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目的分析HBV前S缺失突变病毒株体外复制力及其对表面抗原启动子(surface antigen promoter,SP)Ⅱ转录活性的影响。方法研究对象为119例解放军第三〇二医院的住院患者,包括38例慢性乙型肝炎(慢乙肝)轻中度、40例慢乙肝重度和41例慢加急性肝衰竭。从患者血清中提取HBV DNA,PCR扩增HBV全长基因组,统计前S缺失突变的发生率。挑选代表前S缺失突变株及其相应对照的HBV全长序列克隆至pGEM-Teasy载体中。用BspQⅠ/ScaⅠ双酶切1.0倍HBV基因组,转染HepG2细胞,72 h后检测病毒复制力;用PCR分别扩增含前S缺失突变型和野生型的HBV SPⅡ启动子片段,构建pGL3-SPⅡ双荧光素酶真核报告表达载体,转染HepG2细胞48 h后检测相对荧光素酶活性,分析前S1缺失突变对重叠的SPⅡ荧光素酶表达的影响。结果①HBV基因组前S缺失突变检出率在慢乙肝轻中度、慢乙肝重度和慢加急性肝衰竭3组中逐渐递增,分别为5.3%、12.5%和24.4%,差异有统计学意义(P<0.05);②前S缺失突变病毒株的复制力较相应野生株降低69%;③与野生型相比,前S1缺失突变使重叠的SPⅡ转录活性降低了36%。结论 HBV前S缺失突变发生率随乙肝进展而升高,前S缺失突变株复制力降低,导致重叠的SPⅡ转录活性降低。
Objective To analyze the in vitro replication and its effect on the surface antigen promoter (SP) Ⅱ transcriptional activity of HBV pre-S deletion mutants. Methods The subjects were 119 inpatients of 302 Hospital of PLA, including 38 mild to moderate chronic hepatitis B (CHB), 40 severe chronic hepatitis B and 41 acute plus acute hepatic failure. HBV DNA was extracted from the patient serum and the full-length HBV genome was amplified by PCR. The incidence of pre-S deletion mutation was calculated. The full-length sequence of HBV, representing the pre-S deletion mutant and its corresponding control, was cloned into the pGEM-Teasy vector. The HBV genome was double digested with BspQⅠ / ScaⅠ and transfected into HepG2 cells. The replication of HepG2 cells was detected 72 h later. The HBV SPⅡ promoter fragment containing the pre-S deletion mutant and wild type was amplified by PCR to construct pGL3-SPⅡ The luciferase reporter vector was used to detect the relative luciferase activity in HepG2 cells transfected for 48 h. The effect of pre-S1 deletion mutation on the expression of overlapping SP Ⅱ luciferase was analyzed. Results ① The detection rate of pre-S deletion mutation in HBV genome was gradually increased in moderate to severe chronic hepatitis B, 5.3% in chronic hepatitis B and 12.5% in acute and severe acute liver failure (P <0.05), and the difference was statistically significant (P <0.05). ② The replicative power of the former S deletion mutant strain was 69% lower than that of the corresponding wild strain. ③ Compared with the wild type, the deletion mutation of the former S1 reduced the transcription activity of overlapping SPⅡ by 36%. Conclusions The incidence of pre-S deletion mutations in HBV increases with the progress of hepatitis B, and the replication of pre-S deletion mutants decreases, leading to a decrease in overlapping SPⅡ transcriptional activity.