Bacterial expression, preparation and identification of polyclonal antibody against Escherichia coli FtsZ / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 1117-1125, 2019.
Artículo
en Chino
| WPRIM
| ID: wpr-771816
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 1256 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.
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Plásmidos
/
Proteínas Bacterianas
/
Ensayo de Inmunoadsorción Enzimática
/
Western Blotting
/
Proteínas del Citoesqueleto
/
Escherichia coli
/
Anticuerpos
/
Especificidad de Anticuerpos
Tipo de estudio:
Estudio diagnóstico
Límite:
Animales
Idioma:
Chino
Revista:
Chinese Journal of Biotechnology
Año:
2019
Tipo del documento:
Artículo
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