析預(yù)測該蛋白含有一個(gè)保守的RNA識別模序,理論上是RNA結(jié)合蛋白。將獲得的日本血吸蟲Tsunagi基因的完整ORF亞克隆入原核表達(dá)載體pET28a(+)中,構(gòu)建的重組質(zhì)粒pET28a-SjTsunagi在大腸桿菌中獲得了可溶性表達(dá)。用純化后的融合蛋白免疫小鼠,經(jīng)ELISA鑒定獲得的多克隆抗體效價(jià)為1: 51200,免疫印跡證實(shí)抗體可特異性識別目的蛋白。 為了觀察天然Tsunagi蛋白在日本血吸蟲體內(nèi)的定位,我們首先制備了日本血吸蟲成蟲HE染色切片,掌握了日本血吸蟲成蟲在光學(xué)顯微鏡下的形態(tài)特點(diǎn)后,用間接免疫熒光法觀察到日本血吸蟲Tsunagi蛋白僅在雌蟲卵巢的卵母細(xì)胞內(nèi)高表達(dá)。利用免疫印跡觀察SjTsunagi蛋白在蟲卵、雌蟲、雄蟲和混合成蟲等不同階段的表達(dá)水平,結(jié)果發(fā)現(xiàn)該蛋白也僅在雌蟲階段表達(dá),提示Tsunagi蛋白可能是血吸蟲的性別相關(guān)基因。利用Pull-down和免疫共沉淀實(shí)驗(yàn),證實(shí)了日本血吸蟲Tsunagi和Mago nashi蛋白的相互作用關(guān)系。 本論文率先開展了血吸蟲Tsunagi基因的研究,首次獲得編碼Tsunagi蛋白的基因,成功將Tsunagi基因在大腸桿菌中進(jìn)行表達(dá)并獲得了特異性的多克隆抗體;免疫熒光觀察到天然Tsunagi蛋白在雌蟲的卵母細(xì)胞內(nèi)高表達(dá);免疫印跡發(fā)現(xiàn)該蛋白僅在雌蟲階段表達(dá);Pull-down和免疫共沉淀實(shí)驗(yàn)證實(shí)Tsunagi與Mago nashi蛋白的相互作用,提示這兩種保守表達(dá)的蛋白在日本血吸蟲中可能與模式生物一樣也是性別發(fā)育相關(guān)基因。
[Abstract]:Schistosoma japonicum is one of the most widely distributed and endangered species of human and animal . Schistosoma japonicum is mainly caused by the large amount of egg granulomas and fibrosis in the liver of human and livestock .


Mago nashi gene ( Mago nashi ) is a gene which regulates the androgenism of the male and female in the pattern organism if the sex determination gene of male and female is regulated . The protein product of the gene is highly conserved with Tsunagi ( or Y14 ) protein . In the previous work , we have studied the phenotypic change of Schistosoma japonicum Mago nashi gene ( Accession No . BM735619 ) .


In this paper , a pair of degenerate primers were designed from the cDNA library of Schistosoma japonicum by using 60 amino acid sequences which were highly conserved by the Tsunagi protein . The sequences of amino acids were amplified from the cDNA library of Schistosoma japonicum by using the primers of blastx . Then , a pair of specific primers were designed from the cDNA library of Schistosoma japonicum , and the open reading frame ( ORF ) of the Tsunagi gene of Schistosoma japonicum was amplified from the cDNA library of Schistosoma japonicum and adult worm of Schistosoma japonicum . The deduced amino acid sequence of Tsunagi gene was 55.9 % homology and 67.2 % similarity with the amino acid sequence of Tsunagi protein . The recombinant plasmid pET28a - SjTsunagi was cloned into prokaryotic expression vector pET28a ( + ) .


In order to observe the localization of the native Tsunagi protein in Schistosoma japonicum , we first prepared HE stained sections of Schistosoma japonicum . The expression levels of SjTsunagi protein in different stages of egg , female , male and mixed adults were observed by indirect immunofluorescence .


The research of Tsunagi gene was first carried out in this paper . The gene encoding Tsunagi protein was obtained for the first time . The Tsunagi gene was successfully expressed in E . coli and the specific polyclonal antibody was obtained . The interaction between Tsunagi and Mago nashi protein was confirmed by immuno - fluorescence and the interaction between Tsunagi and Mago nashi protein was confirmed by Western blot .
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R382

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本文編號：1928804