Taq DNA聚合酶及Flap核酸内切酶的玻璃化室温储存方法研究

Storage of Taq DNA Polymerase and FEN1 at Room Temperature by Vitrification

研究对商品化的Taq DNA聚合酶及自表达的重组Flap核酸内切酶1(Flap endonuclease 1,FEN1)进行玻璃化的方法,实现室温(25℃)条件下储存的目的。对购置的商品化的Taq DNA聚合酶使用海藻糖作为赋形剂,对实验室自表达的FEN1使用海藻糖及普鲁兰糖作为赋形剂,采用快速降温以及真空冷冻干燥除水的方法进行玻璃化制备。产品外部放置干燥剂并密封于室温条件下存放,分别对两种玻璃化酶进行时间储存稳定性的考察,对Taq DNA聚合酶采用考察扩增效率与扩增灵敏度的方式表征酶活,对FEN1采用已建立的方法测定酶活性的绝对数值。采用玻璃化产品相对对照品随时间变化有无显著性差异来预计储存时间。时间稳定性考察说明玻璃化Taq DNA聚合酶在室温下可稳定储存至少6个月,其相对于按照说明书中-30℃存放的对照品酶的扩增灵敏度在0 d至第6个月无显著性差异(P>0.05),1年出现后延产生显著性差异(P<0.01)。玻璃化FEN1在室温下可稳定储存至少1个月,其相对于-30℃存放的对照品酶,活性比值在0 d至30 d无显著性差异(P>0.05),第2个月出现下降(P<0.01)。本文建立的方法成功制备了两种玻璃化酶,可用于室温下储存较长时间,便于运输和使用。

To study the method of vitrification of commercialized Taq DNA polymerase and self-expressed recombinant Flap endonuclease 1(FEN1),in order to achieve its room temperature(25 ° C) storage,the commercialized Taq DNA polymerase used the trehalose as excipient,trehalose and pullulan as excipient for FEN1 self-expressed in the laboratory.These two kinds of enzymes both vitrified by rapid cooling and vacuum freeze-drying dewatering.Desiccant was placed outside the product and after sealed,it was stored at room temperature.Time storage stability tests of two kinds of enzymes were investigated.The activity of Taq DNA polymerase was characterized by the efficiency and sensitivity of amplification,the value of enzyme activity was determined for FEN1 by the laboratory established method.The storage time was predicted by investigating the difference between the glass products relative to the reference substances over time,observing whether there is a significant difference.The results of time stability tests showed that the vitrified Taq DNA polymerase could be stored stably at room temperature for at least 6 months.The amplification sensitivity of the vitrified Taq DNA polymerase has no significant difference with that of the control enzyme stored at-30 °C in the instruction from0 day to 6 months(P>0.05),but after 1 year,the efficiency of amplification delayed and indicates a significant difference(**P<0.01).Vitrified FEN1 could be stored stably at room temperature for at least one month.Compared with the control enzyme stored at-30 °C,the activity ratio of vitrified FEN1 has no significant difference between 0 day to 30 days(P>0.05),but the activity ratio decreased in the second month(**P<0.01).Two kinds of vitrified enzymes have been successfully prepared by this method,which can be used for storage at room temperature for a long time,it’s convenient for transportation and application.