强制泛素化乙肝病毒核心抗原融合基因表达质粒的构建

Construction of eukaryotic expression plasmid encoding ubiquitinated hepatitis B virus core protein

目的构建强制泛素化乙肝病毒核心抗原(HBcAg)融合基因真核表达质粒。方法以HBVadr亚型全基因质粒pADR为模板,PCR扩增HBcAg基因片段;无菌操作下分离Balb/C小鼠肝脏,提取mRNA,RT-PCR扩增小鼠泛素基因片段。PCR产物经测序鉴定后双酶切,插入真核表达质粒pcDNA3.1(-),构建重组真核表达质粒Ub-HBcAg-pcDNA3.1(-)并酶切鉴定和基因测序。结果扩增的泛素基因片段和HBcAg基因片段经测序证实序列正确;将上述基因双酶切后插入质粒pcDNA3.1(-)中,构建强制泛素化HBcAg融合基因表达质粒Ub-HBcAg-pcDNA3.1(-);融合基因真核表达质粒Ub-HBcAg-pcDNA3.1(-)经酶切、基因测序等鉴定证实目的基因序列和插入方向正确,与预期结果一致。结论成功构建了强制泛素化乙肝病毒核心抗原融合基因真核表达质粒,为进一步研究强制泛素化HBcAg诱导HBV特异性CTL奠定了实验基础。

【Objective】To construct eukaryotic expression plasmid encoding ubiquitinated hepatitis B virus core antigen. 【Methods】Gene encoding hepatitis B virus core antigen was amplified from the plasmid pADR containing the genome of hepatitis B virus by polymerase chain reaction. Gene encoding mutant ubiquitin was amplified by PCR from genomic DNA obtained from the liver of Balb/C mice. Then, these gene fragments were inserted into pcDNA 3.1 （-） to construct the recombinant plasmid Ub-HBcAg-pcDNA 3.1 （-）.【Results】Gene encoding hepatitis B virus core antigen and gene encoding mutant ubiquitin were amplified. Successfully, the recombinant eukaryotic expression plasmid encoding ubiquitinated hepatitis B virus core antigen was constructed and confirmed by DNA sequence analysis. 【Conclusion】The eukaryotic expression plasmid encoding ubiquitinated hepatitis B virus core antigen was successfully constructed.