一种新的乙型肝炎病毒全基因组克隆技术的建立

A New Method for Amplification and Cloning of the Full-Length HBV Genome

目的建立一种乙型肝炎病毒(HBV)全基因组扩增及克隆的新方法。方法设计含SalⅠ、NotⅠ酶切位点的引物,用一片段PCR法扩增HBV全基因组,经双酶切后将PCR产物克隆入pBlueScript-(SK-)载体,进行HBV全基因组序列测定。结果用此方法成功获得35例HBV全基因组DNA序列。同时以重组质粒为模板进行敏感性和保真性测定,其敏感性为102个初始模板,核苷酸的人为突变率为1.6bp/kb。结论此方法可用于大规模HBV全基因组扩增和序列分析,为HBV的基础和临床研究提供了一种新方法。

Objective To develop a new method for amplification and cloning of a full-length HBV genome. Method Primers containing Sal Ⅰ and Not Ⅰ sites were used in PCR amplification of the HBV genome. The PCR product was cloned into pBlue Script（SK） plasmid and followed by sequencing. Result 35 clones containing the full-length HBV genome were obtained. The sensitivity and fidelity of this cloning method were also analyzed. The mimium unmber of templates was 10^2 and the mutation rate was 1.6 bp/kb. Conclusion This method can be used in a large scale amplification and sequence analysis of the full-length HBV genome.