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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。
Objective To observe the efficiency of hairpin RNA (shRNA) targeting HBV replication and expression in different targets of hepatitis B virus (HBV). Methods According to the design principle of shRNA expression vector, shRNA expression vectors targeting 7 specific and 2 kinds of mutations in HBV P, C, S and X regions were designed and constructed, and co-transfected with 1.3x HBV full-length gene expression vector HepG2 cells were infected with HepG2 cells and the HBV DNA replication intermediates and HBsAg levels were detected by Southern blot and enzyme-linked immunosorbent assay (ELISA) to observe the efficiency of shRNA anti-HBV replication and expression. The results showed that anti-HBV replication and expression were targeted against different target shRNAs of HBV, and inhibition of HBV DNA replication by P1, S2, C2, S1 and X was higher, and the inhibition rate of P1 was as high as 95%. The inhibition of HBsAg expression is similar to the suppression of HBV DNA replication, of which C2, C1 and S2 have a stronger inhibitory effect on HBsAg. Conclusion RNA interference against four HBV open reading frames has an inhibitory effect on HBV replication and antigen expression. Among them, P1 and C2 inhibit HBV replication and expression, respectively.