四川农业大学学报 ›› 2009, Vol. 27 ›› Issue (02): 218-222.

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罗曼鸡白细胞介素18基因的克隆、表达及生物学活性检测

陈红英1, 崔沛2, 崔保安1, 胡功政1, 朱前磊1, 王子馨1   

  1. 1. 河南农业大学 牧医工程学院, 河南 郑州 450002;
    2. 河南省动物疫病预防控制中心, 河南 郑州 450008
  • 收稿日期:2008-06-26 出版日期:2009-06-30 发布日期:2017-02-28
  • 通讯作者: 崔保安(E-mail:baoancui@henau.edu.cn) E-mail:baoancui@henau.edu.cn
  • 基金资助:
    国家"十一五"科技支撑计划专项(2006BAD06A08)

Cloning, Expression and Activity Detection of Chicken Interleukin-18 Mature Protein in Escherichia coli

CHEN Hong-ying1, CUI Pei2, CUI Bao-an1, HU Gong-zheng1, ZHU Qian-lei1, WANG Zi-xin1   

  1. 1. College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China;
    2. Henan Province Center for Animal Diseases Control and Prevention, Zhengzhou 450008, China
  • Received:2008-06-26 Online:2009-06-30 Published:2017-02-28

摘要: 应用RT-PCR技术从罗曼鸡脾淋巴细胞RNA中扩增鸡白细胞介素18(ChIL-18)全基因,并克隆和测序。结果表明,获得了ChIL-18全序列,其大小为597 bp。将其成熟蛋白基因(510 bp)亚克隆到原核表达载体pGEX-6P-1中,转化大肠杆菌BL21(DE3),在IPTG的诱导下表达融合蛋白(GST-ChIL-18)。SDS-PAGE可检测到分子质量为约46 kDa的融合蛋白,Western blot证实该融合蛋白可与鼠抗鸡IL-18单克隆抗体发生特异性反应。用MTT法测定表明,重组蛋白能明显促进鸡T细胞转化的活性。

关键词: 鸡IL-18, 克隆, 原核表达, 活性检测

Abstract: Chicken interleukin-18(ChIL-18) gene was amplified from total RNA extracted from Lohmann chicken splenocyte by reverse transcription-polymerase chain reaction(RT-PCR). PCR products were cloned and sequenced. The results showed that the full-length of chicken IL-18 gene was 597 bp. A prokaryotic expression plasmid of chicken IL-18, pGEX-ChIL-18, was obtained by subcloning the encoding region of the ChIL-18 mature peptide into pGEX-6P-1. The recombinant ChIL-18(rChIL-18) was expressed efficiently in pGEX-ChIL-18-transformed E. coli BL21(DE3) induced by IPTG. The fusion protein was identified by SDS-PAGE and Western blot. The results showed that fusion protein GST-ChIL-18 was about 46 kDa and could react with monoclonal antibody for mouse anti-chicken IL-18. The recombinant protein could significant promote the transformed activity of chicken T-cell by methyl thiazolyl tetrazolium(MTT).

Key words: chicken IL-18, cloning, prokaryotic expression, activity detection

中图分类号: 

  • S852.652