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目的构建绿色荧光蛋白(GFP)与严重急性呼吸综合征(SARS)冠状病毒N蛋白的融合表达载体pEGFP-C1-N以及针对SARS冠状病毒N基因的小发卡型shRNA表达载体pshRNAN,观察shRNA对SARS冠状病毒N基因复制和表达的影响。方法将构建成功的pEGFPC1N与pshRNAN共转染293细胞,于转染后24、48、72h观察GFP表达的强弱,采用Western Blot分别检测GFP与N蛋白的表达,逆转录PCR检测N基因的mRNA。结果psh-RNA-N作用组绿色荧光强度明显弱于未干扰组,Western Blot和逆转录PCR结果也证实pshRNA-N对N基因和N蛋白的表达有明显抑制作用,而无关序列的shRNA无此作用。结论针对SARS冠状病毒N基因的shRNA具有显著和特异的抑制N蛋白表达的作用。
Objective To construct a fusion expression vector pEGFP-C1-N of green fluorescent protein (GFP) and severe coronavirus (SARS) coronavirus N protein and a small hairpin shRNA expression vector pshRNAN against SARS coronavirus N gene. Effect of Coronavirus N Gene Replication and Expression. Methods 293 cells were co-transfected with pEGFPC1N and pshRNAN. The expression of GFP was observed at 24, 48 and 72 hours after transfection. The expression of GFP and N protein was detected by Western Blot. The mRNA of N gene was detected by reverse transcription polymerase chain reaction . Results The green fluorescent intensity of psh-RNA-N group was significantly weaker than that of non-interfering group. The results of Western Blot and reverse transcription PCR also showed that pshRNA-N significantly inhibited the expression of N gene and N protein. effect. Conclusion shRNA targeting the SARS-CoV N gene has a significant and specific inhibitory effect on N protein expression.