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目的:构建HIV-1重组腺病毒疫苗并初步鉴定其免疫原性。方法:用Adeno-X Expression System试剂盒将HIV-1 gagpol基因片段插入到腺病毒载体上,通过脂质体介导将获得的重组腺病毒质粒Adeno-X-gagpol转染至293细胞,使之自主包装成有感染活性的重组腺病毒rAd-gagpol,用western-blotting法鉴定其表达情况;用CsCl密度梯度离心法纯化该重组腺病毒,以5×108pfu/mL的滴度1 mL单次免疫小鼠,15天后测小鼠体液免疫效果。结果:克隆序列与设计相符,重组腺病毒疫苗能稳定表达gagpol蛋白,体液免疫中检测出抗p55和p24的特异性抗体。结论:HIV-1 gagpol重组腺病毒构建成功,具有一定的体液免疫原性。
Objective: To construct HIV-1 recombinant adenovirus vaccine and preliminary identification of its immunogenicity. METHODS: HIV-1 gagpol gene fragment was inserted into adenoviral vector by Adeno-X Expression System kit. The recombinant adenovirus plasmid Adeno-X-gagpol was transfected into 293 cells by liposome mediated The recombinant adenovirus rAd-gagpol was self-packaged and identified by western-blotting. The recombinant adenovirus was purified by CsCl density gradient centrifugation and immunized with 1 mL single immunization with a titer of 5 × 108pfu / mL The mice were tested for humoral immunity after 15 days. Results: The cloned sequences were consistent with the designed ones. The recombinant adenovirus vaccine could stably express gagpol protein, and specific antibodies against p55 and p24 were detected in humoral immunity. Conclusion: The HIV-1 gagpol recombinant adenovirus has been constructed successfully and has certain humoral immunogenicity.