Abstract:

  Objective  To construct a T7 phage display cDNA library from the lung of Microtus
fortis for further screening the schistosomiasis-resistence-related genes of Microtus fortis. Methods
  mRNA was isolated from total RNA extracted from the lungs of Microtus fortis by TRIzol
reagent, and was used to synthesize double strain cDNA by the reverse transcription. Then the
double strain cDNA was given with EcoR I  and Hind I[ adhering ends by ligation with the direc-
tional EcoR I /Hind II linkers and digestion with EcoR i  and Hind  m.  The double strain cDNA
fragments longer than 300 bp in length were fractionated by the Mini Column, and ligated into the
T7 Select l0-3b vector with EcoR I and Hind m adhering ends. After packaging in vitro, the re-
combinant T7 Select l0-3b was transformed into BLT5403 to construct a T7 phage display cDNA li-
brary.  Results   The library constructed here contained l. 5 Xl06 clones and the titer of the amplied
library was l. 1X101z pfu/ml.  The PCR identification results of 100 clones picked at random showed
that 91% clones were recombinant and 90% of recombinant clones contained cDNA fragments
longer than 300 bp in length. Conclusion  A T7 phage display cDNA library from the lung of M/-
crotus fortis is successfully constructed.
   

Key words: Schistosoma japonicum, T7 phage display cDNA Iibrary, Microtus fortis, Lung

摘要：

目的构建东方田鼠肺脏T7噬菌体展示cDNA文库，为筛选东方田鼠抗血吸虫感染抗性相关基因奠定基础。方法  用TRIzol试剂提取东方田鼠肺脏总RNA，分离纯化mRNA，经反转录合成双链cDNA。在双链cDNA末端加上定向EcoR I／HindⅢ接头并用EcoR I和HindⅢ消化，使其两端分别带EcoR I和HindⅢ黏性末端。用Mini Column纯化，收集300 bp以上的双链cDNA片段，再连接于带有EcoR I和Hind Ⅲ末端的T7 Select 10-3b载体，经体外包装后，以BLT5403为受体菌构建T7噬菌体展示cDNA文库。结果  经测定，库容量为1.5×10⁶ pfu，扩增后文库滴度为1.1×10¹²pfu/ml。对从原始文库中随机挑取的100个噬菌斑进行PCR签定，重组率为91%，阳性克隆片段长度分布在200-1 500 bp，其中有90%的插入片段>300 bp。结论成功构建了东方田鼠肺脏T7噬菌体展示cDNA文库。

关键词:  日本血吸虫；T7噬菌体展示cDNA文库；东方田鼠；肺脏