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  

Heterologous expression and purification of organophosphorus hydrolases and assessment of ability to resist ethyl paraoxon poisoning

作　　者：马铭 翟亚楠 王顺业 张中华[2] 李前 高静 MA Ming;ZHAI Yanan;WANG Shunye;ZHANG Zhonghua;LI Qian;GAO Jing(Academy of Military Medical Sciences,Beijing 100850,China;Department of Pharmacy,The 988th Hospital of the Joint Logistic Support Force of PLA,Zhengzhou 450042,China)

机构地区：[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

出　　处：《中国药理学与毒理学杂志》2024年第9期672-680,共9页Chinese Journal of Pharmacology and Toxicology

摘　　要：目的异源表达纯化有机磷水解酶(OPH),初步评价其动物体内抗乙基对氧磷中毒能力。方法构建OPH的大肠杆菌表达菌株,采用镍柱亲和色谱及凝胶过滤色谱进行纯化,对纯化产物进行蛋白质谱鉴定;以乙基对氧磷为底物测定酶活力及其动力学常数K_(m),V_(max),k_(cat)和k_(cat)/K_(m);12只SD大鼠随机分为OPH组和溶剂对照组,OPH组按1 mg·kg^(-1)剂量iv给予OPH溶液,对照组给予等体积生理盐水,给药后2组立刻sc给予2倍半数致死量(LD_(50))乙基对氧磷(0.86 mg·kg^(-1))染毒,观察大鼠状态,对中毒症状进行评分。对存活大鼠每隔24 h重复sc给予2倍LD_(50)乙基对氧磷,根据大鼠存活情况与中毒症状分别绘制生存曲线与症状评分图,评价OPH的抗有机磷中毒能力。结果成功构建了OPH的大肠杆菌表达菌株,经过两步纯化后由十二烷基硫酸钠-聚丙烯酰胺凝胶电泳表征得单一条带,OPH纯度较高,经蛋白质谱测定制备蛋白与目标蛋白序列一致;以乙基对氧磷为底物时,OPH的Km=7.5×10^(-5)mol·L^(-1),V_(max)=2.2×10^(-7)mol·L^(-1)·s^(-1),k_(cat)=158.4 s^(-1),k_(cat)/K_(m)=2.1×10^(6) L·mol^(-1)·s^(-1);对照组大鼠sc给予2倍LD_(50)乙基对氧磷后,出现严重中毒症状,15 min内全部死亡,实验组大鼠在首次染毒后均未出现中毒症状,连续染毒后中毒症状逐渐加重直至第4天全部死亡。结论成功制备高纯度OPH,且OPH在大鼠体内能有效对抗乙基对氧磷中毒。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(K_(m),V_(max),k_(cat) and k_(cat)/K_(m))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×LD_(50) 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×LD_(50) 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,K_(m)=7.5×10^(-5) mol·L^(-1),V_(max)=2.2×10^(-7) mol·L^(-1)·s^(-1),k_(cat)=158.4 s^(-1),k_(cat)/K_(m)=2.1×10^(6) L·mol^(-1)·s^(-1).The rats in the control group showed obvious poisoning symptoms after being given 2×LD_(50) 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 prepare

关 键 词：有机磷化合物 生物清除剂 有机磷水解酶 乙基对氧磷 酶动力学参数 

分 类 号：R99[医药卫生—毒理学]