日本血吸蟲SjShh基因的鑒定分析及SjWnt1蛋白功能的初步研究
發(fā)布時間：2019-03-02 08:10
【摘要】：日本血吸蟲病是由日本血吸蟲引起的一種嚴重的寄生蟲病,危及人類及家畜的健康,影響社會經(jīng)濟的發(fā)展。治療上用藥物單一,存在用藥風(fēng)險。Hedgedge(Hh),Wnt信號通路是與發(fā)育相關(guān)的通路,均可以影響個體的胚胎發(fā)育及維持組織穩(wěn)態(tài)。本文對日本血吸蟲中的Sonic Hedgehog(Shh)基因進行鑒定分析,通過小分子干擾(siRNA)抑制Hh通路中的Shh及wnt通路的wnt1的靶基因,來進一步明確其在日本血吸蟲中的作用,為尋求日本血吸蟲的新的藥靶基因提供思路。1、日本血吸蟲SjShh基因的鑒定分析及不同發(fā)育階段及不同性別蟲體的mRNA的表達利用NCBI檢索Shh的EST序列,并通過RACE技術(shù)得到全基因序列并命名為SjShh基因,并對其進行生物信息學(xué)分析,開放閱讀框(ORF)為2145bp。SjShh蛋白有兩個蛋白結(jié)構(gòu)域,位于氨基端的“Hedge”結(jié)構(gòu)域(HhN)和位于羧基末端的“Hog”結(jié)構(gòu)域(HhC)。對SjShh進行轉(zhuǎn)錄水平結(jié)果的分析,表明SjShh基因在日本血吸蟲的整個發(fā)育階段均起到必不可少的作用�；蜻M行各蟲體階段的mRNA表達水平分析,提示SjShh基因調(diào)控日本血吸蟲的整個發(fā)育過程,在童蟲階段表達量明顯高于成蟲、蟲卵及尾蚴階段,童蟲的18天蟲體轉(zhuǎn)錄水平達到最大,提示SjShh基因?qū)θ毡狙x的生殖系統(tǒng)發(fā)育相關(guān)。非適宜宿主與單性別感染的非正常發(fā)育的蟲體的mRNA轉(zhuǎn)錄水平增高,提示蟲體發(fā)育滯后可以導(dǎo)致Hh通路的轉(zhuǎn)導(dǎo)延遲。2、日本血吸蟲SjShh蛋白的功能研究成功篩選出有效且穩(wěn)定的siRNA,編號為Shh796,并確定最適干擾濃度為1.5OD/ml,進行Shh796 siRNA的體內(nèi)14、24天兩個階段的干擾實驗,結(jié)果表明均有良好的抑制效果,體外SjShh siRNA的干擾試驗中,72小時時抑制效果做好。但是SjShh基因的體內(nèi)的siRNA干擾,對蟲體的形態(tài)和宿主的血常規(guī)沒有明顯的影響。生化指標(biāo)ALT、AST和LDH的減少,表明干擾Hh通路,可以減輕日本血吸蟲對宿主肝臟的損傷。3、日本血吸蟲SjWnt1蛋白的功能研究23天蟲體免疫組化結(jié)果分析,Wnt1蛋白主要分泌在體被下層,腸管周圍和睪丸中,其中睪丸的分布最密集,提示SjWnt1蛋白的功能可能與日本血吸蟲雄蟲的生殖相關(guān)。對SjWnt1進行siRNA干擾,降低其轉(zhuǎn)錄水平,通路的下游基因的mRNA的表達量也出現(xiàn)下調(diào),表明SjWnt1參與Wnt通路的三個轉(zhuǎn)導(dǎo)途徑即Wnt/β-catenin、Wnt/PCP及Wnt/Ca2+信號通路。體外siRNA干擾后蟲體電鏡結(jié)果:皮層變得腫脹,體被出現(xiàn)較大空泡,基底膜溶解,擴散,體棘減少,線粒體激增且伴有嚴重的腫脹,腸道微絨毛脫落,出現(xiàn)斷裂,表明SjWnt1的抑制使日本血吸蟲的表層受損,蟲體能量代謝失衡,蟲體自身的穩(wěn)態(tài)受到影響。本文通過Race技術(shù)獲得了SjShh基因,并對其進行了鑒定分析,通過定量的方法確定了SjShh基因在日本血吸蟲中的表達情況。對SjShh及SjWnt1進行siRNA的體內(nèi)與體外的干擾實驗,為了解SjShh、SjWnt1在日本血吸蟲中的功能作用提供了基礎(chǔ)。
[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).
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.735
本文編號：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).
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.735
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