【摘要】： 血吸蟲病是嚴重危害人民身體健康,阻礙社會經(jīng)濟發(fā)展的重大傳染病。日本血吸蟲致病主要是由于蟲卵在肝臟大量沉積形成的蟲卵肉芽腫及其纖維化�？刂蒲x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之一。 為了解日本血吸蟲Tsunagi蛋白的功能,我們用RNAi技術下調Tsunagi基因的轉錄水平,再觀察干擾作用后蟲體的發(fā)育(尤其是生殖系統(tǒng)發(fā)育)是否與正常蟲體之間存在差異。按照雙鏈RNA(dsRNA)體外合成試劑盒要求,我們從日本血吸蟲童蟲cDNA文庫中擴增出Tsunagi基因,將其片段正、反義鏈連接上T7啟動子,然后把連接有T7啟動子的目的基因的正、反義鏈進行PCR擴增。將PCR產(chǎn)物轉錄成單鏈RNA(ssRNA)并將其純化,最后把一對ssRNA混合合成dsRNA。陰性對照組我們采用試劑盒中提供的基因。在BTX電穿孔儀上,設定參數(shù)為125V電壓、20ms脈沖時值、1次脈沖次數(shù),將dsRNA電穿孔轉染入機械制備的日本血吸蟲童蟲體內,體外培養(yǎng)于電轉后第1, 3, 5天收集童蟲,按TRIzol方法同時提取童蟲的總RNA及總蛋白。經(jīng)實時熒光定量PCR及Western blotting分別檢測Tsunagi基因和蛋白表達水平變化。結果發(fā)現(xiàn)Tsunagi基因的轉錄水平實驗組分別于電穿孔后第1, 3, 5天較陰性對照組下降16%,57%,75%。該基因的蛋白水平在第1, 3, 5天較陰性對照組下降15%,38%,52%。實驗結果證明dsRNA可以特異性的抑制日本血吸蟲靶基因以及蛋白的表達且效果明顯。經(jīng)dsRNA電轉的童蟲注射入小鼠體內,6周后取出蟲體通過一系列處理制成標本,在激光共聚焦顯微鏡下觀察蟲體內部各器官形態(tài)特征及測量蟲體體長、體寬、睪丸長、睪丸寬、睪丸面積、卵巢長、卵巢寬、卵巢面積。結果發(fā)現(xiàn)SjTsunagi dsRNA組中8條中3～4條雄蟲睪丸內有大量精子出現(xiàn),而雌蟲中卵巢,卵黃腺中卻沒有明顯特征變化;測得各項指標經(jīng)SPSS 13.0統(tǒng)計軟件分析陰性對照組與SjTsunagi dsRNA組的體寬、睪丸長、睪丸寬、睪丸面積、卵巢長、卵巢寬、卵巢面積均有顯著性差異。由此結果我們得出Tsunagi基因在日本血吸蟲中是生殖相關基因,在生殖系統(tǒng)器官的正常發(fā)育起一定作用。
[Abstract]:Schistosomiasis is a major infectious disease that harms people's health and hinders social and economic development. Schistosoma japonicum is mainly caused by egg granuloma and fibrosis. Controlling the sex differentiation, sexual maturation and laying eggs of female schistosomiasis is one of the important strategies to control schistosomiasis. In order to understand the function of Tsunagi protein in Schistosoma japonicum, we down-regulated the transcription level of Tsunagi gene by RNAi technique, and then observed whether there were differences between the body development (especially reproductive system development) and normal body after interference. According to the requirement of double-stranded RNA (dsRNA) in vitro synthesis kit, we amplified the Tsunagi gene from the cDNA library of Schistosoma japonicum, and ligated the Tsunagi gene into T7 promoter by sense, antisense chain, and then ligated the target gene with T7 promoter. Antisense strand was amplified by PCR. PCR products were transcribed into single-stranded RNA (ssRNA) and purified. Finally, a pair of ssRNA was mixed to synthesize dsRNA.. In the negative control group, we used the genes provided in the kit. On the BTX electroporation instrument, the parameters of 125V voltage, 20ms pulse time value and the number of pulses were set. The dsRNA electroporation was transfected into the mechanically prepared Schistosoma japonicum child worm and cultured in vitro on the 1st, 3rd and 5th day after electroporation. Total RNA and total protein were extracted by TRIzol method. The expression levels of Tsunagi gene and protein were detected by real-time fluorescence quantitative PCR and Western blotting, respectively. The results showed that the transcription level of Tsunagi gene in the experimental group was 16% lower than that in the negative control group on the 1st, 3rd and 5th day after electroporation. The protein level of the gene was 15% lower than that of the negative control group on the 1st, 3rd and 5th day compared with the negative control group. The results showed that dsRNA could specifically inhibit the expression of target gene and protein of Schistosoma japonicum. The mice were injected with dsRNA electroporated baby worms. After 6 weeks, the worm bodies were taken out and made into specimens by a series of treatments. The morphological characteristics of organs in the body were observed under confocal laser microscope, and the length, width, length of testis and width of testis were measured under confocal laser microscope. Testicular area, ovarian length, ovarian width, ovarian area. The results showed that a large number of spermatozoa were found in the testis of 3 male and 4 males in the SjTsunagi dsRNA group, but there were no obvious changes in the ovaries and yolk glands in the females. The body width, testicular length, testicular width, testicular area, ovary length, ovarian width and ovarian area were significantly different between the negative control group and the SjTsunagi dsRNA group by SPSS 13.0 statistical software. It is concluded that Tsunagi gene is a reproductive related gene in Schistosoma japonicum and plays a certain role in the normal development of reproductive system organs.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R346

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