摘要
为解决现有CRISPR/Cas12b核酸检测试剂需要冷链储运的问题,选取海藻糖、乳糖、蔗糖、普鲁兰多糖、酪蛋白、麦芽糊精、松三糖和甘露醇等8种常用的冻干保护剂,以真空冷冻干燥后相对酶活和物理状态为指标,通过对不同浓度保护剂的单因素试验和正交试验,筛选与优化冻干配方。结果表明,海藻糖、普鲁兰多糖和甘露醇具有较好的冻干保护性,且冻干产物物理性状较好,便于使用。最终确定的冻干配方为海藻糖0.14 g/mL,普鲁兰多糖0.06 g/mL,甘露醇0.06 g/mL。使用该配方制备的CRISPR/Cas12b冻干试剂,初始相对酶活为93.33%,在25℃保存60 d或37℃保存30 d后的相对酶活分别为81.04%和81.28%,具有良好的常温保存性,可用于进一步制备快速核酸检测试剂。
In order to solve the problem of cold chain storage and transportation of existing CRISPR/Cas12b nucleic acid detection reagents,eight commonly used cryoprotectants,including trehalose,lactose,sucrose,pullulan,casein,maltodextrin,raffinose,and mannitol,were selected.The cryoprotectants were then screened and optimized by evaluating the relative enzyme activity and physical state after lyophilizing,using different concentrations of cryoprotectants with single-factor experiments and orthogonal experiments.The results showed that trehalose,pullulan,and mannitol had good lyophilizing protections,and the physical properties of the lyophilized products were good and easy to use.The optimized lyophilized formulation was 0.14 g/mL of trehalose,0.06 g/mL of pullulan,and 0.06 g/mL of mannitol.The CRISPR/Cas12b lyophilized reagent prepared with this formula had an initial relative enzyme activity of 93.33%.After being stored at 25℃for 60 days or at 37℃for 30 days,the relative enzyme activities were 81.04%and 81.28%,respectively.It had good room temperature stability and could be used for further preparation of rapid nucleic acid detection kits.
作者
张胜胜
吴芳
罗志丹
王天琪
黄庆媛
卢辰
ZHANG Shengsheng;WU Fang;LUO Zhidan;WANG Tianqi;HUANG Qingyuan;LU Chen(Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening,Jiangsu Ocean University,Lianyungang 222005,China;Jiangsu Institute of Marine Resources Development,Lianyungang 222005,China)
出处
《江苏海洋大学学报（自然科学版）》
CAS
2023年第3期43-48,共6页
Journal of Jiangsu Ocean University:Natural Science Edition
基金
江苏省研究生科研与实践创新计划项目(KYCX2021-038)。
