基于FRET构建A型肉毒毒素活性检测的高通量方法  

High throughput in vitro screening method for botulinum neurotoxin type A based on FRET technology

作　　者：罗森[1] 郭丽娜[1] 李涛[2] 王琴[2] 王慧[2] Luo Sen;Guo Lina;Li Tao;Wang Qin;Wang Hui(Dept of Pathogenic Microbiology and Immunology,Zunyi Medical and Pharmaceutical College,Zunyi 563006;State Key Laboratory of Pathogens and Biosecurity,Institute of Microbiology and Epidemiology,Academy of Military Medical Science,Beijing 100071)

机构地区：[1]遵义医药高等专科学校细胞生物学与免疫学教研室,遵义563006 [2]军事医学科学院微生物流行病研究所、病原微生物生物安全国家重点实验室,北京100071

出　　处：《安徽医科大学学报》2023年第1期15-21,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:31201352);遵义市科技计划项目[编号:遵市科合HZ字(2019)195号]。

摘　　要：目的利用荧光共振能量转移(FRET)构建针对A型肉毒毒素酶活性检测的高通量体外方法。方法构建只识别A型肉毒毒素的基于双荧光标记底物的重组表达质粒,将其构建的质粒转入大肠杆菌表达系统进行表达,对表达后的荧光标记底物重组蛋白进行纯化和透析并保存备用;利用A型肉毒毒素轻链(ALc)对重组蛋白进行酶切检测活性;对本检测方法的条件进行优化;ALc切割荧光标记底物测定其酶动力学参数K_(m)与K_(cat)值。结果重组表达质粒构建成功,通过大肠杆菌表达系统表达后检测明显出现目的条带,对其纯化后获得的重组蛋白纯度在90%左右,命名重组蛋白为CYA。对CYA通过酶ALc、B型肉毒毒素轻链(BLc)进行酶切鉴定,结果显示CYA只能被ALc酶切产生与预期相符的两个蛋白片段,不能被BLc酶切。对基于FRET底物的条件优化得到:设定滤光片灵敏度在65~110之间;实时动态检测间隔为2 min/次,动态检测时间为30~120 min,底物CYA合适的浓度范围0.5~32μmol/L。CYA在ALc酶作用下随时时间的变化与荧光值528与485的比值作图最小在0.5左右,最大时约为0.9。酶动力学参数测定ALc切割CYA的值K_(cat)值为(5±0.4)s^(-1)和K_(m)为(2.33±0.21)μmol/L。结论成功构建了基于FRET技术的A型肉毒毒素活性检测的高通量体外分析方法。Objective To construct a high-throughput in vitro method for the detection of botulinum toxin A enzyme activity by using fluorescence resonance energy transfer(FRET).Methods Recombinant expression plasmid based on double fluorescent labeled substrate was constructed to recognize botulinum toxin type A only,and the constructed plasmid was transferred into the E.coli expression system for expression.The expressed recombinant protein of fluorescent labeled substrate was purified,dialyzed and stored for standby;The activity of the recombinant protein was detected by digestion of botulinum toxin type A light chain(ALc);The conditions of this detection method were optimized;The enzyme kinetic parameters K_(m)and K_(cat)were determined by cutting the fluorescent labeled substrate with ALc.Results The recombinant expression plasmid was successfully constructed.After being expressed in the E.coli expression system,the target band appeared obviously.The purity of the purified recombinant protein was about 90%.The recombinant protein was named CYA.CYA was identified by enzyme digestion of ALc and Botulinum toxin type B light chain(BLc).The results showed that CYA could only be digested by ALc to produce two protein fragments that were consistent with expectations,but could not be digested by BLc.By optimizing the conditions based on FRET substrate,it was obtained that the filter sensitivity was set between 65-110;The real-time dynamic detection interval was 2 min/time,the dynamic detection time was 30-120 min,and the appropriate concentration range of substrate CYA was 0.5-32μmol/L.The ratio of the time change of CYA at any time under the action of ALc enzyme to the fluorescence value 528 and 485 was plotted to be about 0.5 at the minimum and 0.9 at the maximum.The enzyme kinetic parameters determined that the value of ALC cleaving CYA K_(cat)was(5±0.4)s^(-1)and K_(m)was(2.33±0.21)μmol/L.Conclusion A high-throughput in vitro method for the detection of botulinum toxin type A activity based on FRET technology is successf

关 键 词：FRET 肉毒毒素 高通量检测 

分 类 号：R378.8[医药卫生—病原生物学]