摘要
为研究苦荞金属硫蛋白(FtMT)的生物学功能,制备具有穿膜活性的重组FtMT。采用基因重组技术,分别将FtMT和融合穿膜肽的Tat-FtMT、11R-FtMT编码基因定向克隆至碱性磷酸盐启动子(phoA)分泌型原核表达载体,转化大肠杆菌BL21(DE3)。通过低磷酸盐诱导重组蛋白可溶表达后,经镍柱亲和层析分离纯化,利用SDS-PAGE、Western blot和紫外光谱扫描分析、鉴定。采用电感耦合等离子原子发射光谱仪(ICP-OES)和比色法分析重组FtMT蛋白金属结合特性和羟自由基清除活性。采用免疫荧光法检测穿膜肽介导的FtMT蛋白的穿膜活性,通过MTT细胞增殖试验分析穿膜肽-FtMT融合蛋白对胰岛细胞Ins-1和心肌细胞H9c2在高糖、高脂及缺氧毒性条件下氧化损伤的保护作用。结果表明,获得纯度较高的可溶性FtMT、Tat-FtMT和11R-FtMT重组蛋白,3种重组蛋白具有几乎一致的金属-巯基簇特征吸收峰,具有较强的金属离子结合能力和羟自由基清除活性。穿膜活性试验证实Tat-FtMT和11R-FtMT蛋白较FtMT具有显著的跨膜转运功能(P<0.05),外源穿膜肽-FtMT融合蛋白可通过跨膜进入胞内,显著降低Ins-1和H9c2由高糖、高脂以及缺氧毒性导致的氧化损伤(P<0.05)。试验成功实现了重组穿膜肽-FtMT融合蛋白的表达,在细胞水平分析其抗氧化损伤作用,为FtMT的食药资源开发、应用奠定基础。
In order to further elucidate the physiological function of metallothionein from tartary buckwheat Fagopyrum tataricum(FtMT), recombinant FtMT and CPP-FtMT including 11R-FtMT and Tat-FtMT were produced, which were fused with cell-penetrating peptides(CPPs), by a novel secreted expression method using phoA vector in Escherichia coli(E. coli). Three types of proteins were purified by Ni2+affinity chromatography followed by induction of a low concentration of phosphate and were by identified by SDS-PAGE, Western blot and UV absorption spectrometry. Chemical and functional characteristics of three types of proteins were analyzed including by metal-binding capacity and hydroxyl radical scavenging ability. The results showed three types of proteins have almost the same motif of UV absorption spectrometry and exhibited intact metal binding ability and hydroxyl radical scavenging ability. Cell penetrating ability of CPP-FtMT proteins was investigated using FITC labeled peptide internalization into Ins-1 beta cells and H9c2 cardiomyocytes under fluorescent microscopy. Moreover, Ins-1 beta cells and H9c2 cardiomyocytes were treated with CPP-FtMT to reveal protective effects against glucolipotoxicity-induced and hyperglycemia-induced apoptosis with or without hypoxia, respectively. The results demonstrated CPP-FtMT proteins strongly protected Ins-1 beta cells against oxidative stress and apoptosis induced by glucolipotoxicity with or without hypoxia, and also protected H9c2 cardiomyocytes against hyperglycemiainduced apoptosis with or without hypoxia. These present studies provide a scientific basis for the development and application of FtMT.
作者
何永吉
李云龙
胡俊君
范晓军
李红梅
程哲
He Yongji;Li Yunlong;Hu Junjun;Fan Xiaojun;Li Hongmei;Cheng Zhe(Institute of Functional Food of Shanxi,Shanxi Agricultural University,Taiyuan 030031;Shanxi Key Laboratory of Agro-products Processing,Taiyuan 030031;College of Environmental Science and Engineering,Taiyuan University of Technology,Taiyuan 030024)
出处
《中国食品学报》
EI
CAS
CSCD
北大核心
2021年第10期76-85,共10页
Journal of Chinese Institute Of Food Science and Technology
基金
山西省自然科学基金面上项目(201901D111450)
山西省重点研发计划项目(201703D321026-1)
国家现代农业(燕麦荞麦)产业体系建设专项(CARS-07-E2)。
关键词
苦荞
金属硫蛋白
穿膜肽
抗氧化
糖尿病
Fagopyrum tataricum
metallothionein
cell-penetrating peptide
antioxidant
diabetes mellitus
