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目的 构建针对乙型肝炎病毒 (HBV)核心区的siRNA表达载体pSIHBV/C ,观察其对HBV复制和表达的影响。方法 将构建成功的 pSIHBV/C与 1 3倍HBV真核表达质粒pHBV1 3共转染HepG2细胞 ,转染后 2 4、4 8、72h ,用Abbott试剂检测细胞上清中HBsAg和HBeAg ,并用免疫荧光染色法观察细胞内病毒核心抗原和表面抗原的表达。结果 成功构建了针对HBV核心区的siRNA表达载体 pSIHBV/C ,并发现它能明显抑制HBsAg和HBeAg的分泌 ,转染后第 2天抑制率达高峰 ,分别为 92 %、85 % ,而随机序列的siRNA无此作用。免疫荧光染色结果也证实转染 2 4h后 ,随pSIHBV/C比例的升高 ,其对HBV抗原表达的抑制作用也随之增加 ,当pSIRNA/C与 pHBV1 3比例为 1∶2 0时 ,pSIHBV/C对细胞内HBsAg和HBcAg表达的抑制作用最强。 结论 乙型肝炎病毒核心区的siRNA具有显著和特异的抗HBV复制和表达的作用。
Objective To construct siRNA expression vector pSIHBV / C targeting to hepatitis B virus (HBV) core region to observe its effect on HBV replication and expression. Methods HepG2 cells were cotransfected with pSIHBV / C and pHBV13 co-transfected with HBV DNA. The transfected HepG2 cells were infected with Abbott reagent for detection of HBsAg and HBeAg at 24, 48 and 72 hours after transfection. Fluorescent staining was used to observe the expression of intracellular viral core antigen and surface antigen. Results The siRNA expression vector pSIHBV / C targeting HBV core region was successfully constructed and found to significantly inhibit the secretion of HBsAg and HBeAg. At the second day after transfection, the inhibition rate reached the peak at 92% and 85%, respectively. However, the random sequence SiRNA does not have this effect. Immunofluorescence staining also confirmed that after 24 h of transfection, the inhibition of HBV antigen expression increased with the increase of pSIHBV / C ratio. When the ratio of pSIRNA / C to pHBV13 was 1: 20, pSIHBV / C has the strongest inhibitory effect on the expression of HBsAg and HBcAg in cells. Conclusion siRNA in the core of hepatitis B virus has significant and specific anti-HBV replication and expression.