Prokaryotic expression and polyclonal antibody preparation of TRalphaA in Japanese flounder Paralichthys olivaceus / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 999-1006, 2009.
Article
in Chinese
| WPRIM
| ID: wpr-286610
ABSTRACT
To study the role of the thyroid hormone receptor TRalphaA involved in the process of the metamorphic development of Japanese flounder, we firstly cloned the TRalphaA gene, then ligated into the fusion expression vector pET30a and expressed in Escherichia coli DE3 (BL21) host cells. After induced for 4 h with 1 mmol/L Isopropyl beta-D-Thiogalactoside, the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. The recombinant protein was denatured and purified by His-Bind resin, then renatured through gradient washing on His-bind resin column. After that, polyclonal antibody was prepared by immunizing New Zealand rabbits with purified protein. Dot blotting analysis showed the antibody with the titer of 1200 000 reacted specifically to the expressed recombinant protein. Furthermore, a chromatin immunoprecipitation assay was performed to identify the specific binding between the antibody and TRalphaA in living cells of Japanese flounder. The result showed that thyroid hormone was involved in the alkaline phosphatase (ALP) gene transcriptional regulation through TRalphaA in vivo.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Physiology
/
Flounder
/
Recombinant Fusion Proteins
/
Thyroid Hormone Receptors alpha
/
Alkaline Phosphatase
/
Allergy and Immunology
/
Escherichia coli
/
Genetics
/
Metabolism
/
Metamorphosis, Biological
Limits:
Animals
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2009
Type:
Article
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