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目的将人星状病毒非结构蛋白ns P1a/1基因连接到真核表达载体上,转染人胚肾上皮细胞48 h后检测其表达。方法设计特异性引物PCR扩增人星状病毒非结构蛋白ns P1a/1片段,分别插入真核表达载体pc DNA3.1(+)和p EGFP-N2载体,构建重组表达质粒pc DNA3.1(+)-ns P1a/1-His和p EGFP-N2-ns P1a/1。在转染试剂PEI的介导下将重组表达质粒分别转染293T细胞,转染48 h后分别在荧光显微镜下观察EGFP的表达以及通过Western blot检测ns P1a/1基因的表达。结果重组表达质粒pc DNA3.1(+)-ns P1a/1-His和p EGFP-N2-ns P1a/1构建成功;转染p EGFP-N2-ns P1a/1后48 h能够在荧光显微镜蓝色激发光下观察到较强的黄绿色荧光;转染pc DNA3.1(+)-ns P1a/1-His后48 h收集细胞进行Western blot检测,能够检测到ns P1a/1-His融合报告基因的表达。结论成功构建了人星状病毒非结构蛋白ns P1a/1基因真核表达质粒,并在人胚肾上皮细胞293T细胞获得表达,为进一步深入研究ns P1a/1在人星状病毒抵御宿主细胞抗病毒天然免疫中是否发挥作用奠定了基础。
OBJECTIVE: To construct human eukaryotic expression vector ns P1a / 1, which is a nonstructural protein of human astroviruses, which was transfected into human embryonic kidney epithelial cells for 48 h. Methods Specific primers were designed to amplify ns P1a / 1 non-structural protein of human astrovirus and inserted into pcDNA3.1 (+) and p EGFP-N2 vectors respectively to construct recombinant plasmid pcDNA3.1 +) - ns P1a / 1-His and p EGFP-N2-ns P1a / 1. The recombinant plasmid was transfected into 293T cells under the mediation of transfection reagent PEI. The expression of EGFP was observed under fluorescent microscope 48 h after transfection, and the expression of ns P1a / 1 gene was detected by Western blot. Results The recombinant plasmids pcDNA3.1 (+) - ns P1a / 1-His and p EGFP-N2-ns P1a / 1 were successfully constructed. The transfected pEGFP-N2-ns P1a / Strong fluorescence of yellow-green fluorescence was observed under the excitation of color stimulus; cells were collected 48 h after transfection with pcDNA3.1 (+) - ns P1a / 1-His and Western blot detection could detect ns P1a / 1-His fusion Gene expression. Conclusion The eukaryotic expression plasmid of ns P1a / 1 human non-structural protein ns P1a / 1 was successfully constructed and expressed in human embryonic kidney 293T cells. In order to further study the effect of ns P1a / 1 on the resistance of human astrovirus to host cell Viral innate immunity has played a role in laying the foundation.