Gene cloning and fusion expression of mouse tumor necrosis factor recptor 1 with green fluorescent protein
- VernacularTitle: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
- Author:
Yuwen LI
- Publication Type:Journal Article
- Keywords:
Tumor necrosis factor recptor 1;
Green fluorescent protein;
Fusion protein
- From:
Journal of Medical Postgraduates
2004;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
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.
