【摘要】 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。更多还原

【Abstract】 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 genome of serumal DNA of chronic HBV patients,and PCR product was subcloned 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 full-length sequence of HBcAg 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 development of hepatitis B virus remedial DNA vaccine.更多还原