冻干重组人三突变型低氧诱导因子-1α腺病毒的制备

Preparation of freeze-dried recombinant adenovirus expressing hypoxia inducible factor-1 alpha of triple mutant

目的:研制冻干重组人三突变型低氧诱导因子-1α(HIF-1α)腺病毒。方法:将重组人三突变型HIF-1α腺病毒与不同配比的保护剂按适当比例混合,进行冻干,根据冻干后外观、病毒滴度测定、热稳定性试验、PCR、基因测序等结果,筛选冻干保护剂并评价冻干品质量。结果:冻干腺病毒所携带的目的基因信息无丢失或变异;以10%海藻糖、0.5%明胶、3%山梨醇等成分配制的保护剂作用较好,冻干后腺病毒感染性滴度下降0.33LgPFU/mL;37℃放置3周,滴度下降0.8LgPFU/mL。结论:以合适的保护剂制备冻干重组人三突变型HIF-1α腺病毒能达到较满意的效果。

Objective To prepare freeze-dried recombinant adenovirus expressing hypoxia inducible factor-I alpha （HIF-1α） of triple mutant （Ad-H1F-1α-564/402/803）. Methods Ad-HIF-1α-564/402/803 were mixed with different stabilizers in an appropi：iate propollion and then lyophilized. The optimum stabilizer was selected and the product quality was evaluated according to appearance, virus titer, thermostability, PCR and DNA sequence analysis. Results PCR and gene sequence suggested the correct construction of freeze-dried recombinant adenovirus. The protective agent containing 10% trehalose, 0.5% gelatin, 3% sorbitol had better protecting effects, After lyophilization, the infectious titer of adenovlrus was decreased by 0.33 LgPFU/mL, the titer of lyophilized adenovirus was decreased by 0.8 LgPFU/mL for 3 weeks at 37℃. Conclusions When prepared with proper stabilizer, the freeze-dried recombinant adenovirus expressing HIF-1α of triple mutant can have a good performance.