重组R/Q亚型人血清对氧磷酶1的制备和两者解毒效应比较

Preparation of recombinant human serum paraoxonase 1 R/Q isoenzymes and comparison of their detoxication effect

目的通过基因重组的方法制备人血清对氧磷酶1的R/Q亚型同工酶(rhPON1R/Q192),并比较两种酶作为有机磷中毒解毒剂的效果差异。方法通过昆虫杆状病毒蛋白表达体系获得rhPON1R/Q192。将30只60日龄的雄性昆明种小鼠随机分成染毒对照组、染毒治疗A组、染毒治疗B组,每组10只。将对氧磷按2倍LD50(1.72 mg/kg)的剂量,对各组小鼠采用经口灌胃的方式染毒。而后立即将纯化的rhPON1R192和rhPON1Q192作为解毒剂,按10 U/kg的剂量采用腹腔注射方式分别予A、B染毒治疗组小鼠。连续观察12 h,记录并比较各组小鼠中毒指标的差异,评价rhPON1R/Q192的解毒效果。结果通过基因重组的方法可以大量获得具有对氧磷水解活性的rhPON1R/Q192,其中rhPON1R192的产率为8.34%,比活性为1552.31 U/mg;rhPON1Q192的产率为10.60%,比活性为581.60 U/mg。对照组小鼠在观察期内均出现了中毒症状,死亡率为100%。染毒治疗A、B组小鼠出现中毒症状、De Bleeker评分达到3分及死亡时间均明显延迟(P<0.05);两组的死亡率分别是10%和20%,较对照组明显下降(P<0.05)。A组的各项指标均明显好于B组(P<0.05)。结论通过基因重组方法获取的rhPON1R/Q192,可用作对氧磷中毒的解毒剂,且R亚型同工酶解毒效果更好。

Objective To prepare the isoenzymes R/Q of human serum paraoxonase 1 with polymorphism in 192 locis by gene recombination,and compare their detoxification effects of against organophosphorus.Methods rhPON1R/Q192 was obtained by insect baculovirus protein expression system.Thirty KM mice of 60 days old were randomly divided into three groups:exposure control group,group A(rhPON1R192 treatment group),and group B(rhPON1Q192 treatment group),10 mice in each group.At the dosage of 2 times LD50(1.72 mg/kg),the mice in exposed groups were exposed to paraoxon by intragastric administration,the purified rhPON1R/Q192 was immediately given to the exposed mice by intraperitoneal injection with 10 U/kg of rhPON1R192 or rhPON1Q192.Continuously observe the manifestation of the mice for 12 hours to compare detoxification effect of two isoenzymes.Results The results showed that rhPON1R/Q192 with paraoxonase activity could be obtained in large quantities by Bac-to-Bac method;the yield of rhPON1R192 was 8.34%and its specific activity reached 1552.31 U/mg;the yield of rhPON1Q192 reached 10.60%and its specific activity was 581.60 U/mg.The rhPON1R192 and rhPON1Q192 both have certain detoxification effect against paraoxon,the effect of rhPON1R192 was better than that of rhPON1Q192,near that of natural PON1 isoenzymes with polymorphism in 192 locus.All the exposure controls had poisoning symptoms during observation period,and the mortality rate was 100%;while the onset time of poisoning symptoms in groups A and B were significantly delayed,similarly,the time of death and the time of De Bleeker score reaching three were all delayed,compared with exposure control group;additionally,the mortality rates of them were significantly reduced as well.The comparison of detoxication effect between group A and B showed that rhPON1R192 was better than that of rhPON1Q192.Conclusion The results suggest that rhPON1R/Q192 has good hydrolytic activity on paraoxon,thereby may be used as an antidote in organophosphorus poisoning,and rhPON1R192 has better detoxication effect on organophosphorus.