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目的:体外观察siRNA对HepG22.2.15细胞中乙型肝炎病毒(HBV)SmRNA及HBsAg,HBeAg产生的影响.方法:设计并合成针对HBVS区的三条siRNA,构建含上述siRNA的表达载体pSilencerZY1,pSilencerZY2和pSilencerZY3,pSilencerHK2测序及酶切鉴定后用脂质体介导重组质粒转染HepG22.2.15细胞,用酶免分析法对HepG22.2.15细胞上清中HBsAg,HBeAg进行检测,用逆转录聚合酶链反应检测HBVSmRNA.结果:成功构建了针对HBVS区的siRNA的表达载体,三条siRNA均可程度不同地抑制HepG22.2.15细胞上清中HBsAg,HBeAg的分泌,72h抑制率达高峰,对HBsAg的抑制率分别为81%,29%及78%,对HBeAg的抑制率分别为35%,3%及49%,并有抑制HBVSmRNA的作用.结论:针对HBVS区的siRNA能明显抑制HBV的复制.
OBJECTIVE: To observe the effect of siRNA on the SmRNA, HBsAg and HBeAg production of Hepatitis B virus (HBV) in HepG22.2.15 cells in vitro.Methods: Three siRNAs targeting HBV region were designed and synthesized, and the expression vectors pSilencerZY1, pSilencerZY2 and pSilencerZY3, pSilencerHK2 sequencing and restriction endonuclease identification using liposome-mediated recombinant plasmid transfected HepG22.2.15 cells by enzyme-free analysis of HepG22.2.15 cells supernatant HBsAg, HBeAg were detected by reverse transcription polymerase chain reaction HBVS mRNA was detected.Results: The siRNA vector targeting HBVS region was successfully constructed.All three siRNAs could inhibit the secretion of HBsAg and HBeAg in the supernatant of HepG22.2.15 cells at different degrees, with the highest inhibition rate at 72h and the inhibition rate of HBsAg respectively And 81%, 29% and 78%, respectively, and the inhibition rates of HBeAg were 35%, 3% and 49%, respectively, and inhibited the effect of HBV SmRNA.Conclusion: siRNA targeting HBVS can significantly inhibit HBV replication.