日本血吸虫成虫噬菌体展示cDNA文库的构建与东方田鼠抗血吸虫抗性相关基因筛选

发布时间：2018-01-20 10:28

本文关键词： 日本血吸虫噬菌体展示cDNA文库成虫东方田鼠亲和淘洗　出处：《中国农业科学院》2007年硕士论文　论文类型：学位论文

【摘要】： 日本血吸虫病(Schistosomiasis japonica)是我国一种危害严重的人畜共患寄生虫病。东方田鼠是迄今发现的唯一一种具有抗血吸虫病作用的哺乳动物。如能筛选、克隆出东方田鼠抗血吸虫抗性相关的日本血吸虫基因，可以为开发抗血吸虫疫苗和治疗药物提供新技术、新思路。噬菌体展示技术(phage display)是近年建立和发展起来的利用噬菌体融合表达外源基因的一项新技术，已广泛应用于分子间识别机制的研究。本研究首次应用T7噬菌体展示系统构建了日本血吸虫成虫噬菌体展示cDNA文库，并用东方田鼠阴性血清筛选该文库，以期筛选到与东方田鼠抗血吸虫抗性相关的日本血吸虫基因。方法与结果 1．日本血吸虫成虫噬菌体展示cDNA文库的构建：原始文库的库容量为4.98×10~6pfu，扩增后文库滴度为3.85x 10~(11)pfu／ml。对随机挑取的96个噬菌斑进行PCR鉴定，重组率为93.8％，其中95.6％的插入片段大于300 bp。7种特异性引物均能从文库中钓取到日本血吸虫成虫相关基因。 2．东方田鼠抗血吸虫抗性相关基因的筛选：分离东方田鼠阴性血清，用亲和淘洗的方法筛选文库。经三轮筛选，，阳性克隆得到有效富集，共获得10种目的基因的EST序列，其中7种与GenBank中登陆的日本血吸虫基因序列具有高度相似性，相似性达到99％～100％；1种与其他物种基因有82％相似性，另外2种没有发现显著相似的基因。功能预测结果表明，部分EST编码的蛋白或多肽分子功能主要是结合作用、酶活性及结构蛋白。结论本研究成功构建了日本血吸虫成虫噬菌体展示cDNA文库，并利用东方田鼠阴性血清成功筛选到10种目的基因的EST序列，为深入阐述东方田鼠抗血吸虫分子机理和寻找新候选疫苗抗原分子奠定了基础。
[Abstract]:Schistosomiasis japonica. It is a serious zoonotic parasitic disease in China. Microtus Fortis is the only mammal with anti-schistosomiasis effect found so far. The cloning of Schistosoma japonicum genes related to resistance to Schistosoma japonicum in Microtus Fortis could provide new techniques and new ideas for the development of anti-schistosomiasis vaccine and therapeutic drugs. Phage display is a new technique for expression of foreign genes by phage fusion. T7 phage display system was used for the first time to construct the cDNA library of adult Schistosoma japonicum phage display. The gene of Schistosoma japonicum related to resistance to Schistosoma japonicum was screened by the negative serum of Microtus orientalis. Methods and results 1. Construction of cDNA library of adult Schistosoma japonicum: the capacity of the original library is 4.98 脳 10 ~ (6) PFU. After amplification, the titer of the library was 3.85 x 10 ~ (10) p / ml. 96 randomly selected plaque were identified by PCR, and the recombinant rate was 93.8%. 95.6% of the inserted fragments were more than 300 bp.7 specific primers, all of which could catch the genes related to adult Schistosoma japonicum from the library. 2. Screening of genes related to resistance to Schistosoma japonicum in Microtus Orientalis: the negative sera were isolated and the library was screened by affinity washing method. After three rounds of screening, the positive clones were effectively enriched. A total of 10 EST sequences of target genes were obtained, of which 7 were highly similar to those of Schistosoma japonicum which landed in GenBank, and the similarity reached 99100. One had 82% similarities with other species genes, while the other two had no significant similarity. The functional prediction results showed that some of the protein or polypeptide functions encoded by EST were mainly binding. Enzyme activity and structural protein. Conclusion the phage display cDNA library of adult Schistosoma japonicum was successfully constructed, and the EST sequences of 10 target genes were successfully screened by using the negative serum of Microtus Fortis. It lays a foundation for further expounding the molecular mechanism of Schistosoma japonicum and searching for new vaccine antigen molecules.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383;Q78

【参考文献】