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

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。

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.