两种萤火虫荧光素酶活性检测方法的一致性比较  

The Agreement Study of the Different Methods Used for Detecting the Firefly Luciferase Activity

作　　者：孙志东[1] 孙萍[1] 付秋霞[1] 王怡[1] 周勇[1] 彭剑淳[1] 詹林盛[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《生物技术通讯》2008年第1期80-83,共4页Letters in Biotechnology

摘　　要：目的:比较2种萤火虫荧光素酶活性检测方法的一致性。方法:分别采用化学发光技术(Che)及活体光学成像技术(Bio),从细胞和动物水平检测在转染以萤火虫荧光素酶为报告基因的载体pCI-AAA-Fluc-neo后不同时间点,萤火虫荧光素酶的表达强度。结果:在细胞和动物水平,萤火虫荧光素酶的表达强度均随时间推移逐步递减。在HepG2细胞,萤火虫荧光素酶表达持续96 h,活性从24 h的2781±220 mV(1.6×106±2.3×105光子)降至96 h的49±3.5 mV(6.4×104±2.5×104光子)。在动物水平得到相似的结果,BALB/c小鼠萤火虫荧光素酶表达持续20 d,其活性从1 d的16592±409 mV(1.9×108±3.6×106光子)降至20 d的798±139 mV(3.37×105±3.8×104光子)。通过一致性检验,2种检测方法在细胞和动物水平的直线回归方程分别为lgChe=1.186.lgBio-3.764(r=0.937,P<0.001)和lgChe=0.451.lgBio+0.64(r=0.915,P<0.001);进一步将理论数据与实验数据进行配对t检验,二者无统计学差异(P>0.05)。结论:2种检测方法是一致的;从整个实验过程来看,活体光学成像技术较化学发光法更为简便、直观,可量化地对同一个体连续检测,减少了个体间差异和实验动物用量。Objective： To study the agreement between chemiluminescence assay（Che） and bioluminescence imaging（Bio） used for detecting the firefly luciferase（Fluc） activity. Methods： The methods of chemiluminescence assay and bioluminescence imaging were used to detect the Flue expression level at series time points after transfected with the vector pCIAAA-Fluc-neo in which Fluc was as the report gene. Results： The results indicated that the Flue expression level was decreasing as time going on. In HepG2 cell, the expression of Flue lasted 96 h, the activity of Flue decreased from 2781±220 mV（1.6×10^6±2.3×10^5 photons） at 24 h to 49±3.5 mV（6.4×10^4±2.5×10^4 photons） at 96 h. Similarly in vivo, the expression of Flue of mice lasted 20 d, the activity of Flue decreased from 16592±409 mV（1.9×10^8±3.6×10^6 photons） at 1 d to 798±139 mV（3.37×10^5±3.8×10^4 photons） at 20 d. Through the agreement study, the line regression equations between the two methods at different levels were gotten lgChe=1.186·1gBio-3.764（r=0.937, P〈0.001, cell level） and lgChe=0.451·1gBio＋0.64（r=0.915 P〈0.001, animal level）. Further t-test showed no significant between theory data（gotten from the line regression equations） and experimental data （P〉0.05）. Conclusion： The two methods are concordance with each other. Comparing the two methods, bioluminescence imaging is more convenient and direct. It also can detect the same individual continuously so as to greatly decrease the difference between individuals and usage amounts of animals.

关 键 词：活体光学成像 化学发光 萤火虫荧光素酶 

分 类 号：Q78[生物学—分子生物学] Q503