基于FRET技术构建破伤风毒素和B型肉毒毒素酶类抑制剂高通量体外筛选方法  

High throughput in vitro screening method for tetanus toxin and botulinum neurotoxin type B inhibitors based on FRET technology

作　　者：罗森[1] 丁朋晓[1] 李涛[2] 王琴[2] 王慧[2] 

机构地区：[1]遵义医药高等专科学校病原生物与免疫教研室,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》2018年第5期739-745,共7页Acta Universitatis Medicinalis Anhui

基　　金：遵义市科技计划项目[编号:遵市科合社字(2014)06号];国家自然科学基金(编号:31201352)

摘　　要：目的利用FRET技术构建破伤风毒素和B型肉毒毒素酶类抑制剂高通量体外筛选方法。方法构建针对破伤风毒素和B型肉毒毒素的双荧光标记底物的重组表达质粒,表达并纯化荧光标记底物重组蛋白;利用A型肉毒毒素轻链(ALc),B型肉毒毒素轻链(BLc)和破伤风毒素轻链(TLc)分别对目的蛋白进行酶切反应。对基于双荧光标记底物高通量体外筛选方法的条件进行优化。酶动力学参数分别测定TLc和BLc切割底物荧光标记底物的Km和Kcat值。结果设计并成功构建荧光标记底物的重组表达质粒。表达并纯化后获得纯度为90%左右的重组蛋白,命名为CYVAMP。利用ALc、BLc和TLc分别对CYVAMP进行酶切,CYVAMP能被BLc和TLc识别切割而不能被ALc切割。对基于双荧光标记底物高通量体外筛选方法的条件优化得到:CYVAMP较适宜的范围为0.2~32μmol/L,酶标仪滤光片灵敏度设定的较合适的设置范围在65~110;(对于96孔板)动态实时检测间隔时间2 min较为合适,检测持续时间从30 min到120 min较合适。CYVAMP在内肽酶BLc作用下随反应时间变化与荧光值528与485比值作图最大时在1.5左右,最小在0.5左右。酶动力学参数测定得到BLc酶切CYVAMP的Km和Kcat值分别为(17.6±2.6)μmol/L和(4.16±0.28)s-1。TLc酶切CYVAMP的Km和Kcat值分别为(40.8±3.5)μmol/L和(2.65±0.32)s-1。结论成功构建基于FRET技术的分析法能满足对破伤风毒素和B型肉毒毒素的检测及抑制剂的高通量体外筛选所需。Objective To construct a high-throughput screening method for tetanus toxin and botulinum neurotoxin type B inhibitors by FRET technique. Methods The plasmid was constructed based on the fluorogenic reporters of tetanus toxin and botulinum toxin type B recombinant,expression and purification of recombinant proteins fluorogenic reporters; using ALc,BLc and TLc respectively in response to the target protein. The substrate concentration and enzyme concentration,dynamic detection interval and detection duration were optimized based on fluorogenic reporters molecules. Enzyme kinetic parameters were measured for BLc cleaved substrates with fluorogenic reporters Km and Kcat values. Similarly,the Km and Kcat values of the fluorogenic reporters of TLc cleaved substrates were measured. Results Designed and constructed a fluorogenic reporters recombinant expression plasmid. After the expression and purification,a recombinant protein with a purity of about 90% was obtained and named as CYVAMP,CYVAMP was cleaved with ALc,BLc and TLc,respectively,and CYVAMP was cleaved by BLc and TLc but not by ALc. Optimization of analysis found endopeptidase fluorogenic reporters based on the substrate concentration and enzyme concentration,dynamic detection interval,duration of detection conditions： fluorogenic reporters,peptidase BLc and TLc under the action of time emission wavelength ratio（ 528/485） range was between 0. 5~ 1. 5. The suitable range of fluorogenic reporters was 0. 2 ~ 32 μmol/L; the appropriate dynamic detection interval was 2 min; the continuous detection time was 30 ~ 120 min. The Km and Kcat values of the BLc fluorogenic reporters were determined by enzyme kinetic parameters,respectively（ 17. 6 ± 2. 6） μmol/L and（ 4. 16 ± 0. 28）s^-1. Similarly,the Km and Kcat values of the TLc were（ 40. 8 ± 3. 5） μmol/L and（ 2. 65 ± 0. 32） s^-1 respectively. Conclusion The FRET-based analytical method is successfully established,which can meet the high-throughput in vitro screening of tetanus toxin and botul

关 键 词：FRET 肉毒毒素 破伤风毒素 高通量筛选 

分 类 号：R378.8[医药卫生—病原生物学]