血清中乙型肝炎病毒全基因组及X基因的克隆与鉴定

Cloning and identification of HBV complete genome and X gene from serum

目的:建立从血清样品直接扩增乙型肝炎病毒(HBV)全长DNA的方法,构建HBV全基因组克隆。方法:用长链PCR从血清样品一次性扩增HBV全长DNA,测序鉴定后插入质粒puc19构建含HBV全基因组的重组质粒,再从重组质粒扩增X基因并进行序列分析。结果:经DNA序列测定和酶切分析,获得HBV全基因组克隆,从克隆的HBV全基因组扩增到X基因。结论:从血清中可直接扩增到HBV全基因组DNA,为整体水平研究HBV基因组的变异与其致病及宿主抗感染免疫之间的关系奠定了实验基础。

Objective：To develop a method for amplifying full-length DNA of hepatitis B virus（HBV） from serum samples and construct the complete genome clone of HBV. Methods：The full-length HBV DNA was amplified with long-PCR, the amplified product was identified by sequencing and cloned into pucl9 vector to construct a recombinant plasmid containing the complete genome of HBV,and then the X gene of HBV was amplified from the recombinant plasmid and sequenced. Results：The complete genome clone of HBV was acquired after determination by enzyme digestion and sequence analysis,and the X gene can be amplified from the cloned HBV genome. Conclusion：The complete genome DNA of HBV can be amplified directly from serum samples,which provide the research basis for studying the relations between HBV genome mutation and its pathogenicity and the host anti-infectlon immune in whole level.