【摘要】 目的观察针对乙型肝炎病毒(HBV)不同靶区发夹样RNA(shRNA)抑制HBV复制与表达的效率。方法根据shRNA表达载体设计原则,设计并构建了针对HBV P、C、S和X区7种特异性和2种序列突变的shRNA表达载体,将其与1．3倍HBV全基因表达载体共转染于HepG2细胞,通过Southern blot和酶联免疫吸附法分别检测HBV DNA复制中间体和乙型肝炎表面抗原(HBsAg)水平,来观察shRNA抗HBV复制与表达的效率。结果发现针对HBV不同靶区shRNA均有抗HBV复制和表达效果,以P1、S2、C2、S1和X抑制HBV DNA复制效率较高,P1的抑制率高达95％。HBsAg表达受抑制情况与HBV DNA复制受抑制的程度相似,其中C2、C1和S2对HBsAg的抑制作用较强。结论针对HBV四个开放性读码框进行RNA干扰对HBV复制和抗原表达均有抑制作用,其中P1和C2分别对HBV复制和表达的抑制效率较高。更多还原

【Abstract】 Objective To observe the inhibition of HBV replication and antigen expression by RNA interference aimed at different parts of the HBV genome. Methods Following the rules of shRNA expression vector design and construction, we constructed seven kinds of sequence specific vectors and two kinds of mutant shRNA expression ones. We then cotransfected those shRNA and HBV expression vectors into HepG2 cells using lipofectamine?2000. The level of HBV replication was investigated using Southern blot and the antigen expression using ELISA. Results The replication of HBV DNA was inhibited by many shRNAs, especially the ones against P1, S2, C2, S1 and X. The inhibition rate against P1 was as high as 95%. Results obtained with ELISA showed that the shRNAs targeting C2, C1 and S2 had high rates of inhibition to HBsAg. Conclusion The replication and antigen expression of HBV could be inhibited by shRNAs aimed at four different open read frames, and higher inhibition rates of HBV replication and surface antigen expression could be obtained by P1 and C2, respectively.更多还原