【摘要】 为发现潜在的抗大片吸虫病的候选疫苗分子,控制大片形吸虫病,本研究以大片吸虫成虫为材料,应用SMARTTcDNA文库构建技术,构建了以表达载体λTriplEx2为基础的大片吸虫成虫cDNA表达文库。经测定,库容量为1·08×106PFU/ml ,重组率为96·6 %,扩增后的文库滴度为2·41×109PFU/ml ,插入片段平均大小约为1 000 bp;经大肠杆菌BM25·8质粒化后,从文库中随机挑选40个重组克隆测序,获得32条有效ESTs ;经BLASTX和BLASTn程序检索和分析,发现有9条ESTs代表已知基因, 16条ESTs相似性较低或无匹配,列为新基因。9条已知基因代表了半胱氨酸蛋白酶、卵壳蛋白、钙连接蛋白等三类功能蛋白,其余新基因也暗示与信号传导、蛋白合成、免疫刺激等基因相关,具有潜在的研究价值[动物学报51 (5) : 879 -883 , 2005]。更多还原

【Abstract】 To determine the latent vaccine molecules that resist Fasciola gigantica disease and to provide resources for further study, a high-quality Fasciola gigantica cDNA library was constructed into λ TriplEx2 plasmid using the SMART technique,the result showed that the unamplified library capacity was 1.08×10~6 PFU/ml,the library recombination rate was 96.6%,the titer of the amplified library was 2.41×10~9 PFU/ml and the average inserted cDNA fragment was about 1 000 bp. Then 40 clones were selected to sequence from the BM25.8 plasmids,32 sequences obtained from the plasmids proved useful after editing using biosoftware. After analysing using the BLASTx and BLASTn programs,9 Expressed Sequence Tags (ESTs ) were known-genes,and 16 ESTs whose alignment values were very low or did notmatch known sequences were considered unknown-genes. The 9 ESTs represent cystein protease, eggshell protein and calcium-binding protein.The 16 unknown ESTs included some signal conductive genes, protein translative genes and immune stimulative genes and so on. The Fasciola gigantica cDNA expression library has established the base for research into its bio-elements and mechanism.更多还原