大肠杆菌FtsZ蛋白原核表达及多克隆抗体的制备与鉴定
Bacterial expression, preparation and identification of polyclonal antibody against Escherichia coli FtsZ
投稿时间:2019-01-06  
DOI:  10.13345/j.cjb.190011
中文关键词:大肠杆菌FtsZ蛋白,原核表达,孔雀绿法,免疫荧光,多克隆抗体
英文关键词:Escherichia coli FtsZ protein, bacterial expression, malachite green assay, immunofluorescence assay, polyclonal antibody
基金项目:国家自然科学基金 (Nos. 81503065,81703546),安徽省自然科学基金 (No. 1808085QH265),吉林省科技发展计划 (No. 20160520045JH),皖南医学院博士科研启动基金 (No. RCQD201617),皖南医学院大学生科研资助金 (No. WK2018S54) 资助。
作者单位
陈云雨 1 皖南医学院 药物筛选与评价研究所安徽 芜湖 241002 
牛夏忆 1 皖南医学院 药物筛选与评价研究所安徽 芜湖 241002 
李淼 1 皖南医学院 药物筛选与评价研究所安徽 芜湖 241002 
李霓 2 中国医学科学院-北京协和医学院 药物研究所 天然药物活性物质与功能国家重点实验室北京 100050 
刘晓平 1 皖南医学院 药物筛选与评价研究所安徽 芜湖 241002 
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中文摘要:
      为制备特异性抗大肠杆菌丝状热敏蛋白Z (Escherichia coli filamentous thermosensitive protein Z,Ec-FtsZ)多克隆抗体,将Ec-FtsZ基因进行化学合成后连接pET-22b(+) 表达载体,构建重组质粒Ec-FtsZ-pET-22b(+)。将重组质粒转化到大肠杆菌E. coli BL21(DE3) 中进行Ec-FtsZ原核表达与表达条件优化,以HisTrap层析柱进行Ec-FtsZ的分离纯化,再以孔雀绿法进行Ec-FtsZ GTPase (Guanosine triphosphatase) 活性测定。使用纯化的Ec-FtsZ为抗原免疫大鼠制备多克隆抗体,经酶联免疫吸附测定实验 (Enzyme-linked immunosorbent assay,ELISA)、Western blotting实验和免疫荧光实验鉴定,抗Ec-FtsZ多克隆抗体效价可达1∶256 000且具有良好的抗原特异性。抗Ec-FtsZ多克隆抗体的成功制备为Ec-FtsZ生物学功能研究和生化检测奠定了实验基础。
英文摘要:
      To prepare polyclonal antibody (PcAb) against Escherichia coli filamentous thermosensitive protein Z (Ec-FtsZ), the artificially synthesized gene fragment coding Ec-FtsZ was subcloned into pET-22b(+) plasmid, and Ec-FtsZ protein was expressed in E. coli BL21(DE3) cell under an optimal bacterial expression condition. Then Ec-FtsZ protein was purified by HisTrap affinity chromatography, and the GTPase (Guanosine triphosphatase) activity of purified Ec-FtsZ protein was further analyzed by malachite green assay. Subsequently, the purified Ec-FtsZ protein was used to immunize rat subcutaneously for preparation of anti-Ec-FtsZ PcAb. The results of enzyme-linked immunosorbent assay (ELISA), Western blotting analysis and immunofluorescence assay showed that the titer of PcAb was 1:256 000, and PcAb exhibited a perfect antigenic specificity against purified and endogenous Ec-FtsZ protein. All these data indicated that the anti-Ec-FtsZ PcAb is successfully prepared, which can be used for further cellular function study and biochemical analysis of Ec-FtsZ protein in vivo.
陈云雨,牛夏忆,李淼,李霓,刘晓平.大肠杆菌FtsZ蛋白原核表达及多克隆抗体的制备与鉴定[J].生物工程学报,2019,35(6):1117~1125
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