生物学杂志

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苦参热应激蛋白HSP17.8的体外表达及生物信息学分析

  

  1. 1. 安徽中医药大学 研究生院,合肥 230012; 2. 安徽中医药大学 新安医学教育部重点实验室科研实验中心, 合肥 230038; 3. 安徽中医药大学 药学院, 合肥 230012; 4. 安徽道地药材品质提升协同创新中心, 合肥 230038; 5. 安徽中医药科学院, 合肥 230038
  • 出版日期:2018-02-18 发布日期:2018-02-18
  • 通讯作者: 吴家文,博士,教授,研究方向为中草药分子生物学,E-mail: wujiawen@ahtcm.edu.cn
  • 作者简介:廖怡,硕士,研究方向为中草药分子生物学,E-mail: LIAOYI66@hotmail.com
  • 基金资助:
    安徽省自然科学基金项目(1608085MH177);安徽省留学人员科技活动启动项目(20151x024);安徽中医药大学人才引进科研项目(2015RC002)

Characterization and expression analysis of HSP17.8 protein in Sophora flavescens Ait

  1. 1. Graduate School, Anhui University of Traditional Chinese Medicine, Hefei 230038; 2. Key Laboratory of Xin′an Medicine, Ministry of Education, Anhui University of Traditional Chinese Medicine, Hefei 230038;3. College of Pharmacy, Anhui University of Traditional Chinese Medicine, Hefei 230012; 4. Synergetic Innovation Center of Anhui Authentic Chinese Medicine Quality Improvement, Hefei 230038; 5. Anhui Academy of Chinese Medicine, Hefei 230038, China
  • Online:2018-02-18 Published:2018-02-18

摘要: 利用聚合酶链式反应从苦参植物叶片中扩增出热应激蛋白基因hsp编码区序列,并成功构建了pET22b-hsp原核表达载体,将其转入大肠杆菌BL21(DE3)表达菌株,在不同温度、时间和不同浓度的IPTG诱导下实现了HSP的体外表达,获得了hsp编码区序列,长度为498 bp,编码166个氨基酸,分子质量约17.8 ku的小分子热应激蛋白(HSP17.8)。对此蛋白的物理化学性质、同源进化树、二级结构以及三维结构等生物信息学分析表明:HSP17.8为稳定的亲水性蛋白,其具有多个N-豆蔻酰化位点、磷酸化位点和N-糖基化位点;与羽扇豆应激蛋白HSP的同源性最高,亲缘关系最近;HSP17.8蛋白的结构特点是α螺旋与β折叠依次出现,形成了β1-α1-β2-α2-β3-α3-β4-α4-β5的折叠方式,且同一方向的β折叠被α螺旋包裹在空间结构的内部,构成了立体结构的框架。首次从苦参中成功克隆热应激蛋白基因hsp17.8编码区序列,并在体外得到了其最佳的诱导表达条件,分析了HSP17.8的理化特性、进化关系及结构特点,为该蛋白结构、功能研究奠定了实验基础。

关键词: 热应激蛋白, 聚合酶链式反应, 生物信息学

Abstract: The hsp gene fragment was amplified by polymerase chain reaction (PCR) from the leaves of Sophora Flavescens Ait, and then it was successfully ligated to the prokaryotic expression vector pET22b(+) for protein expression. The expression of hsp was induced with different concentrations of IPTG at different temperature. The coding region of the hsp gene was 498 bp, encoding for a protein of 166 amino acids with an estimated molecular mass of 17.8 ku (HSP17.8). The physicochemical properties, phylogenetic tree, secondary structure and 3D model of HSP17.8 were analyzed by bioinformatics software. The results showed that HSP17.8 was a stable hydrophilic protein with multiple N-myristoylation sites, phosphorylation sites and N-glycosylation sites. The protein had the closest relationship with HSP from Lupinus angustifolius. The 3D structure of HSP17.8 folds into a helix-turn-helix motif on a five-stranded beta-sheet scaffold arranged in the order β1-α1-β2-α2-β3-α3-β4-α4-β5. The hsp17.8 was cloned from Sophorae flavescentis Ait and expressed in vitro for the first time. The study of physicochemical properties, phylogenetic relationships and structure characteristics of HSP17.8 provided a theoretical foundation for the further structural and functional researches of HSP17.8.

Key words:  stress proteins, polymerase chain reaction, bioinformatics