东方田鼠肝脏T7噬菌体展示cDNA文库的构建  

Construction of a T7 phage display cDNA library from liver of Microtus fortis

作　　者：贾人初[1] 孙毅[1] 刘金明[1] 傅志强[1] 苑纯秀[1] 石耀军[1] 陆珂[1] 孙焕[1] 李浩[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

出　　处：《中国预防兽医学报》2008年第1期5-9,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：上海市科学技术委员会科研计划项目(054909002);科技部自然资源平台项目(2005DKA21104)

摘　　要：本项研究的目的是构建东方田鼠肝脏T7噬菌体展示cDNA文库,为筛选东方田鼠抗血吸虫病抗性相关基因奠定基础。用TRIzol试剂提取东方田鼠肝脏总RNA,分离纯化mRNA,经反转录合成双链cDNA。在双链cDNA末端加上EcoRⅠ/HindⅢ定向接头并用EcoRⅠ和HindⅢ酶切,使其两端分别带EcoRⅠ和HindⅢ粘性末端。用Mini Column纯化、收集300bp以上的双链cDNA片段,再连接于带有EcoRⅠ和HindⅢ末端的T7Select10-3b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。经测定,库容量为1.3×107PFU/mL,扩增后文库滴度为1.8×1011PFU/mL。对从原始文库中随机挑取的100个噬菌斑进行PCR鉴定,重组率为91.7%,阳性克隆片段大小分布在200bp～1000bp,其中有95.5%的插入片段大于300bp。用日本血吸虫童虫可溶性抗原对文库进行了初筛,得到了21个ESTs,将这些阳性噬菌体克隆和血吸虫童虫共培养,其中大部分克隆诱导的童虫死亡率比阴性噬菌体对照高出2%～13%。A T7 phage display cDNA library of Microtus fortis was constructed for screening the schistosomiasis-resistence-related gene of Microtus fortis. The mRNA was isolated from livers of Microtus fortis by TRIzol reagent, and used to synthesize the ds-cDNA by reverse transcription. The cDNA fragments longer than 300 bp in length were fractionated by Mini Column, and ligated into the T7 Select 10-3b vertor with EcoR I and Hind Ⅲ adhering ends. After packaged in vitro, the recombinant T7 was transformed into BLT5403 to construct a T7 phage display cDNA library. The library contained 1.3×10^7 clones and the amplied library had a titer of 1.8×10^11 PFU/mL. PCR identification of 100 randomly picked clones showed that 91.7 % clones were recombinant and 95.5 % of recombinant clones contained cDNA fragments longer than 300 bp in length. The library was screened with the soluble extract of schistosomulua and 21 ESTs were obtained. Co-culture of schistosomulua with the screened positive phage resulted in 2 % to 13 % higher mortality than the negative control.

关 键 词：东方田鼠 肝脏 T7噬菌体展示 CDNA文库 

分 类 号：Q782[生物学—分子生物学]