小鼠肿瘤坏死因子受体1基因的克隆及其与绿色荧光蛋白的融合表达

目的：构建小鼠肿瘤坏死因子受体1（TNFR1）和绿色荧光蛋白（GFP）的融合基因真核表达质粒，然后在中国仓鼠卵巢（CHO）细胞表达，为TNFR1的基因干预研究提供简便的判定手段。方法：从小鼠肝组织提取总RNA，RT-PCR扩增出TNFR1基因的cDNA片段，将目的片段克隆至pMD18-T载体。酶切和测序鉴定，然后将目的片段酶切回收后插到GFP表达载体pEGFP-N2中GFP基因序列的上游，构建TNFR1-EGFP融合基因真核表达载体pEGFP-TNFR1，将该重组质粒转染到CHO细胞后24—48h，用免疫组化技术检测TNFR1的表达情况。同时荧光显微镜下观察GFP表达情况。结果：扩增出了长约1360bp的基因片段，并将其克隆至pMD18-T载体，酶切和测序鉴定无误；将pMD-TNFR1中TNFR1基因亚克隆至pEGFP-N2载体，酶切鉴定无误；重组质粒pEGFP-TNFR1转染CHO细胞后，免疫组化染色可见细胞有阳性棕染，同时荧光显微镜下也可观察到绿色荧光蛋白的表达。结论：成功构建了小鼠TNFR1-EGFP融合基因真核表达质粒；重组质粒可在CHO细胞中表达出TNFR1-EGFP融合蛋白，为进一步的TNFR1基因干扰研究奠定了基础。

Gene cloning and fusion expression of mouse tumor necrosis factor recptor 1 with green fluorescent protein

Objection:To construct and characterize TNFR1-GFP fusion protein expression plasmid(pEGFP-TNFR1) and provide a direct and simplified method for assessment of the effect of TNFR1(siRNA) on the TNFR1 gene expression. Methods:Total RNA was extracted from mouse liver.TNFR1 cDNA was amplified by RT-PCR technique and cloned into pMD18-T vector and the sequence was ensured by sequenciog assay.TNFR1 gene was released from pMD-TNFR1 and subcloned into pEGFP-N2 upstream of GFP gene.pEGFP-TNFR1 was analyzed by restrictive enzymatic digestion to ensure the orientation.The plasmid was then transfected into CHO cells,then the fusion protein expression was detected by immunohistochemistry and fluorescent microscope.Rusults:A 1360 bp gene fragment was obtained and coloned into pMD18-T vector,and the sequence was correct.TNFR1 gene was subcloned into pEGFP-N2 vector,and then restriction endonucleases assays showed the correct orientation.24-48 hours after transfection in CHO cells,the expression of TNFR1-GFP fusion protein can be detected by immunohistochemistry and fluorescent microscope.Conclusion:pEGFP-TNFR1 has been constructed successfully.The TNFR1-GFP fusion proteinwas expressed in CHO cells after transtection.It may provide a direct and simplified method for primary assessment of the effect of TNFR1 siRNA on the(TNFR1) gene expression.