| 大肠杆菌FtsZ蛋白原核表达及多克隆抗体的制备与鉴定 |
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| 引用本文： | 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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| 作者姓名： | 陈云雨 牛夏忆 李淼 李霓 刘晓平 |
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| 作者单位： | 1 皖南医学院 药物筛选与评价研究所，安徽 芜湖 241002,1 皖南医学院 药物筛选与评价研究所，安徽 芜湖 241002,1 皖南医学院 药物筛选与评价研究所，安徽 芜湖 241002,2 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 100050,1 皖南医学院 药物筛选与评价研究所，安徽 芜湖 241002 |
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| 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。 |
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| 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。
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| 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 |
| 收稿时间： | 2019/1/6 0:00:00 |
Bacterial expression, preparation and identification of polyclonal antibody against Escherichia coli FtsZ |
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| Yunyu Chen,Xiayi Niu,Miao Li,Ni Li and Xiaoping Liu.Bacterial expression, preparation and identification of polyclonal antibody against Escherichia coli FtsZ[J].Chinese Journal of Biotechnology,2019,35(6):1117-1125. |
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| Authors: | Yunyu Chen Xiayi Niu Miao Li Ni Li and Xiaoping Liu |
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| Institution: | 1 Research Institute for Pharmaceutical Screening & Evaluation, Wannan Medical College, Wuhu 241002, Anhui, China,1 Research Institute for Pharmaceutical Screening & Evaluation, Wannan Medical College, Wuhu 241002, Anhui, China,1 Research Institute for Pharmaceutical Screening & Evaluation, Wannan Medical College, Wuhu 241002, Anhui, China,2 State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China and 1 Research Institute for Pharmaceutical Screening & Evaluation, Wannan Medical College, Wuhu 241002, Anhui, China |
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| Abstract: | 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. |
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| Keywords: | Escherichia coli FtsZ protein bacterial expression malachite green assay immunofluorescence assay polyclonal antibody |
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