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

【摘要】： 血吸蟲(chóng)病(Schisosomiasis)是由血吸蟲(chóng)寄生于人體所引起的一種分布廣泛、危害嚴(yán)重的人獸共患寄生蟲(chóng)病。血吸蟲(chóng)是吸蟲(chóng)中罕見(jiàn)的雌雄異體形式,在血吸蟲(chóng)發(fā)育過(guò)程中,雌雄蟲(chóng)合抱是雌蟲(chóng)發(fā)育成熟的前提,雌雄蟲(chóng)間信息物質(zhì)的傳遞通過(guò)合抱實(shí)現(xiàn)。因此,如果能夠找出這種雌雄蟲(chóng)間傳遞的信息物質(zhì),對(duì)研究血吸蟲(chóng)雌雄蟲(chóng)之間的相互作用、血吸蟲(chóng)生殖發(fā)育機(jī)理以及對(duì)抗血吸蟲(chóng)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(lái)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(xiàng)新技術(shù),已廣泛應(yīng)用于分子間識(shí)別機(jī)制的研究。 本研究用融合表達(dá)的日本血吸蟲(chóng)抱雌溝蛋白篩選日本血吸蟲(chóng)成蟲(chóng)噬菌體展示cDNA文庫(kù),隨機(jī)選取獲得的陽(yáng)性噬菌體克隆進(jìn)行測(cè)序、生物信息學(xué)分析,并對(duì)其中部分陽(yáng)性克隆進(jìn)行了免疫預(yù)防效果評(píng)估。 經(jīng)過(guò)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(jī)挑取陽(yáng)性噬菌體克隆進(jìn)行測(cè)序以及生物信息學(xué)分析,共獲得25個(gè)有效EST序列,其中20個(gè)與已知基因或表達(dá)序列標(biāo)簽同源,5個(gè)EST序列與已知基因或表達(dá)序列標(biāo)簽均無(wú)同源性,為新的表達(dá)序列標(biāo)簽。對(duì)20個(gè)有效編碼蛋白的功能預(yù)測(cè)結(jié)果顯示:10個(gè)EST序列為卵殼蛋白相關(guān)基因與血吸蟲(chóng)卵的發(fā)育相關(guān),3個(gè)EST序列為核糖體蛋白相關(guān)編碼基因序列,7個(gè)EST序列編碼蛋白與信號(hào)受體顆粒的亞基同源。 將篩選得到的陽(yáng)性噬菌體克隆進(jìn)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(chóng)尾蚴。結(jié)果顯示:展示日本血吸蟲(chóng)SJCHGC06360蛋白(19號(hào)克隆)噬菌體免疫組和展示兩種未知蛋白噬菌體(6號(hào)克隆和18號(hào)克隆)聯(lián)合免疫組獲得了顯著的免疫預(yù)防效果,分別誘導(dǎo)了40.53%和31.14%的減蟲(chóng)率、35.31%和44.65%的肝臟減卵率。抗體水平檢測(cè)結(jié)果顯示:免疫組均取得了較高的特異性抗體。 本項(xiàng)研究首次用日本血吸蟲(chóng)抱雌溝蛋白篩選日本血吸蟲(chóng)成蟲(chóng)噬菌體展示cDNA文庫(kù),獲得了多個(gè)陽(yáng)性克隆,并通過(guò)免疫預(yù)防實(shí)驗(yàn)發(fā)現(xiàn)SJCHGC06360蛋白和兩種未知蛋白在血吸蟲(chóng)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(xué)位授予單位】：新疆農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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本文編號(hào)：1445908