【作者】 罗洪林；

【导师】 黄维义；

【作者基本信息】 广西大学 ， 预防兽医学， 2005， 硕士

【摘要】 大片形吸虫(Fasciola gigantica)主要寄生在牛、羊等反刍动物肝脏,是我国华东、华南地区的优势虫种,给畜牧业和人民身体健康带来严重危害,但目前还没有有效的药物和疫苗来控制。本文通过构建大片形吸虫成虫的cDNA文库并获取部分表达序列标签(EST),旨在发现潜在的抗大片形吸虫的候选疫苗分子,为防大片形吸虫病奠定基础。 本研究应用SMART方法构建了高质量的大片形吸虫成虫cDNA文库,经测定,库容量为1.08×10~6 Pfu/mL,重组率为96.6%。扩增后的文库滴度为2.41×10~9Pfu/mL,插入片段平均大小约为1000bp;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;从所获得的ESTs中挑选了一条作为目标序列,运用生物信息学方法并结合相关软件,利用网络数据库进行了电子克隆(in siloco cloning),获得了长度为1920bp的重叠群(contig),开放阅读框(ORF)长930bp,编码310个氨基酸,预测为卵壳蛋白,在抗生殖免疫方面发挥重要作用。对此重叠群设计引物,更多还原

【Abstract】 Fasciola gigantica always parasitizes the ruminants and mainly distributes the east and south of china,and always be harmful to the yield-power of stockbreeding and the health of people,but so far there are no efficacious drugs and vaccines to control its developmemt,so, in order to find out the latent vaccine molecules to resist it,the cDNA expression library from adult Fasciola gigantica was constructed,and the EST mothed was used to analyse the obtainted sequences.A high-quality Fasciola gigantica cDNA library was constructed by using the SMART technique, the result showed that the unamplified library capacity was 1.08 X106 Pfu/ml, the library recombination rate was 96.6%,the titer of the amplied library was 2.41 X 10~9Pfu/ml and the average inserted cDNA fragment was about 1000bp. Then 40 clones were selected to sequence from the BM25.8 plasmids,32 sequences obtainted from the plasmids were proved to be useful after editing using some biosoftwares.After analysising by using the BLASTx and BLASTn programs,9ESTs were proved to be the known-genes, and 16 ESTs whose aligment value were very low or which suited sequence were not found were proved to be unknown-genes.The results also showed that the 9 ESTs represent the cystein protease-, eggshell protein and calcium-binding protein,the other 16 unknown ESTs included some signal conductive genes protein translative genes and immune stimulative genes and so on. One EST was choosed to in silico cloning by using the bioinformatic and some biosoftwares on internet,one contig whose length was 1920bp% ORF was 930 bp and coded 310 amino acidwas obtained ,and was predicted to be eggshell protein gene.In order to prove the dependability of the in silico cloning method ,one primer which intend to amplify to 758bp was designed along the contig, the experiment cloned the target sequence successfully and the sequencing result was identical to the target sequence absolutely.It also beared out the cDNA library can provide the resources for cloning new genes.The study showed that the Fasdola gigantica cDNA library was constructed firstly and the strategy of how to clone the gene by in silico cloning mothed based the internet databases efficiently was explored,and had important implications for further studies on controling Fasdola gigantica diseases.更多还原