High throughput in vitro screening method for botulinum neurotoxin type A based on FRET technology
10.19405/j.cnki.issn1000-1492.2023.01.004
- Author:
Sen Luo
1
;
Lina Guo
1
;
Tao Li
2
;
Qin Wang
2
;
Hui Wang
2
Author Information
1. Dept ofPathogenic Microbiology and Immunology,Zunyi Medical and Pharmaceutical College , Zunyi 563006
2. State Key Laboratory ofPathogens and Biosecurity, Institute ofMicrobiology and Epidemiology, Academy ofMilitary Medical Science , Beijing 100071
- Publication Type:Journal Article
- Keywords:
FRET;
botulinum toxin;
high throughput screening
- From:
Acta Universitatis Medicinalis Anhui
2023;58(1):15-21
- CountryChina
- Language:Chinese
-
Abstract:
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 proteinwas detected by digestion of botul inum 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 realtime 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 - 32 μmol/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 successfully constructed. Key words FRET;botulinum toxin;high throughput screening
- Full text:2024070419002477315基于FRET构建A型肉毒毒素活性检测的高通量方法_罗森.pdf
