人硫氧还蛋白1在大肠杆菌中的重组表达及其对高糖导致的人脐静脉内皮细胞损伤的保护作用  

Prokaryotic expression of human thioredoxin-1 in Escherichia coli and its protective effect on the damage in human vascular endothelial cell induced by high glucose concentration in vitro

作　　者：罗红[1] 朱颜鑫 赵兵兵[2] 智妍[1] 兰露莎 江滟[2,3] 

机构地区：[1]贵州医科大学实验动物中心,贵州贵阳550004 [2]贵州医科大学附属医院微生物免疫科,贵州贵阳550004 [3]贵州医科大学附属医院临床研究中心,贵州贵阳550004

出　　处：《华西药学杂志》2017年第3期266-269,共4页West China Journal of Pharmaceutical Sciences

基　　金：贵州省科教青年英才培养工程项目[黔省专合字(2012)161号]

摘　　要：目的克隆人硫氧还蛋白1(h TRX1)基因并构建其原核表达载体,获得重组人硫氧还蛋白1(rh TRX1),评价rh TRX1对高糖导致的人脐静脉内皮细胞(HUVEC)损伤的保护作用。方法采用逆转录PCR方法扩增h TRX1基因片段,插入p ET22b(+)质粒并转化大肠杆菌Rosetta-gami(2),获得工程菌Rosetta-gami(2)-p ET22ab(+)/h TRX1。用SDS-PAGE电泳和Western blot鉴定重组蛋白的正确性,用镍亲和层析纯化重组蛋白。采用高糖制备HUVEC损伤模型,MTT比色法检测HUVEC的存活率,生化方法测定HUVEC中乳酸脱氢酶(LDH)的外漏率以及细胞上清液中一氧化氮(NO)的水平。结果构建的工程菌及其产生的重组蛋白rh TRX1正确。与正常对照组比较,高糖损伤组HUVEC的存活率降低、LDH外漏率明显升高,NO的水平明显地降低。不同剂量rh TRX1处理组HUVEC的存活率升高、LDH的外漏率明显降低,NO的水平明显地升高。结论成功克隆并在大肠杆菌中表达rh TRX1、获得结构正确的rh TRX1,rh TRX1对高糖诱导的HUVEC损伤具有保护作用。OBJECTIVE To obtain the recombinant human thioredoxin - 1 （ rhTRX1 ） peptide through prokaryotic expression system, and to evaluate the protective effect of rhTRX1 on the damage in HUVEC induced by high glucose concentration in vitro. METHODS The human thioredoxin - 1 （ hTRX1 ） gene was amplified using reverse transcription - PCR and the prokaryotic expression plasmid pET22ab（ ＋ ）/hTRX1 was constructed, and the plasmid was transferred into Escherichia coli Rosetta -gami （2）. HUVEC were divided into six groups, control group, high glucose group,low dose group, medium dose group, high dose group and rhTRX1 group. Cell viability was detected by MTT assay. The activity of lactic dehydrogenize （LDH） both in medium and cell was examined, and the content of nitric oxide （NO） in medium was measured. RESULTS The Rosetta - gami （ 2 ） - pET22ab （ ＋ ）/hTRX1 was successfully constructed.Compared with high glucose group,HUVEC viabilily and NO content of rhTRXI was increased.and tile I.DH leakage rate of rhTRX1 was decrease. CONCLUSION The recombinant human thioredoxin - 1 peptide is suec＇essfully obtained.and rhTRXI has protective effect on the damage in HUVEC induced by high glucose concentralit,n in vitro.

关 键 词：人硫氧还蛋白 基因克隆 原核表达 重组蛋白 脐静脉内皮细胞 高糖损伤 体外实验 保护作用 

分 类 号：R96[医药卫生—药理学]