摘要
目的构建乙型肝炎病毒(HBV)核心启动子(CP)荧光素酶表达载体,探讨HBVCP变异对下游基因转录活性的影响。方法 2005年5月至2008年3月于解放军302医院就诊的慢性乙型肝炎(CHB)及慢加急性肝衰竭(ACLF)患者各40例,采集血清提取HBV DNA,采用巢式PCR法扩增HBVCP片段,克隆至pGEM-TEasy载体。挑选含有CP相关变异位点的质粒,经KpnⅠ/BglⅡ双酶切后,构建pGL3-CP荧光素酶真核报告质粒,并采用定点突变方法获得野生型质粒,将两者同时转染肝癌细胞系HepG2,48h后进行荧光素酶检测。结果患者临床资料经统计分析后显示,CHB患者HBeAg阳性率和HBV DNA载量均高于ACLF患者(P<0.01),而TBIL含量则低于ACLF患者(P<0.01);对CP区热点突变频率分析后发现:G1764A/C1766T/T1768A三联突变在CHB患者中为0.0%(0/40),在ACLF患者中为12.5%(5/40,P<0.05)。细胞转染结果显示:典型CP双联突变A1762T/G1764A病毒株的启动子活性为相应野生株的1.67倍,而G1764A/C1766T/T1768A三联突变病毒株的启动子活性为相应野生株的1.43～1.80倍。结论 HBeAg阳性率、TBIL水平、HBV DNA载量与乙型肝炎重症化相关,HBVCP中存在的A1762T/G1764A、G1764A/C1766T/T1768A突变有顺式激活下游基因转录活性的作用。
Objective To construct the luciferase reporter gene recombinant plasmid of hepatitis B virus （HBV） core promoter （CP）,and investigate the influence of HBV CP mutations on its transcriptional activation. Methods Sera of patients with chronic hepatitis B （CHB） and acute-exacerbation of chronic liver failure （ACLF） （40 each） admitted to 302 Hospital of PLA from May 2005 to Mar. 2008 were collected. HBV DNA was extracted to amplify fragment of HBV CP by nest PCR,which was cloned into pGEM-T Easy vector. The recombinant pGL3-CP plasmids and the counterpart wild-type plasmids generated by site-directed mutagenesis were transfected into HepG2 cells with FuGENE HD transfection reagents. The activity of luciferase in transfected HepG2 cells was detected at 48 hours post-transfection. Results The analysis of clinical features showed that HBeAg positive rate and HBV DNA load were significantly higher and TBIL level was significantly lower in CHB patients than in ACLF patients （P0.01）. The frequency of G1764A/C1766T/T1768A triple mutation was significantly higher in ACLF patients （5/40,12.5%） than in CHB patients （0/40,0.0%,P0.05）. The expressions of luciferase in HepG2 cells transfected with CP A1762T/G1764A double mutants and CP G1764A/C1766T/T1768A triple mutants were 1.67-fold and 1.43―1.80-fold compared with CP counterpart wild-type viruses on average,respectively. Conclusions HBeAg positive rate,HBV DNA load and TBIL level may be associated with the severity of hepatitis B. HBV CP A1762T/G1764A double mutations and G1764A/C1766T/T1768A triple mutations may up-regulate the transcriptional activation of HBV CP.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2010年第8期954-957,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家重点基础研究发展计划课题(2007CB512803)
北京市自然科学基金重点课题(7091006)
国家“十一五”传染病重大专项子课题(2008ZX10002-005-6,2008ZX10002-011)
关键词
肝炎
乙型
慢性
突变
启动区
反式激活
hepatitis B
chronic
mutation
promoter regions
trans-activation
