Chen Yunyu, Niu Xiayi, Li Miao, Li Ni, Liu Xiaoping
Research Institute for Pharmaceutical Screening & Evaluation, Wannan Medical College, Wuhu 241002, Anhui, China.
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.
Sheng Wu Gong Cheng Xue Bao. 2019 Jun 25;35(6):1117-1125. doi: 10.13345/j.cjb.190011.
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.
为制备抗大肠杆菌丝状热敏蛋白Z（Ec-FtsZ）的多克隆抗体（PcAb），将人工合成的编码Ec-FtsZ的基因片段亚克隆至pET-22b(+)质粒中，并在最佳细菌表达条件下于大肠杆菌BL21(DE3)细胞中表达Ec-FtsZ蛋白。然后通过HisTrap亲和层析法纯化Ec-FtsZ蛋白，并采用孔雀石绿测定法进一步分析纯化后的Ec-FtsZ蛋白的GTP酶（鸟苷三磷酸酶）活性。随后，用纯化后的Ec-FtsZ蛋白皮下免疫大鼠以制备抗Ec-FtsZ PcAb。酶联免疫吸附测定（ELISA）、蛋白质免疫印迹分析和免疫荧光测定结果显示，PcAb的效价为1:256 000，且PcAb对纯化的和内源性Ec-FtsZ蛋白均表现出良好的抗原特异性。所有这些数据表明已成功制备出抗Ec-FtsZ PcAb，可用于进一步研究Ec-FtsZ蛋白在体内的细胞功能及进行生化分析。
