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目的:干扰素rhIFN-α2B体外诱导人胚胎肾细胞系293T细胞获得IFITM3基因,观察IFITM3基因在293T细胞中的表达与定位。方法:利用干扰素rhIFN-α2B体外诱导293T细胞获得IFITM3基因,采用RT-PCR技术扩增获得干扰素诱导的跨膜蛋白IFITM3全长cDNA,扩增产物连接至pMD18-T载体上,测序正确后,将IFITM3基因亚克隆至真核表达载体pVAX1中获得重组质粒pV-IFITM3,通过转染293T细胞进行表达与定位研究。结果:RT-PCR和Western blot结果表明,获得的目的基因IFITM3能够表达,且具有良好抗原活性;激光共聚焦显微镜观察显示,IFITM3基因表达后主要分布于细胞膜上。结论:成功获得IFITM3基因,并验证了其表达。该研究为基于IFITM3的抗病毒药物以及探讨其抗病毒机制研究奠定了坚实基础。
OBJECTIVE: The IFITM3 gene was induced in human embryonic kidney cell line 293T by interferon rhIFN-α2B, and the expression and localization of IFITM3 gene in 293T cells were observed. Methods: IFITM3 gene was induced by interfering rhIFN-α2B in 293T cells in vitro. The full-length IFITM3 cDNA of interferon-induced transmembrane protein was amplified by RT-PCR. The amplified product was ligated into pMD18-T vector and sequenced correctly The IFITM3 gene was subcloned into the eukaryotic expression vector pVAX1 to obtain the recombinant plasmid pV-IFITM3, which was transfected into 293T cells for expression and localization. Results: The results of RT-PCR and Western blot showed that IFITM3 gene was expressed and had good antigen activity. Confocal laser scanning microscopy showed that IFITM3 gene mainly distributed on the cell membrane after IFITM3 gene expression. Conclusion: The IFITM3 gene was successfully obtained and its expression verified. This study lays a solid foundation for the study of IFITM3-based antiviral drugs and their antiviral mechanisms.