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

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

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。

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 eDNA was amplified by RT-PCR technique and cloned into pMD18-T vector and the sequence was ensured by sequencing 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.