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构建针对乙型肝炎病毒(HBV)核心区或表面抗原编码区的siRNA表达载体pSI-C和pSI-S,在体外HBV复制细胞模型中观察对HBV复制和表达的影响.将pSI-C,pSI-S与1.3倍HBV真核表达质粒pHBV共转染HepG2细胞,分别在转染后24,48和72h,观察pSI-C和pSI-S对细胞上清和胞内病毒抗原表达的影响,并用分子杂交方法从病毒复制、转录和翻译水平检测目的基因的表达情况.实验结果表明,pSI-C及pSI-S能明显抑制细胞内HBsAg和HBcAg的合成,呈序列特异性和剂量依赖性的特点,而对照组siRNA无抑制作用.pSI-C比pSI-S抑制作用更强,转染后第2天对HBsAg和HBeAg抑制率达高峰,分别为92%和85%.Southern和Northern杂交结果表明,HBVsiRNA能降低目的基因转录和病毒复制中间体的表达水平,提示针对乙型肝炎病毒核心区和表面抗原编码区的siRNA能有效抑制HBVRNA分子转录和病毒复制,最终降低病毒抗原表达,这为运用RNAi技术进行HBV基因治疗提供了新的思路.
To construct siRNA expression vectors pSI-C and pSI-S targeting HBV core region or surface antigen coding region, and to observe the effect on HBV replication and expression in HBV replication cell model in vitro.PSI-C, pSI -S and 1.3-fold HBV eukaryotic expression plasmid pHBV were co-transfected into HepG2 cells at 24, 48 and 72 hours after transfection to observe the effect of pSI-C and pSI-S on the expression of the virus supernatant and intracellular virus antigen, Hybridization method to detect the expression of the target gene from the level of virus replication, transcription and translation.Experimental results show that pSI-C and pSI-S can significantly inhibit the synthesis of intracellular HBsAg and HBcAg in a sequence-specific and dose-dependent manner, While the control siRNA showed no inhibitory effect.pSI-C showed a stronger inhibitory effect than pSI-S, and the highest inhibition rates of HBsAg and HBeAg reached 92% and 85% respectively on the second day after transfection.The results of Southern and Northern blot showed that, HBVsiRNA can reduce the expression of target gene transcription and virus replication intermediates, suggesting that siRNA targeting core region and surface antigen of hepatitis B virus can effectively inhibit HBV RNA molecule transcription and virus replication, and ultimately reduce viral antigen expression, Technology for HBV gene Treatment provides a new way of thinking.