抱雌沟蛋白筛选日本血吸虫成虫噬菌体展示cDNA文库的研究

发布时间：2018-01-19 23:21

本文关键词： 日本血吸虫抱雌沟蛋白噬菌体展示cDNA文库筛选阳性噬菌体克隆免疫　出处：《新疆农业大学》2009年硕士论文　论文类型：学位论文

【摘要】： 血吸虫病(Schisosomiasis)是由血吸虫寄生于人体所引起的一种分布广泛、危害严重的人兽共患寄生虫病。血吸虫是吸虫中罕见的雌雄异体形式,在血吸虫发育过程中,雌雄虫合抱是雌虫发育成熟的前提,雌雄虫间信息物质的传递通过合抱实现。因此,如果能够找出这种雌雄虫间传递的信息物质,对研究血吸虫雌雄虫之间的相互作用、血吸虫生殖发育机理以及对抗血吸虫疫苗研究有非常重要的意义。噬菌体展示技术是新近发展起来的,将外源肽或蛋白与特定噬菌体衣壳蛋白融合,并展示于噬菌体表面的一项新技术,已广泛应用于分子间识别机制的研究。本研究用融合表达的日本血吸虫抱雌沟蛋白筛选日本血吸虫成虫噬菌体展示cDNA文库,随机选取获得的阳性噬菌体克隆进行测序、生物信息学分析,并对其中部分阳性克隆进行了免疫预防效果评估。经过三轮筛选后,噬菌体由第一轮的1.8×10-6pfu增加到第三轮的1.6×10-4pfu,富集了将近90倍,成功的筛除了低亲和力和非特异性结合的噬菌体。随机挑取阳性噬菌体克隆进行测序以及生物信息学分析,共获得25个有效EST序列,其中20个与已知基因或表达序列标签同源,5个EST序列与已知基因或表达序列标签均无同源性,为新的表达序列标签。对20个有效编码蛋白的功能预测结果显示:10个EST序列为卵壳蛋白相关基因与血吸虫卵的发育相关,3个EST序列为核糖体蛋白相关编码基因序列,7个EST序列编码蛋白与信号受体颗粒的亚基同源。将筛选得到的阳性噬菌体克隆进行功能分组后,选择5种噬菌体展示抗原,以每只1013pfu免疫BALB/c小鼠并攻击感染日本血吸虫尾蚴。结果显示:展示日本血吸虫SJCHGC06360蛋白(19号克隆)噬菌体免疫组和展示两种未知蛋白噬菌体(6号克隆和18号克隆)联合免疫组获得了显著的免疫预防效果,分别诱导了40.53%和31.14%的减虫率、35.31%和44.65%的肝脏减卵率。抗体水平检测结果显示:免疫组均取得了较高的特异性抗体。本项研究首次用日本血吸虫抱雌沟蛋白筛选日本血吸虫成虫噬菌体展示cDNA文库,获得了多个阳性克隆,并通过免疫预防实验发现SJCHGC06360蛋白和两种未知蛋白在血吸虫生殖发育方面具有重要作用,且是较为理想的候选疫苗抗原。
[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.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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