【作者】 王晓楠；

【导师】 沈际佳；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2008， 硕士

【摘要】 血吸虫病是一种分布广泛、危害严重的人兽共患寄生虫病。血吸虫致病主要是由于性成熟雌虫所产虫卵在人畜肝脏大量沉积形成的虫卵肉芽肿和纤维化,而沉积在肠壁的虫卵排出体外是该病流行传播的重要途径。另一方面,血吸虫雌雄合抱是雌虫生殖系统正常发育和产卵的前提条件,雌虫的发育成熟又是雄虫依赖性的。因此,控制血吸虫性别分化、性成熟及雌虫产卵成为防治血吸虫病的重要策略。Mago nashi基因在模式生物如果蝇和秀丽隐杆线虫中是调节雌雄同体性别决定基因即决定生殖细胞雄性化作用的基因,该基因的蛋白产物与Tsunagi(或Y14)蛋白结合成复合物共同行使功能。已有实验报道这两种蛋白在进化过程中均高度保守表达。本课题组在前期工作中已经用RNAi方法研究了日本血吸虫Mago nashi基因(登录号BM735619)干扰后血吸虫的表型变化,发现部分实验组雄虫睾丸小叶中的精母细胞表现出泛雄性化表型。但是日本血吸虫Tsunagi基因至今未有报导,本文旨在获得日本血吸虫Tsunagi基因序列并对该基因的功能进行初步鉴定。根据遗传密码的简并性,本文利用黑腹果蝇和秀丽隐杆线虫Tsunagi蛋白高度保守的60个氨基酸序列,使用blastp,blockmarker,CodeHop等网络工具及数据库设计一对简并引物,从日本血吸虫成虫cDNA中扩增出了一段长180bp的PCR产物,测序后经blastx检索GenBank,发现这段基因的氨基酸序列与黑腹果蝇和秀丽隐杆线虫Tsunagi蛋白的氨基酸序列高度同源。然后利用5’端和3’端锚定PCR方法分别从日本血吸虫童虫cDNA文库中扩增出日本血吸虫Tsunagi蛋白基因上、下游的未知序列。再根据锚定PCR产物序列设计一对特异性引物,并在引物5’端分别引入BamⅠ和XhoⅠ酶切位点,从日本血吸虫童虫cDNA文库和日本血吸虫成虫cDNA中均扩增出了日本血吸虫Tsunagi基因的开放阅读框(Open reading frame, ORF)。经基因测序分析后该cDNA长度为531bp,编码177个氨基酸,蛋白的理论分子量为20.078 KD,等电点为6.195。SjTsunagi基因同源性比对后发现其编码的氨基酸序列与其它物种的Tsunagi蛋白有很高的相似性:与黑腹果蝇Tsunagi蛋白的氨基酸序列有55.9%的同源性和67.2%的相似性;与秀丽隐杆线虫Tsunagi蛋白的氨基酸序列有40.6%的同源性和51.4%的相似性。蛋白功能分析预测该蛋白含有一个保守的RNA识别模序,理论上是RNA结合蛋白。将获得的日本血吸虫Tsunagi基因的完整ORF亚克隆入原核表达载体pET28a(+)中,构建的重组质粒pET28a-SjTsunagi在大肠杆菌中获得了可溶性表达。用纯化后的融合蛋白免疫小鼠,经ELISA鉴定获得的多克隆抗体效价为1: 51200,免疫印迹证实抗体可特异性识别目的蛋白。为了观察天然Tsunagi蛋白在日本血吸虫体内的定位,我们首先制备了日本血吸虫成虫HE染色切片,掌握了日本血吸虫成虫在光学显微镜下的形态特点后,用间接免疫荧光法观察到日本血吸虫Tsunagi蛋白仅在雌虫卵巢的卵母细胞内高表达。利用免疫印迹观察SjTsunagi蛋白在虫卵、雌虫、雄虫和混合成虫等不同阶段的表达水平,结果发现该蛋白也仅在雌虫阶段表达,提示Tsunagi蛋白可能是血吸虫的性别相关基因。利用Pull-down和免疫共沉淀实验,证实了日本血吸虫Tsunagi和Mago nashi蛋白的相互作用关系。本论文率先开展了血吸虫Tsunagi基因的研究,首次获得编码Tsunagi蛋白的基因,成功将Tsunagi基因在大肠杆菌中进行表达并获得了特异性的多克隆抗体;免疫荧光观察到天然Tsunagi蛋白在雌虫的卵母细胞内高表达;免疫印迹发现该蛋白仅在雌虫阶段表达;Pull-down和免疫共沉淀实验证实Tsunagi与Mago nashi蛋白的相互作用,提示这两种保守表达的蛋白在日本血吸虫中可能与模式生物一样也是性别发育相关基因。更多还原

