晕船易感大鼠脑干组织消减cDNA文库的构建
Construction of subtracted cDNA library of seasickness susceptibile rat brain stem
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摘要:目的 筛选晕船易感大鼠脑干组织的晕船易感性相关基因, 为建立晕船易感人群预测方法提供基础数据.方法 以晕船易感大鼠为实验组, 以非易感大鼠为驱动组, 抽提脑干组织mRNA, 利用抑制消减杂交技术(SSH), 通过2次抑制性PCR反应得到二者之间差异表达的cDNA.将PCR产物与pGEM-T载体连接, 构建消减cDNA文库, 将连接产物用电击法转化大肠杆菌进行文库扩增.对所得克隆进行PCR扩增鉴定.结果 构建具有高消减效率的消减cDNA文库, 差异表达的cDNA得到富集, 文库中包含470个白色克隆(高表达260个、低表达210个), 其中442个有插入片段, 片段大小主要在250~650bp之间.结论 利用抑制消减杂交技术成功构建了晕船易感大鼠脑干组织差异表达基因cDNA文库, 为研究晕船易感特异表达基因奠定了基础.Abstract:Objective Isolation of genes related to seasickness susceptibility in rat's brain stem with suppression subtractive hybridization was helpful in establishing a method of predicting people seasickness suscept ibility.Methods the mRNA were extracted from simulated seasickness susceptible and insusceptible rats brain stem.Suppr ession subtractive hybridization (SSH)method was used for isolating differentially expressed cDNA.After two times of suppression PCR, the cDNA fragments were inserted into T-vectors to set up the subtracted library, amplification of the library was carried out with transformation of E.coli by high votage electroperfor mation.The clones were amplified by PCR and identified.Results the subtracted library contained 470 clones(260 high expressed, 210 low expressed)including 442 clones containing 250-650 bp inserts.Conclusion the subtracted cDNA library of simulated seasickness susceptible rat's br ain stem was successfully constructed.It was helpful in the study of new and specific genes expressed in seasickness.
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