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目的构建含HBsAg基因的重组复制缺陷型腺病毒疫苗载体,观察小鼠抗HBsAg免疫反应。方法以pEcob-6为模板,PCR扩增目的基因HBsAg,腺病毒穿梭载体PAd-track-cmv与HBsAg经T4连接酶连接,重组为PAd-track-cmv-HBs穿梭质粒;腺病毒骨架载体质粒PAd-Easy-1再与PAd-track-cmv-HBs在大肠杆菌BJ-5183中同源重组PAd-Easy-1-HBs质粒,PAd-Easy-1-HBs脂质体介导转染293细胞,包装HBsAg重组腺病毒。30只小鼠随机分成3组,分别肌肉注射PAd-Easy-1(载体组)、HBsAg重组腺病毒(实验组)、PBS(正常对照组),免疫2次。2月后取小鼠血清检测抗HBsAg抗体反应。结果PAd-track-cmv-HBs穿梭质粒经上海基康生物技术公司进行DNA测序,结果与预期结果相同,含有完整的HBsAg目的基因。PAd-Easy-1-HBs用Pac-1酶切,Pac-1能将载体切成两个片段,大片段约为30kb,小片段约为3kb。HBsAg重组腺病毒组抗HBsAg的OD值明显升高,与正常对照组比较,差异有统计学意义(P<0.05)。与PAd-Easy-1载体对照组比较,差异有统计学意义(P<0.05)。结论HBsAg腺病毒疫苗载体构建成功,且能够诱导小鼠产生抗HBsAg。
Objective To construct a recombinant replication-deficient adenovirus vaccine containing HBsAg gene and observe the anti-HBsAg immunoreactivity in mice. Methods The target gene HBsAg was amplified by PCR using pEcob-6 as a template. The adenovirus shuttle vector PAd-track-cmv and HBsAg were ligated by T4 ligase and recombined into the PAd-track-cmv-HBs shuttle plasmid. The adenoviral backbone vector plasmid PAd -Easy-1 and PAd-track-cmv-HBs in E. coli BJ-5183 homologous recombination plasmid PAd-Easy-1-HBs, PAd-Easy-1-HBs liposome mediated 293 cells transfected, packaging HBsAg recombinant adenovirus. Thirty mice were randomly divided into three groups and were intramuscularly injected with PAd-Easy-1 (vector group), HBsAg recombinant adenovirus (experimental group) and PBS (normal control group) respectively. After 2 months to take the mouse serum anti-HBsAg antibody response. Results The PAd-track-cmv-HBs shuttle plasmid was sequenced by Shanghai JIKEN Biotechnology Co., Ltd. The result was the same as expected and contained the complete HBsAg gene. PAd-Easy-1-HBs was digested with Pac-1. Pac-1 was able to cut the vector into two fragments, a large fragment of about 30 kb and a small fragment of about 3 kb. The OD value of anti-HBsAg of HBsAg recombinant adenovirus group was significantly higher than that of normal control group (P <0.05). Compared with PAd-Easy-1 vector control group, the difference was statistically significant (P <0.05). Conclusion The HBsAg adenovirus vaccine vector was successfully constructed and could induce anti-HBsAg in mice.