含拉米夫定耐药基因的乙型肝炎病毒全基因组载体的构建及鉴定

Construction of full-length genome hepatitis B virus plasmid with lamivudine drug-resistance gene

目的建立含拉米夫定耐药基因的乙型肝炎病毒(HBV)全基因组载体,为进一步建立耐药细胞模型奠定基础。方法采用基因扩增、重组和克隆的方法,提取HBV-DNA进行扩增,插入pBluescript-SK(-)载体,酶切和测序鉴定重组质粒含有HBV-DNA全基因组后,以该重组质粒的多聚酶逆转录酶区为模板,设计引入突变位点的引物,聚合酶链反应(PCR)进行定点突变,突变产物经DpnⅠ消化后,转化到感受态细胞DH5α,最后将提取的突变质粒进行测序鉴定。结果凝胶电泳分析血清HBV-DNA扩增可见3.2kb条带,HBV-DNA和pBluescript-SK(-)载体连接后可见6.9kb目的条带,该重组质粒双酶切后可见3.2kb和2.9kb目的条带,突变PCR产物经DpnⅠ消化后可见明显条带。突变前质粒测序显示含有HBV基因型为C型血清型为adr的全基因组,突变后测序证实存在HBV逆转录酶173、180、204位点的预期碱基突变。结论通过测序鉴定,成功构建了拉米夫定耐药HBV全基因组载体。

Objective To construct lamivudine drug-resistance recombinant plasmids containing full-length DNA of hepatitis virus B. Methods Full-length HBV-DNA was extracted and amplified by polymerase chain reaction （PCR）, Then the PCR product was cloned into pBluescript-SK（-） vector. Primers were designed according to the reverse transcription gene sequence of HBV polymerase and mismatches were introduced into primers. Sitedirected mutagenesis of HBV polymerase gene eodon was conducted using PCR. Mutants were detached from the PCR product and trsanformed into competent DHSa. Plasmid was extacted from the bacteria and sequenced. Results The agarose gel electrophoresis showed 3.2 kb brand of HBV-DNA amplification, and 6.9 kb brand could be seen after HBV-DNA was ligated with pBlueseript-SK（-） vector. Brands of 3.2 kb and 2.9 kb can be seen after the reeombiant plasmid was digested with Not Ⅰ and Sal Ⅰ, and brand of 6. 9 kb can be seen when the mutants were digested with Dpn Ⅰ. The results of DNA sequencing confirmed that the genotype and serotype of the HBV-DNA in reeombiant plasmid is C and adr respectively, and the base sequences of the mutant genes were completely concordant with experiment design. Conclusion Lamivudine drug-resistanse recombinant plasmids were successfully constructed, whieh can be used in further research.