体外培养细胞内乙肝病毒共价闭合环状DNA检测方法的建立

A method for detecting intracellular HBV covalently closed circular DNA in cultured cells in vitro

目的:建立一种体外培养细胞内乙肝病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)的检测方法。方法:Southern blot检测HBV复制中间体,以确认HepAD38、HepG2.2.15和P+P-细胞是否支持HBV复制;用Hirt法分别提取细胞内的去蛋白DNA(protein-free DNA,PF DNA),经加热变性处理,EcoRⅠ酶切,然后用Southern blot检测HBV cccDNA;用同样方法检测不同培养时间的HepAD38细胞内cccDNA,观察培养时间对cccDNA积累量的影响。结果:用Southern blot可在HepAD38、HepG2.2.15和P+P-细胞中检测到HBV复制中间体;Hirt法提取物经变性和酶切处理,可有效地将cccDNA与其他形式HBV DNA区分开;HepAD38、HepG2.2.15和P+P-细胞内均可检测到HBV cccDNA;HepAD38细胞在铺满后继续培养的时间较长时,其细胞内cccDNA的量较多。结论:成功建立了体外培养细胞内HBV cccDNA检测方法,该方法结合Hirt提取法和地高辛探针Southern blot方法,能可靠、准确地检测HBV cccDNA。

Objective ： To establish a method for detecting hepatitis B virus （HBV） covalently closed circular DNA （cccDNA） in cul- tured cells in vitro. Methods：Southern blot was used to detect the HBV replication intermediates from HepAD38, HepG2.2.15 and P＋ P- cell lines to confirm their capability of supporting HBV replication. Extraction of protein-free DNA（PF DNA） was carried out with a modified Hirt extraction procedure from HepAD38, HepG2.2.15 and P＋P- cells, which were cultured for several days after the cells growing to confluence. The cccDNA was thermally denatured and then subjected to EcoR I digestion before Southern blot analysis. Dynamic changes of HBV cccDNA in HepAD38 cells at different growth time were examed. Results：HBV replication intermediates were detectable in HepAD38,HepG2.2.15 and P＋P- cells by Southern blot. HBV cccDNA extracted from HepAD38,HepG2.2.15 and P＋P- cells can be effectively distinguished from other forms of HBV DNA in Southern blot. A larger amount of HBV cccDNA was detected when HepAD38 cells grew more time after confluence. Conclusions：A method for detecting intracellular HBV cccDNA is suc- cessfully established. This method combines Hirt extraction procedure with digoxigenin-probe-based Southern blot, allowing us detect HBV cccDNA accurately and faithfully.