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目的脆性X相关蛋白(FXR1P)是一种RNA结合蛋白,本研究构建了其N-端结构域、KH结构域的酵母三杂交诱饵载体并转化酵母菌株L40 ura3/pHyblex/Zeo-MS2,为筛选这2种结构域的相互作用RNA作基础。方法根据Genebank提供的序列设计引物,以pYESTrp3/FXR1质粒作为模板,扩增分别包含FXR1P N-端结构域、KH结构域的编码区序列,连接到用于酵母三杂交文库筛选的pYESTrp3质粒,得到诱饵质粒,导入大肠杆菌Top10扩增,筛选阳性克隆并提取其中的重组质粒,再转化酵母菌L40 ura3/pHyblex/Zeo-MS2。结果经过测序验证,该重组诱饵质粒构建成功,转化酵母菌之后对宿主菌无毒性,无自激活现象。结论成功构建了能用于酵母三杂交文库筛选的FXR1P蛋白N-端结构域、KH结构域酵母三杂交诱饵载体,为研究这两个结构域的RNA结合功能奠定了基础。
Purpose Fragile X-related protein (FXR1P) is an RNA-binding protein. In this study, a yeast three-hybrid bait vector with N-terminal domain and KH domain was constructed and transformed into yeast strain L40 ura3 / pHyblex / Zeo- The two domains interact with RNA as a basis. Methods Primers were designed according to the sequences provided by Genebank. The pYESTrp3 / FXR1 plasmid was used as a template to amplify the coding region of the N-terminal domain and KH domain of FXR1P, respectively, and ligated into pYESTrp3 plasmid for screening of yeast three-hybrid library. The bait plasmid was introduced into Escherichia coli Top10 to amplify, the positive clones were screened and the recombinant plasmids were extracted, and then the yeast L40 ura3 / pHyblex / Zeo-MS2 was transformed. Results After sequencing, the recombinant bait plasmids were successfully constructed and transformed into yeast without any toxicity to the host bacteria. Conclusion The N-terminal domain of FXR1P and the yeast three-hybrid bait vector of KH domain were successfully constructed for the screening of yeast three-hybrid library, which laid the foundation for the study of the RNA binding function of these two domains.