人SNW1基因的克隆、表达及其调控IFN-β表达的初步研究

Clone and expression of human SNW1 and study about its regulatory activity on IFN-β expression

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。

This study designed to investigate the modulation of SNW1 on the expression of IFN-β expression.Human SNW1 gene was amplified using c DNA from 293 T cells as template and then the gene was cloned to p CMV-N-Flag eukaryotic expression vector. SNW1 expression plasmid was identified by restrictive enzyme digestion and DNA sequencing; the expression of SNW1 was determined by transient transfection and immunoblotting;the localization of SNW1 was determined by immune fluorescence assay. Regulation of IFNB promoter activity was determined using dual luciferase reporter system with or without Se V infection. Data showed that a specific band of SNW1 about 1.6 kb was amplified by using PCR. Restrictive enzyme digestion and DNA sequencing results proved that SNW1 was cloned into p CMV-N-Flag. Flag-SNW1 was about 70 k D and mainly localized in nucleus.Overexpression of SNW1 significantly enhanced the IFNB promoter activity during Se V infection, however, inhibited the basal IFNB promoter activity. In conclusion, transcription of IFN-β could be significantly regulated by SNW1.