Heterologous expression and purification of organophosphorus hydrolases and assessment of ability to resist ethyl paraoxon poisoning
10.3867/j.issn.1000-3002.2024.09.004
- VernacularTitle:有机磷水解酶异源表达纯化及抗乙基对氧磷中毒能力初步评价
- Author:
Ming MA
1
,
2
;
Yanan ZHAI
;
Shunye WANG
;
Zhonghua ZHANG
;
Qian LI
;
Jing GAO
Author Information
1. 军事医学研究院,北京 100850
2. 中国人民解放军联勤保障部队第988医院药剂科,河南 郑州 450042
- Keywords:
organophosphorus compounds;
bioscavenger;
organophosphorus hydrolase;
ethyl paraoxon;
enzyme kinetic parameter
- From:
Chinese Journal of Pharmacology and Toxicology
2024;38(9):672-680
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE To heterologously express and purify bioscavenger organophosphorus hydrolase(OPH),and evaluate its ability to resist ethyl paraoxon poisoning in vivo.METHODS The Escherichia coli expression strain of OPH was constructed and purified by Ni-column affinity chroma-tography and gel filtration chromatography,and the purified product was identified by mass spectrometry.The enzyme activity and kinetic constants(Km,Vmax,kcat and kcat/Km)were measured using ethyl paraoxon as the substrate.Twelve SD rats were randomly divided into the experimental group and control group.The experimental group was given 1 mg·kg-1 of OPH solution by iv administration while the control group was given the same volume of normal saline.After administration,the two groups were immedi-ately sc administration with 2×LD50 ethyl paraoxon(0.86 mg·kg-1).The state of the rats was observed and the poisoning symptoms were scored.The survival rats were given 2×LD50 ethyl paraoxon every 24 h,and the survival curve and symptom score chart were drawn according to survival and poisoning symp-toms of the rats to evaluate the anti-organophosphorus poisoning ability of OPH.RESULTS The expres-sion strain of OPH in E.coli was successfully constructed.After two-step purification,a single band of OPH was obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis,indicating that OPH had high purity,and the prepared protein sequence was consistent with the target protein.For OPH,Km=7.5×10-5 mol·L-1,Vmax=2.2×10-7 mol·L-1·s-1,kcat=158.4 s-1,kcat/Km=2.1×106 L·mol-1·s-1.The rats in the control group showed obvious poisoning symptoms after being given 2×LD50 ethyl paraoxon,and all rats died with in 15 min.The rats in the experimental group did not show poisoning symptoms after the first exposure,and the poisoning symptoms gradually deepened after continuous exposure until all the rats died on the 4th day.CONCLUSION OPH with high purity is successfully prepared in this study,and OPH could effectively resist ethyl paraoxon poisoning in rats.
