乙肝病毒核心抗原强制泛素化DNA疫苗的构建

Construction of DNA vaccine encoding ubiquitinated HBcAg against hepatitis B virus

目的构建乙型肝炎病毒核心抗原(HBcAg)强制泛素(Ub)化的DNA疫苗表达质粒。方法以BALB/c小鼠肝脏mRNA为模板,RT-PCR扩增Ub单体基因片段,并将其羧基端第76位氨基酸突变为丙氨酸;以HBVpADR为模板,PCR扩增HBcAg基因片段,并根据N末端法则将其第1位氨基酸突变为精氨酸;利用重组PCR技术拼接Ub-HBcAg融合基因,纯化后连接pUCm-T载体,酶切鉴定正确后将Ub-HBcAg片段插入真核表达质粒pcDNA3.1(-),构建重组真核表达质粒Ub-HBcAg-pcDNA3.1(-),并行酶切鉴定和基因测序。结果构建的HBcAg强制Ub化融合基因DNA疫苗经基因测序证实目的基因插入方向正确,突变与预期相符;其余基因序列与GenBank发布序列完全一致。结论成功构建了HBcAg强制Ub化融合基因DNA疫苗。

Objective To construct DNA vaccine encoding ubiquitinated hepatitis B virus core antigen （HbcAg） against hepatitis B virus.Methods Gene encoding mutant ubiquitin （Ub） was amplified by RT-PCR from the liver of BALB/c mice,and HBcAg gene fragment was amplified from the plasmid pADR containing the genome of hepatitis B virus by PCR.Ub-HBcAg fusion gene was spliced by recombinant PCR.After purification and collection,Ub-HBcAg was connected with pUCm-T vector to construct the recombinant plasmid pUCm-Ub-HBcAg.After identification of pUCm-Ub-HBcAg by enzyme digestion analysis,Ub-HBcAg was inserted into eukaryotic expression plasmid pcDNA3.1（-） to construct the recombinant eukaryotic expression plasmid Ub-HBcAg-pcDNA3.1（-）,which was identified through sequencing.Results Gene encoding HbcAg and gene encoding mutant Ub were amplified.Ub-HBcAg fusion gene eukaryotic expression plasmid Ub-HBcAg-pcDNA3.1（-） was confirmed through sequencing.Fusion gene Ub-HBcAg was inserted into the right direction of plasmid pcDNA3.1（-）,the mutant gene fragment was confirmed to accord to expectation,and there was no other difference between the target gene and published gene in GenBank.Conclusion DNA vaccine encoding ubiquitinated HBcAg against hepatitis B virus has been successfully constructed.