日本血吸虫SjShh基因的鉴定分析及SjWnt1蛋白功能的初步研究
发布时间：2019-03-02 08:10
【摘要】：日本血吸虫病是由日本血吸虫引起的一种严重的寄生虫病,危及人类及家畜的健康,影响社会经济的发展。治疗上用药物单一,存在用药风险。Hedgedge(Hh),Wnt信号通路是与发育相关的通路,均可以影响个体的胚胎发育及维持组织稳态。本文对日本血吸虫中的Sonic Hedgehog(Shh)基因进行鉴定分析,通过小分子干扰(siRNA)抑制Hh通路中的Shh及wnt通路的wnt1的靶基因,来进一步明确其在日本血吸虫中的作用,为寻求日本血吸虫的新的药靶基因提供思路。1、日本血吸虫SjShh基因的鉴定分析及不同发育阶段及不同性别虫体的mRNA的表达利用NCBI检索Shh的EST序列,并通过RACE技术得到全基因序列并命名为SjShh基因,并对其进行生物信息学分析,开放阅读框(ORF)为2145bp。SjShh蛋白有两个蛋白结构域,位于氨基端的“Hedge”结构域(HhN)和位于羧基末端的“Hog”结构域(HhC)。对SjShh进行转录水平结果的分析,表明SjShh基因在日本血吸虫的整个发育阶段均起到必不可少的作用。基因进行各虫体阶段的mRNA表达水平分析,提示SjShh基因调控日本血吸虫的整个发育过程,在童虫阶段表达量明显高于成虫、虫卵及尾蚴阶段,童虫的18天虫体转录水平达到最大,提示SjShh基因对日本血吸虫的生殖系统发育相关。非适宜宿主与单性别感染的非正常发育的虫体的mRNA转录水平增高,提示虫体发育滞后可以导致Hh通路的转导延迟。2、日本血吸虫SjShh蛋白的功能研究成功筛选出有效且稳定的siRNA,编号为Shh796,并确定最适干扰浓度为1.5OD/ml,进行Shh796 siRNA的体内14、24天两个阶段的干扰实验,结果表明均有良好的抑制效果,体外SjShh siRNA的干扰试验中,72小时时抑制效果做好。但是SjShh基因的体内的siRNA干扰,对虫体的形态和宿主的血常规没有明显的影响。生化指标ALT、AST和LDH的减少,表明干扰Hh通路,可以减轻日本血吸虫对宿主肝脏的损伤。3、日本血吸虫SjWnt1蛋白的功能研究23天虫体免疫组化结果分析,Wnt1蛋白主要分泌在体被下层,肠管周围和睾丸中,其中睾丸的分布最密集,提示SjWnt1蛋白的功能可能与日本血吸虫雄虫的生殖相关。对SjWnt1进行siRNA干扰,降低其转录水平,通路的下游基因的mRNA的表达量也出现下调,表明SjWnt1参与Wnt通路的三个转导途径即Wnt/β-catenin、Wnt/PCP及Wnt/Ca2+信号通路。体外siRNA干扰后虫体电镜结果:皮层变得肿胀,体被出现较大空泡,基底膜溶解,扩散,体棘减少,线粒体激增且伴有严重的肿胀,肠道微绒毛脱落,出现断裂,表明SjWnt1的抑制使日本血吸虫的表层受损,虫体能量代谢失衡,虫体自身的稳态受到影响。本文通过Race技术获得了SjShh基因,并对其进行了鉴定分析,通过定量的方法确定了SjShh基因在日本血吸虫中的表达情况。对SjShh及SjWnt1进行siRNA的体内与体外的干扰实验,为了解SjShh、SjWnt1在日本血吸虫中的功能作用提供了基础。
[Abstract]:Schistosoma japonicum is a serious parasitic disease caused by Schistosoma japonicum, which endangers the health of human and domestic animals and affects the development of social economy. Hedgedge (Hh), Wnt signaling pathway is a developmental pathway which can affect the embryonic development of individuals and maintain tissue homeostasis. In this paper, the Sonic Hedgehog (Shh) gene in Schistosoma japonicum was identified and analyzed, and the role of (siRNA) in Schistosoma japonicum was further clarified by interfering with the target gene of Shh in Hh pathway and wnt1 in wnt pathway. 1. The identification and analysis of SjShh gene of Schistosoma japonicum and the expression of mRNA in different developmental stages and genders of Schistosoma japonicum were carried out by NCBI to search the EST sequence of Shh. The whole gene sequence was obtained by RACE and named as SjShh gene. The open reading frame (ORF) (ORF) had two protein domains of 2145bp.SjShh protein, which was analyzed by bioinformatics. The "Hedge" domain (HhN) at the amino end and the "Hog" domain (HhC). At the carboxyl end The analysis of transcription level of SjShh showed that SjShh gene played an essential role in the whole development stage of Schistosoma japonicum (Schistosoma japonicum). The results showed that SjShh gene regulated the whole developmental process of Schistosoma japonicum, and the expression level of SjShh gene was significantly higher than that of adult worm at the stage of larval and cercariae. The transcription level of 18-day-old worm reached the maximum at the stage of egg and cercariae. These results suggest that SjShh gene is related to the reproductive system development of Schistosoma japonicum. The increased level of mRNA transcription in unsuitable host and unisexual developing worms suggests that delayed development of the worm may lead to delayed transduction of the Hh pathway. 2. The functional study of Schistosoma japonicum SjShh protein successfully screened out the effective and stable siRNA, numbered Shh796, and determined the optimal interference concentration of 1.5 OD / ml. The interference experiments were carried out in Shh796 siRNA for 14 days and 24 days. The results showed that all of them had good inhibition effect. In the interference test of SjShh siRNA in vitro, the inhibition effect was good at 72 hours. However, the siRNA interference of SjShh gene had no obvious effect on the morphology of the worm and the blood routine of the host. The decrease of ALT,AST and LDH indicated that interference of Hh pathway could reduce the damage of Schistosoma japonicum to the host liver. 3. The function of SjWnt1 protein of Schistosoma japonicum was studied by immunohistochemistry in 23 days. The Wnt1 protein is mainly secreted in the lower layer of the body, around the intestine and in the testis, in which the distribution of the testis is the most dense, suggesting that the function of SjWnt1 protein may be related to the reproduction of the male Schistosoma japonicum. The expression of mRNA in downstream genes of SjWnt1 was down-regulated by siRNA interference, which indicated that SjWnt1 was involved in three transduction pathways of Wnt pathway, namely Wnt/ 尾-catenin,Wnt/PCP and Wnt/Ca2 signaling pathways. After interference of siRNA in vitro, the results of electron microscope showed that the cortex became swollen, the body appeared large vacuoles, the basement membrane dissolved, diffused, the body spine decreased, the mitochondria proliferated and accompanied with severe swelling, the intestinal microvilli fell off and appeared to break. The results showed that the inhibition of SjWnt1 damaged the surface layer of Schistosoma japonicum, the energy metabolism of Schistosoma japonicum was unbalanced, and the homeostasis was affected. The expression of SjShh gene in Schistosoma japonicum (Schistosoma japonicum) was determined by quantitative analysis and identification of SjShh gene by Race technique. The in vivo and in vitro interference tests of siRNA on SjShh and SjWnt1 provide a basis for understanding the function of SjShh,SjWnt1 in Schistosoma japonicum (Schistosoma japonicum).
