重组猪尿酸氧化酶基因突变及其对聚乙二醇化的影响

Recombinant pig uric acid oxidase gene mutation and itseffect on PEGization

目的对重组猪尿酸氧化酶(recombinant porcine urate oxidase,r PUOX)进行突变改造,以增加rPUOX上可供聚乙二醇(polyethylene glycol,PEG)耦合的赖氨酸(Lys)残基数,探究PEG修饰对改造前后的酶液在SPF鸡体内的药效和抗原屏蔽作用的影响。方法对纯化的基因改造前后的r PUOX进行PEG化修饰,修饰蛋白静脉注射SPF鸡,共4次,每次间隔7 d。实验分为20×5K PEG-rPUOX-215组、20×5K PEG-rPUOX组和空白对照组(0.9%生理盐水)。于鸡翼根静脉处采血,用血糖尿酸测试仪及尿酸测试条测定尿酸浓度,酶反应-紫外分光光度法检测尿酸酶活性,间接ELISA法检测免疫原性。结果 20×5K PEG-r PUOX-215和20×5K PEG-rPUOX均获得充分修饰,且前者修饰效果更好。第1、2次注射,降低鸡血尿酸幅度20×5K PEG-rPUOX-215组高于20×5K PEG-rPUOX组约11%和6%。3次注射药效维持时间20×5K PEG-rPUOX组相较于20×5K PEG-rPUOX-215组逐次降低0、24和48 h。20×5K PEG-rPUOX-215组抗体产生速率相对较慢,且14 d抗体阳性率低于20×5K PEG-rPUOX组约14.3%。结论 20×5K PEG-rPUOX-215修饰后药效学作用更显著,药物半衰期延长,且免疫原性相对较低,为研制低免疫原性的rPUOX及实现临床上多次给药提供了新的方法和思路。

Objective To modify recombinant porcine urate oxidase(rPUOX)by gene mutation so as to increase the lysine(Lys)residues which may be coupled with polyethylene glycol(PEG),and investigate the influences of PEG modification on the efficacies and antigen shielding of rPUOX and rPUOX-215 in SPF chickens.Methods Purified rPUOX before and after gene mutation were modified with PEG,with which SPF chickens were injected i.v.for 4 times each at an interval of 7 d.The chickens were divided into 20×5 K PEG-r PUOX-215,20×5 K PEG-rPUOX and blank control groups(0.9%normal saline).The venous blood samples of chickens were collected,and determined for uric acid(UA)concentration by blood glucose and uric acid tester and uric acid test strip,for uric acid enzyme activity by enzymatic reaction-ultraviolet spectrophotometry,and for immunogenicity by indirect ELISA.Results Both PEG-rPUOX-215 and PEG-rPUOX were fully modified,while the former was better decorated.Compared with those in 20×5 K PEGr PUOX group,the chicken blood uric acid concentrations in 20×5 K PEG-r PUOX-215 group after the 1 st and the 2 nd injections decreased by about 11%and about 6%respectively.However,and durations of efficacy of 20×5 K PEGr PUOX after the 1 st,2 nd and 3 rd injections were 0,24 and 48 h shorter than those of 20×5 K PEG-rPUOX-215.The antibody production rate in 20×PEG-r PUOX-215 group was relatively low,and the 14 d antibody-positive rate was about14.3%lower than that in 20×PEG-rPUOX group.Conclusion The pharmacodynamic effect of 20×5 K PEG-rPUOX-215 was improved,while the half-life was prolonged,and the immunogenicity was relatively low,which provided a novel route for the development of a low immunogenic rPUOX and the realization of clinical multiple drug administration.