AQP5重组腺病毒载体构建及其临床作用

目的 构建AQP5的重组腺病毒载体,并验证其在干燥综合征中的治疗作用.方法 采用RT-PCR方法扩增AQP5基因,经酶切插入穿梭质粒pAdTrack-CMV,形成重组质粒pAdTrack-CMV-AQP5.经Pmel酶切线性化后与pAdeasy-1同源重组,重组质粒pAdeasy-AQP5经Pacl酶切,经脂质体转染入HEK293细胞中包装获得病毒.经过体外检测确认病毒表达后,于NOD小鼠颌下腺周注射病毒,观察其对唾液分泌量的影响.结果 经PCR和测序证实重组pAdeasy-AQP5质粒构建成功.并可见其在293细胞中表达;初步实验研究发现,其能缓解NOD小鼠的唾液腺分泌功能异常.结论 成功构建了AQP5的重组腺病毒载体,初步证实了pAdeasy-AQP5在干燥综合征中的治疗作用.

Construction of recombinant adenovirus vector of AQP5 gene

Objective To construct recombinant adenovirus vector of AQP5 gene. Methods Mouse AQP5 cDNA was amplified by RT-PCR and cloned into shuttle vector pAdTrack-CMV; the recombinant plasmid pAdTrack-CMV-AQP5 was linealized with Pmel and recombined with pAdeasy-1. The identified recombinant adenovirus plasmid pAdeasy-AQP5 was digested with Pacl and transfected to 293 cells to pack adenovirus particles. The recombinant adenovirus production was injected into NOD mice at submandibular gland and the secretion volume of saliva was measured. Results pAdeasy-AQP5 was constructed successfully; and fluorescent microscopy showed its expression in 293 cells. Injection of recombinant adenovirus in NOD mice increased the secretion of saliva. Conclusion The recombinant adenovirus vector of AQP5 gene has been constructed successfully, and in vivo study indicates that it may have potential therapeutic effect for Sicca syndrome.