本文關(guān)鍵詞： 日本血吸蟲抱雌溝蛋白 噬菌體展示cDNA文庫 篩選 陽性噬菌體克隆 免疫　出處：《新疆農(nóng)業(yè)大學》2009年碩士論文　論文類型：學位論文

【摘要】： 血吸蟲病(Schisosomiasis)是由血吸蟲寄生于人體所引起的一種分布廣泛、危害嚴重的人獸共患寄生蟲病。血吸蟲是吸蟲中罕見的雌雄異體形式,在血吸蟲發(fā)育過程中,雌雄蟲合抱是雌蟲發(fā)育成熟的前提,雌雄蟲間信息物質(zhì)的傳遞通過合抱實現(xiàn)。因此,如果能夠找出這種雌雄蟲間傳遞的信息物質(zhì),對研究血吸蟲雌雄蟲之間的相互作用、血吸蟲生殖發(fā)育機理以及對抗血吸蟲疫苗研究有非常重要的意義。噬菌體展示技術(shù)是新近發(fā)展起來的,將外源肽或蛋白與特定噬菌體衣殼蛋白融合,并展示于噬菌體表面的一項新技術(shù),已廣泛應用于分子間識別機制的研究。 本研究用融合表達的日本血吸蟲抱雌溝蛋白篩選日本血吸蟲成蟲噬菌體展示cDNA文庫,隨機選取獲得的陽性噬菌體克隆進行測序、生物信息學分析,并對其中部分陽性克隆進行了免疫預防效果評估。 經(jīng)過三輪篩選后,噬菌體由第一輪的1.8×10-6pfu增加到第三輪的1.6×10-4pfu,富集了將近90倍,成功的篩除了低親和力和非特異性結(jié)合的噬菌體。隨機挑取陽性噬菌體克隆進行測序以及生物信息學分析,共獲得25個有效EST序列,其中20個與已知基因或表達序列標簽同源,5個EST序列與已知基因或表達序列標簽均無同源性,為新的表達序列標簽。對20個有效編碼蛋白的功能預測結(jié)果顯示:10個EST序列為卵殼蛋白相關(guān)基因與血吸蟲卵的發(fā)育相關(guān),3個EST序列為核糖體蛋白相關(guān)編碼基因序列,7個EST序列編碼蛋白與信號受體顆粒的亞基同源。 將篩選得到的陽性噬菌體克隆進行功能分組后,選擇5種噬菌體展示抗原,以每只1013pfu免疫BALB/c小鼠并攻擊感染日本血吸蟲尾蚴。結(jié)果顯示:展示日本血吸蟲SJCHGC06360蛋白(19號克隆)噬菌體免疫組和展示兩種未知蛋白噬菌體(6號克隆和18號克隆)聯(lián)合免疫組獲得了顯著的免疫預防效果,分別誘導了40.53%和31.14%的減蟲率、35.31%和44.65%的肝臟減卵率。抗體水平檢測結(jié)果顯示:免疫組均取得了較高的特異性抗體。 本項研究首次用日本血吸蟲抱雌溝蛋白篩選日本血吸蟲成蟲噬菌體展示cDNA文庫,獲得了多個陽性克隆,并通過免疫預防實驗發(fā)現(xiàn)SJCHGC06360蛋白和兩種未知蛋白在血吸蟲生殖發(fā)育方面具有重要作用,且是較為理想的候選疫苗抗原。
[Abstract]:Schisosomiasis (Schisosomiasis) is a widely distributed species caused by the parasitism of Schistosoma japonicum in human body. Schistosoma japonicum is a rare form of androgeny in trematodes. During the development of Schistosoma japonicum, female and male conjunctions are the premise of female development and maturation. The transmission of information material between male and female worms is realized by conjunctions. Therefore, if we can find out the information material transmitted between male and female worms, we can study the interaction between male and female worms of Schistosoma japonicum. The mechanism of Schistosoma japonicum reproductive development and the study of anti-schistosomiasis vaccine are very important. Phage display technology is recently developed, fusion of exogenous peptide or protein with specific phage capsid protein. A new technique on phage surface has been widely used in the study of molecular recognition mechanism. In this study, the cDNA library of adult Schistosoma japonicum phage display was screened by fusion expressed Schistosoma japonicum female sulcus protein. The positive phage clones were selected randomly for sequencing and bioinformatics analysis. And some of the positive clones were evaluated for the effect of immunoprophylaxis. After three rounds of screening, the phage concentration increased from 1.8 脳 10 -6 PFU in the first round to 1.6 脳 10 -4 pfuu in the third round, which enriched the phage by nearly 90 times. In addition to the low affinity and non-specific binding phage, 25 effective EST sequences were obtained by random selection of positive phage clones for sequencing and bioinformatics analysis. Among them, 20 were homologous with known gene or expression sequence tags, and 5 EST sequences had no homology with known gene or expression sequence tags. The results of functional prediction of 20 effectively encoded proteins showed that 10 EST sequences were ovalbumin related genes associated with the development of Schistosoma japonicum eggs. Three EST sequences were ribosomal protein-related coding genes, and seven EST sequences were homologous to the subunits of signal receptor granules. Five kinds of phage display antigens were selected after functional grouping of the selected positive phage clones. BALB/c mice were immunized with 1013 PFU and infected with cercariae of Schistosoma japonicum. The results showed that the SJCHGC06360 protein (clone 19) of Schistosoma japonicum was displayed. Bacteriophage immunization group and phage display two unknown proteins (clone 6 and clone 18) combined immunization group obtained a significant immunoprophylaxis effect. The worm reduction rates of 40.53% and 31.14% were induced, respectively. The results of 35.31% and 44.65% liver oocyte reduction rate and antibody level showed that higher specific antibodies were obtained in the immunized group. In this study, the phage display cDNA library of adult Schistosoma japonicum was screened with Schistosoma japonicum female sulcus protein for the first time, and several positive clones were obtained. It was found that SJCHGC06360 protein and two unknown proteins play an important role in the reproductive development of Schistosoma japonicum and are ideal candidate vaccine antigens.
【學位授予單位】：新疆農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

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