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

doi:10.7506/spkx1002-6630-201309037

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

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.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. 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/Abstract

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

中图分类号:

Q786

陈明1,2，徐幸莲2，周光宏2，汤晓艳1,*，袁飞3，陈爱亮1. 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[J]. 食品科学, 2013, 34(9): 180-184.

CHEN Ming1,2，XU Xing-lian2，ZHOU Guang-hong2，TANG Xiao-yan1,*，YUAN Fei3，CHEN Ai-liang1. Expression, Purification and Polyclonal Antibody Preparation of the Flagellin Gene fliC of Salmonella typhimurium[J]. FOOD SCIENCE, 2013, 34(9): 180-184.

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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

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

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