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.735
本文编号：2432865
[Abstract]:Schistosoma japonicum is a serious parasitic disease caused by Schistosoma japonicum, which endangers the health of human and domestic animals and affects the development of social economy. Hedgedge (Hh), Wnt signaling pathway is a developmental pathway which can affect the embryonic development of individuals and maintain tissue homeostasis. In this paper, the Sonic Hedgehog (Shh) gene in Schistosoma japonicum was identified and analyzed, and the role of (siRNA) in Schistosoma japonicum was further clarified by interfering with the target gene of Shh in Hh pathway and wnt1 in wnt pathway. 1. The identification and analysis of SjShh gene of Schistosoma japonicum and the expression of mRNA in different developmental stages and genders of Schistosoma japonicum were carried out by NCBI to search the EST sequence of Shh. The whole gene sequence was obtained by RACE and named as SjShh gene. The open reading frame (ORF) (ORF) had two protein domains of 2145bp.SjShh protein, which was analyzed by bioinformatics. The "Hedge" domain (HhN) at the amino end and the "Hog" domain (HhC). At the carboxyl end The analysis of transcription level of SjShh showed that SjShh gene played an essential role in the whole development stage of Schistosoma japonicum (Schistosoma japonicum). The results showed that SjShh gene regulated the whole developmental process of Schistosoma japonicum, and the expression level of SjShh gene was significantly higher than that of adult worm at the stage of larval and cercariae. The transcription level of 18-day-old worm reached the maximum at the stage of egg and cercariae. These results suggest that SjShh gene is related to the reproductive system development of Schistosoma japonicum. The increased level of mRNA transcription in unsuitable host and unisexual developing worms suggests that delayed development of the worm may lead to delayed transduction of the Hh pathway. 2. The functional study of Schistosoma japonicum SjShh protein successfully screened out the effective and stable siRNA, numbered Shh796, and determined the optimal interference concentration of 1.5 OD / ml. The interference experiments were carried out in Shh796 siRNA for 14 days and 24 days. The results showed that all of them had good inhibition effect. In the interference test of SjShh siRNA in vitro, the inhibition effect was good at 72 hours. However, the siRNA interference of SjShh gene had no obvious effect on the morphology of the worm and the blood routine of the host. The decrease of ALT,AST and LDH indicated that interference of Hh pathway could reduce the damage of Schistosoma japonicum to the host liver. 3. The function of SjWnt1 protein of Schistosoma japonicum was studied by immunohistochemistry in 23 days. The Wnt1 protein is mainly secreted in the lower layer of the body, around the intestine and in the testis, in which the distribution of the testis is the most dense, suggesting that the function of SjWnt1 protein may be related to the reproduction of the male Schistosoma japonicum. The expression of mRNA in downstream genes of SjWnt1 was down-regulated by siRNA interference, which indicated that SjWnt1 was involved in three transduction pathways of Wnt pathway, namely Wnt/ 尾-catenin,Wnt/PCP and Wnt/Ca2 signaling pathways. After interference of siRNA in vitro, the results of electron microscope showed that the cortex became swollen, the body appeared large vacuoles, the basement membrane dissolved, diffused, the body spine decreased, the mitochondria proliferated and accompanied with severe swelling, the intestinal microvilli fell off and appeared to break. The results showed that the inhibition of SjWnt1 damaged the surface layer of Schistosoma japonicum, the energy metabolism of Schistosoma japonicum was unbalanced, and the homeostasis was affected. The expression of SjShh gene in Schistosoma japonicum (Schistosoma japonicum) was determined by quantitative analysis and identification of SjShh gene by Race technique. The in vivo and in vitro interference tests of siRNA on SjShh and SjWnt1 provide a basis for understanding the function of SjShh,SjWnt1 in Schistosoma japonicum (Schistosoma japonicum).
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.735
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