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

作　　者：陈明[1,2] 徐幸莲[2] 周光宏[2] 汤晓艳[1] 袁飞[3] 陈爱亮[1] 

机构地区：[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

出　　处：《食品科学》2013年第9期180-184,共5页Food Science

基　　金：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)

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。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.

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 

分 类 号：Q786[生物学—分子生物学]