【Abstract】 Schistosomiasis is known as a debilitating disease in human. The life cycle of schistosomes is complex and involves both a snail intermediate host and a mammalian definitive host in which they become adult worms that are reproductively active for years. The eggs produced by female worms are the agent of schistosomiasis transmission. Meanwhile, the pathogenesis of this disease is the granulomatous reaction evoked by the soluble egg antigens. Controlling sexual maturation, sexual dimorphism and labour division may be effective in prevention of schistosomiasis.Mago nashi was a strict maternal-effect gene important for germ plasm assembly during the physiological development of the Drosophila embryo and also plays important roles in cellular differentiation. The protein encoded by Mago nashi gene interacts avidly and specifically with the Tsunagi protein (also known as Y14) among divergent organisms. In Drosophila oogenesis, both Tsunagi and Mago nashi are required for interpreting the posterior follicle cell-to-oocyte signal to define the major body axes and to localize components necessary for determination of the primordial germ cells. Moreover, the Caenorhabditis elegans homologue Ce-Y14 is essential for embryonic morphogenesis and regulation of germline sex switching. Previous studies in other laboratories have also shown the significance of Tsunagi in sexual development and its conservation among species. We have previously isolated the Mago nashi gene (GenBank accession number BM735619) from a Schistosomulum japonicum cDNA library and investigated its function in schistosomes by RNAi method. However, no information is available regarding the characterization of Tsunagi in Schistosoma japonicum. We hypothesized that because schistosomes undergo sexual differentiation during development, it is probable that the parasites have similar pathways to achieve this. Moreover, because Tsunagi proteins are ubiquitous and highly conserved, we reasoned that a Tsunagi homologue is also present in S. japonicum. This paper describes the identification and preliminary characterization of the Tsunagi protein from S. japonicum.For this purpose, we have isolated the partial gene of Tsunagi protein from adult cDNA of S. japonicum using a degenerate PCR strategy. This strategy employed the CODEHOP primer design algorithm. The sequence amplified from degenerate PCR was about 180bp, displaying strong identity to other Tsunagi proteins. The remaining cDNA sequence, including the 5’and 3’regions, was recovered by anchored PCR from a cDNA library of Schistosomulum japonicum. The whole sequence of the SjTsunagi cDNA containing ORF was amplified from the schistosomulum cDNA library and the adult cDNA, respectively. It encoded a protein of 177 amino acids with an estimated molecular weight of 20.078 KD and isoelectric point of pH6.195. The gene has 55.9% identity and 67.2% similarity at the predicted amino acid level to Drosophila melanogaster and has 40.6% identity and 51.4% similarity to C. elegans. Amino acid sequence analysis and alignment showed significant similarity to other Tsunagi proteins, including the conservation of an RNA recognition motif. The encoding ORF was amplified and inserted into pET-28a expression vector and then pET-28a-SjTsunagi recombinant protein was expressed as a 6xHis-tag fusion protein. Soluble recombinant protein used for in vitro experiments was purified by affinity column chromatography. Specific antiserum was obtained from the mice immunized with the protein. Mouse antiserum (1: 51200) was used to detect the recombinant protein by indirect ELISA. To investigate the distribution of SjTsunagi in tissue, immunofluorescent staining was performed on cryosections prepared from adult worms. SjTsunagi was most abundant in the ovary of female worms. Immunoblot analyses was used to determine the proteins extracted from eggs, adult females, adult males and mixed sex parasites. Proteins extracted from adult females and mixed sex parasites probed with anti-SjTsunagi polyclonal antiserum identified a single protein with an apparent molecular weight of 20 KD and revealed that SjTsunagi protein is detected in adult females only. Besides, by pull-down and co-immunoprecipitation, we therefore concluded that the interaction of Tsunagi with the mago nashi gene product is also conserved in S. japonicum. Taken together, these results suggest that SjTsunagi might be a gender-associated protein in schistosomes.更多还原