插入CpG序列的乙肝病毒核心抗原基因真核表达质粒的构建及在树突状细胞中的表达

Construction of eukaryotic expression vector containing hepatitis B virus C antigen gene and CpG motifand identification of expressed protein in dendritic cells

目的:构建pEGFP-N1/CpG-HBcAg(ISS)真核表达载体,探讨乙肝病毒核心抗原(HBcAg)在树突状细胞(DC)中的表达,为研制乙肝治疗性疫苗奠定基础。方法:根据HBcAg基因序列,设计合成两对引物,在引物中引入针对人敏感的CpG基序和不含CpG的片段,用PCR方法从慢性乙型肝炎患者血清HBV DNA中扩增出HBcAg基因片段,将扩增产物与pEGFP-N1连接,构建重组体pEGFP-N1/CpG-HBcAg,进行酶切、PCR及测序鉴定;分离人外周血单个核细胞(PBMC),体外诱导分化为DC,通过脂质体将重组质粒pEGFP-N1/CpG-HBcAg和空载体分别转染DC,Western blot检测HBcAg在DC的表达。结果:HBcAg基因体外扩增产物大小为530bp。所构建的pEGFP-N1/CpG-HBcAg经双酶切及PCR鉴定,与预期片段的大小相符。测序结果与GenBank中收录的HBcAg全长序列一致,表明pEGFP-N1/CpG-HBcAg真核表达体构建正确;PBMC体外成功刺激分化为DC,HBcAg可在DC中表达。结论:成功构建了真核重组表达载体pEGFP-N1/CpG-HBcAg,且可在DC中表达,为CpG的功能研究和乙型肝炎治疗性疫苗的研制奠定基础。

Objective： To construct the recombinant eukaryotic expression vector pEGFP-N1/CpG-HBcAg （ISS）, and to express HBcAg protein in dendritic cells, which provided the basic material for the development of hepatitis B virus remedial DNA vaccine. Methods： Two couples of primers were designed for PCR according to the known sequence of HBcAg. The CpG fragments sensitive to human and Non-CpG fragments were pulled into primers. The HBcAg gene was amplified by PCR from gehome of serumal DNA of chronic HBV patients, and PCR product was subcloncd into eukaryotic expression vector pEGFP- N1. The constructed pEGFP-N1/CpG-HBcAg was identified by restricting enzyme digestion analysis, PCR amplifying, and DNA sequencing; peripheral blood monocytes （PBMC） isolated from blood of healthy individuals and differentiated into immature DCs. Transfection of pEGFP-N1/CpG-HBcAg in dendritic cells was accomplished using lipofectamine. The expression of HBcAg protein was verified by Western blot. Results： The size of amplified HBcAg gene was 530bp. Restriction enzyme digestion, PCR amplifying and DNA sequencing confirmed that pEGFP-N1/CpG-HBcAg had been constructed successfully. The harvested fulllength sequence of HBeAg gene was identical with that registered in the GenBank. PBMC were successfully differentiated into immature DCs and HBcAg protein could express in DCs. Conclusion： The pEGFP-N1/CpG-HBcAg （ISS） has been successfully constructed, and HBcAg protein could expresse in DCs, it provides the basic material for further study the function of CpG and the devdopment of hepatitis B virus remedial DNA vaccine.