HBsAg及B7-1双表达重组逆转录病毒载体的构建

Construction of HBsAg and B7-1 Coexpression Ritroviral Vector

【目的】为探索共刺激分子B7 1增强HBsAg真核表达载体基因免疫的效果 ,并用于消除乙肝免疫耐受。【方法】用高保真PCR法扩增目的基因片段B7 1及带接头和启动子的IRES HBs基因片段 ,亚克隆到pBluescriptKS+载体中测序 ,再克隆到逆转录病毒载体PLXSN中。【结果】所扩增的目的基因片段经测序证实 ,未发现序列有改变。先将B7 1通过EcoRⅠ、XhoⅠ插入plxsn中 ,构建成 plxsn B7 1,再用 XhoⅠ、BamHⅠ将IRES HBs插入 ,构建成plxsn B7 1 HBs。【结论】成功构建了共表达HBsAg及B7 1抗原的重组逆转录病毒载体 ,可行体外真核基因表达 ,研究目的抗原表达的特性 ,或行基因免疫 ,研究B7 1对HBsAg免疫反应的影响 ,进一步可用于防治乙肝的研究。

Objective To investigate the enhanced role of B7-1 in gene immunization of eukaryotic vector which expressing HBsAg, and the possibility of it's use in elimination of immunotolerance to hepatitis B virus. Method B7-1 and IRES-HBs with linker and promoter sequence prior to HBs gene were amplified with high fidelity PCR, then subcloned into plasmid pBluescript KS +, which was used to sequence. The eukaryotic expression vector was constructed by inserting B7-1 and IRES-HBs into retroviral vector plxsn. Result No change was found in the sequences of target genes amplified by high fidelity PCR compared with the known sequences. Plxsn-B7-1-HBs was constructed by first inserting B7-1 into plxsn with EcoRⅠ/XhoⅠ, then IRES-HBs was inserted in the same way with XhoⅠ/BamHⅠ. Conclusion The retroviral vector which expressing B7-1 and HBsAg simultaneously was constructed successfully. It may be used to study the character of target genes expression in cell lines in vitro. Also it may be used to conduct gene immunization and investigate the role of B7-1 on HBsAg immune response, which may be further used for prevention and therapy of hepatitis B.