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目的克隆人SNW1基因并构建其真核表达载体,验证SNW1基因的表达和细胞定位,探索SNW1基因对IFN-β转录的调控作用。方法以来自293T细胞的c DNA为模板PCR扩增人SNW1基因并克隆至真核表达载体p CMV-N-Flag上,对重组质粒进行酶切鉴定和测序;通过瞬时转染和免疫印迹验证SNW1在细胞中的表达;通过免疫荧光确定SNW1在细胞内的定位;利用IFNB启动子荧光素酶双报告系统和仙台病毒(Se V)初步检测SNW1对IFN-β转录的调控作用。结果 PCR扩增出约1.6 kb的条带,大小与预期相符;酶切结果表明SNW1成功克隆至PCMV-N-Flag,进一步测序验证SNW1序列正确;Flag-SNW1蛋白大小约Mr70 000,主要定位于细胞核中;在Se V感染条件下过量表达SNW1可以显著增强IFN-β的表达,而过量表达的SNW1对IFN-β的本底表达有一定的抑制作用。结论成功克隆并构建了SNW1真核表达载体,初步显示SNW1对IFN-β的转录具有较强的调控作用。
Objective To clone human SNW1 gene and construct its eukaryotic expression vector to verify the expression of SNW1 gene and cellular localization and to explore the regulatory effect of SNW1 gene on IFN-β transcription. Methods The human SNW1 gene was amplified by PCR from c DNA of 293T cells and cloned into the eukaryotic expression vector pCMV-N-Flag. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. The transient expression of SNW1 In the cells. The intracellular localization of SNW1 was determined by immunofluorescence. The IFNB promoter luciferase dual reporter system and Sendai virus (Se V) were used to detect the regulatory effect of SNW1 on IFN-β transcription. The results of PCR amplification of the approximately 1.6 kb band was consistent with the expected size. The results of digestion showed that SNW1 was successfully cloned into PCMV-N-Flag. The sequence of SNW1 was confirmed by sequencing. The size of Flag-SNW1 protein was about 70 000, In the nucleus, overexpression of SNW1 under Se V infection significantly enhanced the expression of IFN-β, whereas overexpression of SNW1 inhibited the background expression of IFN-β. Conclusion The SNW1 eukaryotic expression vector was successfully cloned and constructed. It showed that SNW1 had a strong regulatory effect on the transcription of IFN-β.