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/Hind Ⅲ接头并用EcoR Ⅰ和Hind Ⅲ消化,使其两端分别带EcoR Ⅰ和Hind Ⅲ黏性末端。用Mini Column纯化,收集300bp以上的双链cDNA片段,再连接于带有EcoR和Hind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1.5×106pfu,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鉴定,重组率为91%,阳性克隆片段长度分布在200-1500bp,其中有90%的插入片段〉300bp。结论成功构建了东方田鼠肺脏T7噬菌体展示cDNA文库。
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 HindⅠ adhering ends by ligation with the directional EcoR Ⅰ /HindⅠ linkers and digestion with EcoR Ⅰ and Hind Ⅱ. The double strain cDNA fragments longer than 300 bp in length were fractionated by the Mini Column, and ligated into the T7 Select 10-3b vector with EcoR Ⅰ and Hind Ⅰ adhering ends. After packaging in vitro, the recombinant T7 Select 10-3b was transformed into BLT5403 to construct a T7 phage display cDNA library. Results The library constructed here contained 1.5 × 10^6 clones and the titer of the amplied library was 1.1 × 10^12 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 Microtus fortis is successfully constructed.
出处
《中国血吸虫病防治杂志》
CAS
CSCD
2007年第4期252-256,共5页
Chinese Journal of Schistosomiasis Control
基金
科技部自然资源平台项目(2005DKA21104)
上海市科学技术委员会科研计划项目(054909002)
作者简介
贾人初（1982-），男，硕士研究生。研究方向：寄生虫分子生物学
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