日本血吸虫成虫噬菌体展示cDNA文库的构建  

Construction of Phage Display cDNA Library from Adult Worms of Schistosoma japonicum

作　　者：孙毅[1] 贾人初[1] 刘金明[1] 苑纯秀[1] 石耀军[1] 陆珂[1] 傅志强[1] 孙焕[1] 蔡幼民[1] 林矫矫[1] 

机构地区：[1]中国农业科学院上海兽医研究所,国家防治动物血吸虫病专业实验室,农业部动物寄生虫学重点开放实验室,上海200232

出　　处：《中国寄生虫学与寄生虫病杂志》2007年第5期406-410,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：上海市科学技术委员会科研计划项目(No.054909002);科技部自然资源平台项目(No.2005DKA21104)~~

摘　　要：目的构建日本血吸虫成虫T7噬菌体展示cDNA文库。方法用Trizol试剂提取日本血吸虫成虫总RNA,分离纯化mRNA,经随机引物反转录合成双链cDNA。在双链cDNA末端加上定向EcoRⅠ/HindⅢ接头,再用EcoRⅠ和HindⅢ消化,使其成为两端分别带有EcoRⅠ和HindⅢ粘性末端的双链cDNA。收集300bp以上的双链cDNA片段,与T7Select 10-3b载体连接,经体外包装后,以大肠埃希菌BLT5403为宿主菌构建T7噬菌体展示cDNA文库。通过滴度测定及PCR技术鉴定文库质量,用7种特异性引物以PCR法从文库中钓取目的基因以检测文库的代表性。结果原始文库的库容量为4.98×106pfu,扩增后文库滴度为3.85×1011pfu/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300bp。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。结论构建了日本血吸虫成虫T7噬菌体展示cDNA文库。Objective To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum. Methods Total RNA was extracted from adult worms of S. japonicum by Trizol reagent and mRNA was isolated from the total RNA. The ds cDNA was synthesized by reverse transcription using random primer. Directional EcoR Ⅰ / HindⅢ linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoR Ⅰ and Hind Ⅲ, which resulted in ds cDNA with EcoR Ⅰ and Hind Ⅲ adhering ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector. After packaging in vitro, the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display eDNA library. Plaque assay and PCR were used to evaluate the library. Seven known objective genes of S. japonicum were screened by PCR to detect the representation of the library. Result Primary library capacity was 4.98×10^6 pfu, and the titer of amplified library was 3.85×10^11 pfu/mL. The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%, in which 95.6% inserted eDNA fragments were longer than 300 bp in length. All the seven known objective genes of S.japonicum were amplified from the library. Conchlsion The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

关 键 词：日本血吸虫 噬菌体展示cDNA文库 成虫 

分 类 号：R383.24[医药卫生—医学寄生虫学] R392.1[医药卫生—基础医学]