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目的:设计和构建乙型肝炎病毒(HBV)X基因(HBx)特异性的小干扰RNA(siRNA)体内表达载体,筛选抑制X基因表达的有效siRNA,并初步探讨其对HBV复制功能的影响。方法:以X基因为目的基因,以产生siRNA质粒载体pSilencerTM-U6为表达模板,细胞内转录合成3条siRNA,并构建携带荧光素酶报告基因的重组质粒载体plucF-HBx。将重组质粒载体plucF-HBx与产生siRNA的质粒pSilencerTM-U6共转染HepG2细胞,检测荧光素酶活性以筛选出抑制荧光素酶表达的有效siRNA,逆转录聚合酶链反应检测HBx mRNA的表达,进一步证实siRNA对HBx表达的抑制效果,然后将siRNA转染HepG2.2.15,酶联免疫吸附法和荧光定量PCR检测siRNA对HBV复制的影响。结果:合成的3条siRNA中有1条抑制荧光素酶表达,抑制效率为76.3%,并能特异性抑制HBV的复制。结论:成功构建并筛选到针对X基因表达的有效siRNA质粒,为HBV的基因治疗奠定基础。
OBJECTIVE: To design and construct HBx-specific small interfering RNA (siRNA) expression vector in vivo to screen for effective siRNAs that inhibit the expression of X gene, and to explore its effect on HBV replication. Methods: The X gene was used as the target gene to generate the siRNA plasmid vector pSilencerTM-U6 as the expression template. Three siRNAs were transcribed intracellularly, and the recombinant plasmid vector, plucF-HBx, carrying the luciferase reporter gene was constructed. HepG2 cells were cotransfected with recombined plasmid vector plucF-HBx and siRNA-producing plasmid pSilencerTM-U6, luciferase activity was detected to select effective siRNAs for inhibiting luciferase expression, HBx mRNA expression was detected by reverse transcription polymerase chain reaction, Further confirmed the inhibitory effect of siRNA on HBx expression, and then siRNA was transfected into HepG2.2.15, and the effect of siRNA on HBV replication was detected by enzyme-linked immunosorbent assay and fluorescence quantitative PCR. Results: One of the 3 synthesized siRNAs inhibited luciferase expression with an inhibitory efficiency of 76.3% and could specifically inhibit HBV replication. Conclusion: The siRNA plasmid targeting to the expression of X gene was successfully constructed and screened, which laid the foundation for the gene therapy of HBV.