中国畜牧兽医 ›› 2017, Vol. 44 ›› Issue (9): 2587-2592.doi: 10.16431/j.cnki.1671-7236.2017.09.007

• 生物技术 • 上一篇    下一篇

猪白细胞介素18基因的克隆及真核表达重组质粒的构建

石昂, 时庆贺, 李新果, 王玉国, 杨利, 李双双, 陈陆, 王川庆   

  1. 河南农业大学牧医工程学院, 郑州 450002
  • 收稿日期:2017-05-24 出版日期:2017-09-20 发布日期:2017-09-22
  • 通讯作者: 陈陆 E-mail:chenluhau@126.com
  • 作者简介:石昂(1993-),男,河南南阳人,硕士生,研究方向:分子生物学,E-mail:834476857@qq.com
  • 基金资助:

    国家自然科学基金(31272567、31772781);河南省高校科技创新人才支持计划(14HASTIT022)

Cloning and Eukaryotic Expression Plasmid Construction of Porcine Interleukin-18 Gene

SHI Ang, SHI Qing-he, LI Xin-guo, WANG Yu-guo, YANG Li, LI Shuang-shuang, CHEN Lu, WANG Chuan-qing   

  1. College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China
  • Received:2017-05-24 Online:2017-09-20 Published:2017-09-22

摘要:

为获得表达猪白细胞介素18(interleukin-18,IL-18)的真核表达重组质粒,试验通过RT-PCR从猪脾脏、肺脏和淋巴结组织中扩增猪IL-18全基因并定向克隆到真核表达载体pZJ-1,测序分析和酶切鉴定正确后,转染至293T细胞中,并通过RT-PCR和Western blotting分别在基因和蛋白水平检测IL-18的表达。结果表明,试验成功构建了真核表达重组质粒pZJ-IL-18,且可以在293T细胞中表达IL-18基因。Western blotting试验证实,猪IL-18多克隆抗体能与约17 ku的表达产物发生特异性反应,表明IL-18能正确表达且具有反应原性。本试验构建了表达猪IL-18基因的真核表达质粒,并在293T细胞中瞬时表达,为进一步研究IL-18的功能奠定基础。

关键词: 猪; IL-18基因; 真核表达载体; 克隆

Abstract:

To obtain recombinant eukaryotic expression plasmid of porcine interleukin-18(IL-18), the whole gene of porcine IL-18 gene was amplified from porcine spleen, lung and lymph nodes by RT-PCR and cloned into eukaryotic expression vector pZJ-1. The recombinant expression plasmid pZJ-IL-18 were identified by enzyme digestion and sequencing analysis, and was transfected into 293T cells.The expression of IL-18 was detected by RT-PCR and Western blotting in both gene and protein levels. The results showed that the eukaryotic expression plasmid of porcine IL-18 was constructed and could express transiently in 293T cells. Western blotting result confirmed that porcine IL-18 polyclonal antibody could react specifically with approximately 17 ku expression products,and indicated that IL-18 could express correctly and be responsive.This study constructed the eukaryotic expression plasmid of porcine IL-18 gene which could express transiently in 293T cells, and laid the foundation for studying function of IL-18.

Key words: porcine; IL-18 gene; eukaryotic expression vector; clone

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