化学发光法检测体外HBV复制  

Analysis of Hepatitis B Virus Replication in Vitro by Chemiluminescent Detection Method

作　　者：崔静[1] 胡接力[1] 张文露[1] 王媛媛[1] 李毅[1] 黄爱龙[1] 

机构地区：[1]重庆医科大学病毒性肝炎研究所,感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《生物技术》2009年第2期44-46,共3页Biotechnology

基　　金：国家自然科学基金项目资助(No.30600520)

摘　　要：目的:建立化学发光法检测体外HBV复制水平的方法,研究其灵敏度和稳定性。方法:用HBV DNA重组质粒pCH9转染到人肝癌细胞株HepG2和Huh7中,5d后收集细胞并抽提其HBV复制中间体DNA,转印后以地高辛标记HBV DNA为探针进行杂交,用化学发光法检测杂交结果,同时进行探针灵敏度的检测。结果:转染后HepG2和Huh7细胞提取HBV DNA中检测出较强的复制中间体的信号,分别为松散环状DNA(rcDNA),双链线性DNA(dslDNA),单链DNA(ssDNA),探针检测的灵敏度可达到1pg,接近同位素法检测的灵敏度。整个实验重复3次获得同样结果。结论:成功建立了稳定的化学发光法检测体外HBV复制水平的方法。Objective：To develop a chemiluminescent detection method for analysis of hepatitis V virus （HBV） replication level in vitro, and observe its stability and sensitivity. Method： HBV recombinant plasmid （pCH9） was transfected into human hepatoma cell lines HepG2 and Huh7. Cells were harvested 5 days posttransfection and intracellular HBV replication intermediates were extracted. Southern blot assays for HBV replication intermediates were performed by digoxigenin-labeled probe and chemiluminescent detection. Result： High levels of HBV replication intermediates DNA were detected by Southern blot in the HepG2 and Huh7 cell lines respectively. The digoxigenin-labeled probe showed a sensitivity near to that of radiolabeled probe, detecting as little as 1 pg of HBV DNA. Conclusion： A stable chemiluminescent detection method for analysis of HBV replication level in vitro is successfully established.

关 键 词：HBV DNA 化学发光法 复制 

分 类 号：R512.62[医药卫生—内科学]