Gene cloning and fusion expression of mouse tumor necrosis factor recptor 1 with green fluorescent protein / 医学研究生学报

ABSTRACT

ObjectionTo 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.RusultsA 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.