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目的评价基于载体的靶向乙型肝炎病毒(HBV)C基因的小发卡RNA(shRNA)对耐拉米夫定HBV的抑制作用,探讨新的抗病毒治疗手段。方法从临床耐拉米夫定患儿中提取耐药HBV基因组,经高保真重复延伸及序列拼接后测序,纯化并克隆到pGEM-T Easy TA载体,再经酶切消化,连接入pcDNA3.1载体,构建表达耐药HBV的表达载体pcDNA-HBV-1;根据GenBank收录HepG2细胞系中HBV基因全序列,设计构建针对HBV C基因区的3条siRNA的表达载体pSilencer4.1/HBV-C1,C2,C3。经退火连接入pSilencer-CMV4.1载体。中量超纯提取质粒,定量后与转染试剂siPort XP-1及pcDNA-HBV-1共转染人肝癌细胞系HepG2细胞。分别设立正常HepG2组(未转染组)、载体阴性对照组及共转染组。在共转染后的不同时间点,应用Western blot检测细胞中乙肝病毒表面抗原(HBsAg)蛋白、乙肝病毒e抗原(HBeAg)蛋白的表达;应用实时荧光定量PCR定量检测细胞上清中HBV DNA拷贝的变化。结果构建的耐药HBV表达载体用于转染HepG2细胞,在转染后的第7天至下次传代前该细胞中可检测到稳定表达的HBV DNA,HBsAg、HBeAg可检出。Western blot结果:在转染的第3天载体pSilencer4.1/HBV-C1、pSilencer4.1/HBV-C2、pSilencer4.1/HBV-C3均可抑制HBV的复制与表达,其中pSilencer4.1/HBV-C2抑制效应尤其显著,在转染后第3天对HBsAg、HBeAg的抑制率分别为(83.53±0.48)%、(86.12±0.83)%。定量PCR检测显示特异性siRNA可以显著抑制HBV-DNA,可以使DNA拷贝数降低102个数量级。结论基于载体的特异性siRNA在体外可抑制HepG2细胞中耐药HBV的复制和表达。该特异性siRNA可作为耐拉米夫定HBV的一种有效的基因治疗措施。
OBJECTIVE: To evaluate the inhibitory effect of vector-based small hairpin RNA (shRNA) targeted to hepatitis C virus (HBV) C gene on HBV resistant to lamivudine and to explore new means of antiviral therapy. Methods The resistant HBV genome was extracted from children with clinical resistance to lamivudine and then purified by high fidelity repeat extension and sequencing, purified and cloned into pGEM-T Easy TA vector. After digested by restriction enzyme, the recombinant plasmid was ligated into pcDNA3.1 According to the complete sequence of HBV gene in HepG2 cell line, GenBank was used to construct the expression vector pSilencer4.1 / HBV-C1 for three siRNAs against HBV C gene region. C2, C3. The pSilencer-CMV4.1 vector was annealed. The plasmid was extracted by ultrapure and transfected into human hepatocellular carcinoma HepG2 cells by co-transfection with siPort XP-1 and pcDNA-HBV-1. The normal HepG2 group (untransfected group), vector negative control group and co-transfection group were established respectively. Western blot was used to detect the expression of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) protein at different time points after co-transfection. Quantitative detection of HBV DNA copies in the cell supernatant by real-time fluorescence quantitative PCR The change. Results The constructed drug-resistant HBV expression vector was used to transfect HepG2 cells. Stably expressing HBV DNA, HBsAg and HBeAg were detected in the cells from the 7th day after transfection to the next passage. Western blot results: On the 3rd day of transfection, the vectors pSilencer4.1 / HBV-C1, pSilencer4.1 / HBV-C2, pSilencer4.1 / HBV-C3 all inhibited the replication and expression of HBV. The inhibitory effects of HBsAg and HBeAg on the 3rd day after transfection were (83.53 ± 0.48)% and (86.12 ± 0.83)%, respectively. Quantitative PCR detection showed that specific siRNA can significantly inhibit HBV-DNA, can reduce the number of DNA copies 102 orders of magnitude. Conclusion Carrier-based siRNA can inhibit the replication and expression of drug-resistant HBV in HepG2 cells in vitro. This specific siRNA can be used as an effective gene therapy for HBV resistant to lamivudine.