食品科学 ›› 2013, Vol. 34 ›› Issue (9): 180-184.doi: 10.7506/spkx1002-6630-201309037

• 生物工程 • 上一篇    下一篇

鼠伤寒沙门氏菌鞭毛蛋白FliC的原核表达、纯化及其多克隆抗体的制备

陈 明1,2,徐幸莲2,周光宏2,汤晓艳1,*,袁 飞3,陈爱亮1   

  1. 1.中国农业科学院农业质量标准与检测技术研究所,农业部农产品质量安全重点实验室,北京 100081;
    2.南京农业大学食品科技学院,肉品加工与质量控制教育部重点实验室,江苏 南京 210095;
    3.中国检验检疫科学研究院,北京 100123
  • 收稿日期:2012-05-10 修回日期:2013-04-10 出版日期:2013-05-15 发布日期:2013-05-07
  • 通讯作者: 汤晓艳 E-mail:txycaas@126.com
  • 基金资助:

    国家现代农业产业技术体系建设专项(CARS-42);国家自然科学青年基金项目(31000799);
    “十二五”国家科技支撑计划项目(2012BAD28B03)

Expression, Purification and Polyclonal Antibody Preparation of the Flagellin Gene fliC of Salmonella typhimurium

CHEN Ming1,2,XU Xing-lian2,ZHOU Guang-hong2,TANG Xiao-yan1,*,YUAN Fei3,CHEN Ai-liang1   

  1. 1. Key Laboratory of Agri-food Safety and Quality, Ministry of Agriculture, Institute of Quality Standards and Testing Technology
    for Agri-products, Chinese Academy of Agricultural Sciences, Beijing 100081, China;2. Key Laboratory of Meat Processing and
    Quality Control, Ministry of Education, College of Food Science and Technology, Nanjing Agricultural University,
    Nanjing 210095, China;3. Chinese Academy of Inspection and Quarantine, Beijing 100123, China
  • Received:2012-05-10 Revised:2013-04-10 Online:2013-05-15 Published:2013-05-07
  • Contact: TANG Xiao-yan E-mail:txycaas@126.com

摘要:

利用PCR扩增出鼠伤寒沙门氏菌鞭毛蛋白基因fliC,连接到原核表达载体pET28a(+)上,测序鉴定后转化至大肠杆菌BL21(DE3)pLysS感受态细胞中,构建了原核表达系统。经1mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导及Ni-NTA纯化后,得到了带6×His纯化标签的分子质量大小约为54.6kD的表达产物,和预测蛋白大小一致,且大部分表达产物以可溶形式存在? 利用Western-blotting进一步鉴定,结果表明,该蛋白能与抗 His 标签的单抗发生特异性反应。用纯化的融合蛋白免疫BALB/c小鼠,获得多克隆抗体,并对抗血清的效价和特异性进行检测,结果表明,多抗的效价较高,特异性较好。

关键词: 鼠伤寒沙门氏菌, 鞭毛蛋白FliC, 原核表达, 纯化, 鉴定, 多克隆抗体

Abstract:

In this study, the flagellar gene fliC of Salmonella typhimurium was amplified by PCR from the Salmonella
genome, and cloned into the prokaryotic expression vector pET28a(+). After being sequenced, the recombinant plasmid
pET28a-fliC was transformed into strain BL21(DE3)pLysS, and FliC protein was expressed by the recombinant strain after
IPTG(1 mmol/L) induction. By Ni-NTA purification, the 6×His-tagged proteins were extracted. The molecular weight of
the induced protein was about 54.6 kD as expected. And most of the expressed products were soluble. Western-blotting
analysis indicated that the expressed proteins had a specific reactivity with the monoclonal anti-His-Tag antibody. Polyclonal
antibodies were obtained via immunizing BALB/c mice with the purified protein, and the results showed that the polyclonal
antibodies had high titer and good specificity.

Key words: Slamonella typhimurium, flagellar, prokaryotic expression, purification, identification, polyclonal antibodies

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