汉逊酵母活性及活力荧光素二乙酸酯-碘化丙啶双荧光染色检测方法的建立及应用  

Development and application of a method for determination of activity and vitality of Hansenula polymorpha by carboxyfluorescein diacetate-propidium iodide dua fluorescent staining

作　　者：王玢 徐世友 杨俊杰 周晖国 雷清 WANG Bin;XU Shiyou;YANG Junjie;ZHOU Huiguo;LEI Qing(Lanzhou Institute of Biological Products,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《中国生物制品学杂志》2023年第11期1347-1352,共6页Chinese Journal of Biologicals

基　　金：甘肃省科技重大专项(17ZD2FA007)。

摘　　要：目的建立用于检测汉逊酵母活性和活力的荧光素二乙酸酯(carboxyfluorescein diacetate,CFDA)-碘化丙啶(propidium iodide,PI)双荧光染色法。方法通过单因素试验,优化CFDA的染色时间(5、10、20、40、60、120 min)、工作液浓度(50、100、200、300μmol/L)及PI的染色时间(5、10、15、20、25 min)、工作液浓度(2、10、20、30μmol/L)。采用最适条件检测理论存活率为0、25%、50%、75%和100%的汉逊酵母样品,荧光显微镜下观察荧光信号,ImageJ软件分析灰度值;计数存活及死亡细胞个数,计算实际存活率和实际死亡率。同时分析实际灰度值、实际存活率及实际死亡率与相应理论值的相关性。采用建立的CFDA-PI双荧光染色法检测3批汉逊酵母培养物的活性及活力。结果CFDA和PI的最佳染色时间为60和5 min,最佳工作液浓度为200和2μmol/L。不同理论存活率汉逊酵母样品的实际灰度值、实际存活率及实际死亡率与相应理论值均具有显著相关性(R^(2)=0.9983~0.9992,P<0.05)。3批汉逊酵母培养物活性及活力3次检测值的CV分别为3.20%~4.03%和1.10%~2.27%。结论成功建立了用于检测汉逊酵母活性和活力的CFDA-PI双荧光染色法,可用于相关研究中汉逊酵母活性和活力的快速检测。Objective To develop a method for determination of activity and viability of Hansenula polymorpha by dual fluorescent staining with carboxyfluorescein diacetate(CFDA)and propidium iodide(PI).Methods The time durations(5,10,20,40,60 and 120 min for CFDA,5,10,15,20 and 25 min for PI)and working solution concentrations(50,100,200and 300μg for CFDA,2,10,20 and 30μmol/L for PI)for the dual fluorescent staining were optimized by single factor test.Under the optimal condition,the H.polymorpha samples at theoretical survival rates of 0,25%,50%,75% and 100% were determined,of which the fluorescent intensity was observed under fluorescent microscope,and the gray value was analyzed by ImageJ software.The live and dead cells were counted,based on which the actual survival and death rates were calculated.Meanwhile,the relationships of actual gray value,actual survival rate and actual death rate to the corresponding theoretical values were analyzed.Activity and viability of three batches of cultured H.polymorpha were detected by CFDA-PI dual fluorescence staining.Results The optimal time durations for staining with CFDA and PI were 60 and 5 min,while the optimal working solution concentrations were 200 and 2μmol/L,respectively.The actual gray value,actual survival rate and actual death rate of H.polymorpha samples at various theoretical survival rates were significantly correlated to the corresponding theoretical values(R^(2)=0.9983~0.9992,P<0.05).The CVs of activity and viability values in three detections of three batches of H.polymorpha culture were 3.20%~4.03%and 1.10%~2.27%,respectively.Conclusion The CFDA-PI dual fluorescent staining was successfully developed,which may be used for determination of activity and vitality of H.polymorpha.

关 键 词：汉逊酵母 荧光素二乙酸酯 碘化丙啶 荧光染色 活性 活力 

分 类 号：R392[医药卫生—免疫学]