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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 clone the human TRX1 gene and construct its prokaryotic expression vector to obtain rh TRX1 and evaluate the effect of rh TRX1 on human umbilical vein endothelial cells (HUVEC) induced by high glucose The protective effect. Methods The hTRX1 gene fragment was amplified by RT-PCR and inserted into p ET22b (+) plasmid and transformed into E.coli Rosetta-gami (2) to obtain the engineered bacteria Rosetta-gami (2) -p ET22ab (+) / h TRX1. The correctness of the recombinant protein was confirmed by SDS-PAGE electrophoresis and Western blot, and the recombinant protein was purified by nickel affinity chromatography. The HUVEC injury model was prepared by high glucose. The survival rate of HUVEC was determined by MTT colorimetric assay. The leakage rate of lactate dehydrogenase (LDH) and the level of nitric oxide (NO) in HUVEC were determined by biochemical methods. Results Construction of engineered bacteria and their recombinant protein rh TRX1 was correct. Compared with the normal control group, the survival rate of HUVEC in high glucose group was decreased, the rate of LDH leakage was significantly increased, and the level of NO was significantly decreased. The survival rate of HUVECs in different doses of rh TRX1 treatment group increased, the leakage rate of LDH decreased significantly, the level of NO increased significantly. Conclusion rh TRX1 was successfully cloned and expressed in E. coli. Rh TRX1 with the correct structure was obtained. Rh TRX1 could protect HUVEC induced by high glucose.