生物学杂志 ›› 2019, Vol. 36 ›› Issue (6): 42-.doi: 10.3969/j.issn.2095-1736.2019.06.042

• 研究报告 • 上一篇    下一篇

黄鳝白细胞介素1β基因在大肠杆菌中的重组表达及活性分析 #br#

  

  1. 长江大学 生命科学学院, 荆州 434025
  • 出版日期:2019-12-18 发布日期:2019-12-12
  • 通讯作者: 李伟,博士,副教授,研究方向为鱼类分子生物学, E-mail: wetli@yangtzeu.edu.cn
  • 作者简介:杨龙,硕士研究生,研究方向为水生生物学,E-mail: 2656194281@qq.com
  • 基金资助:
    湖北省科技创新创业服务能力建设专项项目(2017BEC006)

Prokaryotic expression and bioactivity analysis of IL-1β in swamp eel #br#

  1. College of Life Sciences, Yangtze University, Jingzhou 434025, China
  • Online:2019-12-18 Published:2019-12-12

摘要:

白细胞介素1β(IL-1β)在免疫调节和炎症反应中起着重要作用。为了解黄鳝IL-1β基因的功能,以黄鳝肝脏cDNA为模板,PCR扩增IL-1β基因后将其亚克隆至pET28a表达载体中。将序列测定验证过的重组质粒转化大肠杆菌BL21(DE3),筛选阳性重组菌进行IPTG诱导后Ni2+亲和柱分离纯化获得重组蛋白rIL-1β;用SDS-PAGE电泳和Western Blot检测rIL-1β。按2 μg/kg剂量对黄鳝进行腹腔注射rIL-1β,用荧光定量PCR分析注射rIL-1β对肝脏IL-1β、NF-κB、AKR和Nrf2等基因表达的影响。结果表明:成功构建了黄鳝IL-1β基因的原核表达载体并实现了重组蛋白的分离纯化;重组的rIL-1β蛋白具有较好的生物活性,腹腔注射rIL-1β显著提高了肝脏IL-1β和NF-κB基因的表达水平,却对AKR和Nrf2基因的表达有明显的抑制作用。研究为进一步深入探索鱼类IL-1β基因在免疫系统中的功能提供了参考资料。


关键词: 白细胞介素1β, 黄鳝, 活性分析, 重组表达

Abstract:

IL-1β plays crucial roles in mediating immune regulatory and inflammatory responses. In order to investigate the biological function of swamp eel IL-1β, gene-specific primers were synthesized and used for amplifying the cDNA fragments. Subsequently, the purified fragments were subcloned into pET-28a. The recombinant vector was sequenced and transformed into BL21(DE3). In addition, a recombinant strain was induced with IPTG and then Ni-NTA affinity chromatography was performed to isolate the rIL-1β protein. Moreover, SDS-PAGE and Western blotting were carried out to analyze the recombinant protein. Finally, qRT-PCR was performed to investigate the effects on expression of several genes in liver after intraperitoneal injection of rIL-1β at a dose of 2 μg/kg. Results displayed that the prokaryotic expression vector was successfully constructed as well as rIL-1β protein was efficiently isolated with obvious activity. Intraperitoneal injection of rIL-1β upgraded the expression of IL-1β and NF-κB and inhibited the expression of AKR and Nrf2 in liver of swamp eel. All above results provided us a good opportunity to further our study on the function of fish IL-1β in immune system.


Key words: IL-1β, swamp eel, bioactivity analysis, recombinant expression

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