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;朱颜鑫;赵兵兵;智妍;兰露莎;江滟;
Author：
收稿日期： 年卷（期）页码：2017,32(03):-266-269
期刊名称：华西药学杂志
Journal Name：WEST CHINA JOURNAL OF PHARMACEUTICAL SCIENCES
关键字：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
Key words：
基金项目：贵州省科教青年英才培养工程项目[黔省专合字(2012)161号]
中文摘要
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。
参考文献
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