贮存温度对携带HGF基因的重组腺病毒Ad-HGF稳定性的影响

Effect of storage temperature on stability of recombinant adenovirus carrying hepatocyte growth factor gene

目的 :探讨不同温度对重组腺病毒Ad_HGF保存时间的影响。方法 :以含 10 %甘油的Hanks液保存Ad_HGF(携带人肝细胞生长因子基因 )、Ad_GFP(携带绿色荧光蛋白基因 ) ,用快速细胞病变 (CPE)法、ELISA方法、流式细胞仪分别检测在不同贮存温度 (- 6 0℃ ,- 2 0℃ ,4℃ ,2 5℃和 37℃ )、不同保存时间的重组腺病毒对其感染滴度、目的基因表达和转染细胞效率的影响。结果 :- 6 0℃保存最长时间 4 4个月 ,其转染效率、HGF蛋白表达、病毒滴度未见明显下降 ;- 2 0℃保存 12个月各指标未见明显变化 ;4℃冰箱保存 2个月 ,其各项指标已有所下降 ,但不是很明显 ,而保存 7个月时 ,其转染效率、HGF蛋白表达量及病毒滴度均明显下降 ;2 5℃和 37℃保存 ,除病毒滴度外 ,其余指标呈逐日下降趋势。结论 :该病毒保存于 - 2 0℃以下性能是稳定的 ,可以满足临床贮存和应用条件。

Objective:To investigate the effect of storage temperature and storage time on stability of recombinant adenovirus carrying hepatocyte growth factor gene (Ad-HGF).Methods:Ad-HGF and Ad-GFP samples in Hanks solution containing 10% glycerin were stored at different temperatures(-60℃,-20℃,4℃,25℃ and 37℃).The virus infectivity, gene expression and transfection efficiency of the samples were examined at different storage times by cytopathogenic effect (CPE), ELISA and flow cytometer methods. Results: There was nearly no decrease in levels of the three parameters at -60℃ for 44 months and at -20℃ for 12 months. Although the levels were reduced at 4℃ for 2 months, the reduction was not remarkable, but there was a more rapid decline in virus titer, gene expression and transfection efficiency after storing for 7 months. By storing at 25℃ and 37℃, the levels of gene expression and transfection efficiency were decreasing day by day except for infection titer.Conclusion:The activity of recombinant adenovirus was stable when it was stored at temperatures below -20℃ for a rather long time.