维甲酸核受体RARα真核表达载体的构建、突变纠正及表达

目的构建维甲酸核受体RARα真核表达载体,并检测其在人肺腺癌细胞A549中表达。方法从小鼠巨噬细胞RAW264.7中提取总RNA,以RT-PCR法扩增RARαcDNA,克隆至真核表达载体pDsRed1-C1中,测序结果显示RARα第1040位A→G,导致其编码蛋白的氨基酸发生改变。通过二次PCR将其纠正,重组载体RedC1-RARα转化大肠埃希菌Top10,筛选阳性克隆做酶切及测序鉴定。脂质体瞬时转染A549细胞,在荧光显微镜下观察RARα的表达。RT-PCR法检测RARα的mRNA水平表达。结果通过RT-PCR及二次PCR得到RARαcDNA,构建其真核表达载体,脂质体瞬时转染A549细胞得到了成功表达,RARα基因产物定位于细胞核内。结论成功构建维甲酸核受体RARα真核表达载体,且证实RARα编码蛋白定位于细胞核内,本研究结果为进一步探讨结核分枝杆菌固有免疫机制奠定了基础。

Construction,point mutation correction and expression of eukaryotic expression vectors for retinoic acid nuclear receptor

Objective To construct the eukaiyotic expression vector containing letinoic acid nuclear receptor. Method The coding sequence of RARα gene was amplified from the total RNA of murine macrophage RAW264.7 cell by RT-PCR. The gene was cloned into pDsRed1-C1. The mutants, (A→G in RARα) , resulted in the change of RARα protein. Correction of mutations was performed by a two-step PCR reaction. The correct recombinant vector was transformed into E. coli Top10 and was identified by restrict endonuclease digestion and sequencing. The RedC1-RARα was transiently trans-fected into AS49 cell. The RARa was visualized directly with fluorescence microscope, and the expression of RARα gene was detected by RT-PCR. Result By RT-PCR and two-step PCR, we obtained RARa. The eukaryotic expression plasmid RedC1-RARα was constructed. The protein was successfully expressed in targeted cells and its localization was in nucleus. Conclusion The eukaryotic expression vector RedC1-RARα has been successfully constructed. The protein can be expressed in A549 cell and located in nucleus. It provides a good basis for the further research on the innate immunology a-gainst tuberculosis.