文章摘要
薛 一,孙 静,刘梦兰,黄 琼,杜新丽,张日华,刘 云.S100A16基因的原核表达和多克隆抗体制备[J].南京医科大学学报,2012,(10):1376~1380
S100A16基因的原核表达和多克隆抗体制备
Prokaryotic expression of S100A16 and preparation of its polyclonal antibody
投稿时间:2012-05-07  
DOI:10.7655
中文关键词: S1000A16  原核表达  多克隆抗体
英文关键词: S100A16  prokaryotic expression  polyclonal antibody
基金项目:江苏省科技支撑项目(BE2011802);南京医科大学第一附属医院创新团队工程;上海市糖尿病重点实验室开放课题(SHKLD-KF-1105)
作者单位
薛 一 南京医科大学第一附属医院老年医学内分泌科,江苏 南京 210029 
孙 静 南京医科大学第一附属医院老年医学内分泌科,江苏 南京 210029 
刘梦兰 南京医科大学第一附属医院老年医学内分泌科,江苏 南京 210029 
黄 琼 南京医科大学第一附属医院老年医学内分泌科,江苏 南京 210029 
杜新丽 南京医科大学第一附属医院老年医学内分泌科,江苏 南京 210029 
张日华 南京医科大学第一附属医院老年医学内分泌科,江苏 南京 210029 
刘 云 南京医科大学第一附属医院老年医学内分泌科,江苏 南京 210029 
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中文摘要:
      目的:构建S100A16原核表达载体,制备S100A16蛋白多克隆抗体并初步鉴定?方法:RT-PCR扩增得到小鼠肝脏组织的S100A16基因,将此片段克隆到带有His标签的原核表达载体pET-28a多克隆位点,并转化大肠杆菌BL21(DE3)?IPTG诱导融合蛋白表达,以亲和层析的方法纯化His-S100A16融合蛋白?纯化蛋白经SDS-PAGE电泳鉴定后,免疫新西兰白兔制备抗血清?分别采用ELISA和Western blot方法检测抗体效价和特异性?结果:经双酶切和核酸序列分析证实成功构建pET-28a-S100A16原核表达质粒?考马斯亮兰染色结果证实IPTG可有效诱导融合蛋白表达?用纯化融合蛋白免疫新西兰白兔得到的S100A16抗体血清,经ELISA和Western blot检测结果显示该抗体效价高,特异性好?结论:构建带有His标签的S100A16原核表达载体,并获得高纯度His-S100A16融合蛋白及其多克隆抗体,为进一步研究S100A16生物学功能奠定基础?
英文摘要:
      Objective:To construct the prokaryotic plasmid expressing the S100A16 and prepare the rabbit polyclonal antibody against S100A16. Methods:S100A16 gene was amplified by RT-PCR and cloned into the expression vector pET-28a,then the recombinant plasmid was transformed into E. coli BL21 (DE3) and induced to express recombinant protein with IPTG. The fusion protein was further purified by affinity chromatography and identified by SDS-PAGE. The rabbit was immunized with fusion protein to produce polyclonal antibody,and the sensitivity and specificity of the antibody was evaluated by enzymelinked immunosorbent assay (ELISA) and Western blot. Results:The recombinant expression plasmid has been successfully constructed,which was confirmed by the restriction enzyme digestion and DNA sequencing analysis. The recombinant protein could be expressed effectively,and the antibody was specific and effective. Conclusion:The S100A16 prokaryotic expression plasmid was constructed,and polyclonal antibody directed against this protein has been successfully prepared,which will provide a useful tool for the further research of S100A16.
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