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《Journal of Medical Postgraduates》 2007-01
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Gene cloning and fusion expression of mouse tumor necrosis factor recptor 1 with green fluorescent protein

LI Yu-wen~(1),ZHU Chuan-Long~(2),NING Qin~(2),LUO Xiao-ping~(1) (1.Department of Pediatrics;2.Department of Infectious Disease,Tongji Hospital of Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,Hubei,China)  
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.
【Key Words】： Tumor necrosis factor recptor 1 Green fluorescent protein Fusion protein
【Fund】： 国家杰出青年基金资助项目(批准号:30225040);; 国家重点基础研究发展计划(973)项目(批准号:2005CB522507)
【CateGory Index】： R346
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