摘要
目的通过基因工程技术生产的豆豉血栓溶解酶,研究其安全性及溶栓功效。方法采用酪蛋白平板从豆豉中分离获得具有纤溶活性的候选菌株,利用基因工程技术将其AprE9912D基因在大肠杆菌BL21(DE3)-pDE1中过表达AprE9912D重组蛋白。经过Ni-NTA纯化柱和透析纯化AprE9912D重组蛋白,体外采用纤维蛋白平板、溶解混合血栓考察溶栓作用,体内采用动静脉旁路血栓模型进行功效评价。同时对其安全性进行初步评估。结果经过分子鉴定,筛选出具有纤溶酶活性的菌株是贝莱斯芽孢杆菌9912D(Bacillus velezensis 9912D)。序列分析发现AprE9912D基因的开放阅读框有382个氨基酸,属于S8家族碱性丝氨酸蛋白,使用SWISS-MODEL同源预测AprE9912D蛋白3D模型。经过Ni-NTA柱纯化,AprE9912D纤溶酶蛋白相对分子质量约为36000。体外测得AprE9912D纤溶酶酶活力为442.6 kU/mg,2.0 kU/mL AprE9912D纤溶酶就能有效溶解混合血栓,其溶解率为40.0%。在体内,4.0 kU/kg能显著抑制动静脉旁路血栓形成,抑制率为32.9%。体外溶血显示AprE9912D纤溶酶93.0 U/mL时,其溶血率为3.33%;测得小鼠静脉注射AprE9912D纤溶酶半数致死量(LD50)>102.6 kU/kg。结论AprE9912D纤溶酶具有活性较强、安全性较高的特点,具有进一步开发的广阔前景。
Objective To study safety and thrombolytic effect of Douchi thrombolytic enzyme produced by genetic engineering technology.Methods A candidate strain with fibrinolytic activity was isolated and obtained from Douchi by casein plate.AprE9912D gene of the candidate strain was expressed in Escherichia coli BL21(DE3)-pDE1 to produce the recombinant protein AprE9912D by genetic engineering technology.This protein was purified by Ni-NTA column and dialysis,the fibrin plate and dissolution of the mixed thrombus were using for observing the thrombolytic effect in vitro;The rat arteriovenous shunt thrombosis model in vivo was used for efficacy evaluation.At the same time,the safety was evaluated preliminarily.Results The strain with fibrinolytic activity from screening was Bacillus velezensis 9912D by molecular identification.Sequence analysis indicated that the open reading framework of AprE9912D gene had 382 amino acids,and belonged to a S8 family Alkaline serine protease;three-dimensional structure of AprE9912D protein was predicted by SWISS-MODEL.The protein molecular weight of AprE9912D fibrinolytic enzyme was 36000 after purification by Ni-NTA column.Fibrinolytic activity of AprE9912D enzyme in vitro was 442.6 kU/mg;2.0 kU/mL AprE9912D fibrinolytic enzyme could dissolve mixed thrombus effectively,and the thrombolytic ratio was 40.0%.In vivo,4.0 kU/kg AprE9912D fibrinolytic enzyme could significantly inhibit thrombosis in the rat arteriovenous shunt thrombosis model,the inhibition ratio was 32.9%.In vitro,the hemolysis test showed that when the concentration of the AprE9912D fibrinolytic enzyme was 93.0 U/mL,the hemolysis ratio was 3.33%.The median lethal dose(LD50)of the AprE9912D fibrinolytic enzyme was greater than 102.6 kU/kg by iv.Conclusion The AprE9912D fibrinolytic enzyme has stronger activity and higher safety.It would have broad prospects for further development.
作者
张秀
谭正怀
马苗苗
李晓媛
周雪
ZHANG Xiu;TAN Zheng-huai;MA Miao-miao;LI Xiao-yuan;ZHOU Xue(Sichuan Key Laboratory of TCM Quality Evaluation,Evaluation Platform of Pharmacodynamics of TCM for Major Disease Prevention and Treatment,Sichuan Academy of Chinese Medicine Sciences,Chengdu 610041,China)
出处
《中草药》
CAS
CSCD
北大核心
2021年第2期367-377,共11页
Chinese Traditional and Herbal Drugs
基金
四川省科技厅基本业务专项(A-2018N-27)
四川省中医药管理局科学技术研究专项项目(2018QN040)。
关键词
贝莱斯芽孢杆菌
过表达
安全性
溶栓活性
血栓溶解酶
基因工程技术
大肠杆菌
Bacillus velezensis
overexpression
safety
fibrinolytic activity
thrombolytic enzyme
genetic engineering technology
Escherichia coli
