+10 Gz重复暴露大鼠脑差异表达基因cDNA文库的构建

目的构建＋10Gz重复暴露大鼠脑差异表达基因的消减cDNA文库。方法本实验用SD大鼠，分别提取暴露组与对照组的总RNA，并分离纯化mRNA，应用抑制性消减杂交技术分离＋10GI重复暴露大鼠脑差异表达基因eDNA片段并建立消减eDNA文库；利用PCR对随机挑选的75个白色菌落进行插入片段的验证，对其中70个克隆进行eDNA斑点杂交验证。结果所构建的eDNA文库扩增后包含约400个白色克隆和100个兰色克隆，随机挑选75个白色克隆入质粒载体后共获得70个阳性克隆。结论应用抑制性消减杂交技术成功构建了＋10Gz重复暴露大鼠脑差异表达基因消减eDNA文库，为进一步筛选和克隆脑损伤相关基因奠定了基础。

Construction and identification of a differentially expresses genes subtracted cDNA library in rats brain repeated exposed to +10 Gz

Objective To construct a differentially expressed genes subtracted cDNA library in rats brain exposed to 10 Gz repeatly.Methods In this study,SD rats served as experimental subjects.Total RNA were extracted from the rat brain of 10 Gz exposure group and those of control group.A cDNA subtractive library,which had high subtractive efficiency of repeated 10 Gz exposures in rat brain was constructed with suppression subtractive hybridization(SSH).Positive clones were screened by differential screen technique.Results The amplified library contained approximately 400 white bacteria clones and 100 blue bacteria clones.Random analysis of 75 clones with nested PCR method were identified Seventy positive clones from SSH pools.Conclusion A differentially expressed genes subtracted cDNA library of rats brain with repeated Gz exposures is constructed by SSH.The library is a highly efficient one and lays solid foundation for screening and cloning new and specific genes related to rat brain damage induced by repeated Gz exposures.