Bacterial expression, preparation and identification of polyclonal antibody against Escherichia coli FtsZ.

Yunyu Chen; Xiayi Niu; Miao LI; Ni LI; Xiaoping Liu

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Bacterial expression, preparation and identification of polyclonal antibody against Escherichia coli FtsZ.

Author: Yunyu CHEN ¹ ; Xiayi NIU ¹ ; Miao LI ¹ ; Ni LI ² ; Xiaoping LIU ¹
Author Information

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.
Publication Type:Journal Article
Keywords: Escherichia coli FtsZ protein; bacterial expression; immunofluorescence assay; malachite green assay; polyclonal antibody
MeSH: Animals; Antibodies; Antibody Specificity; Bacterial Proteins; Blotting, Western; Cytoskeletal Proteins; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Plasmids; Rats
From: Chinese Journal of Biotechnology 2019;35(6):1117-1125
CountryChina
Language:Chinese
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.