Cloning, prokaryotic expression of cattle Ghrelin gene and biological activity detection of the expressed protein / 生物工程学报
Chinese Journal of Biotechnology
; (12): 23-28, 2009.
Article
en Zh
| WPRIM
| ID: wpr-302860
Biblioteca responsable:
WPRO
ABSTRACT
The cDNA of cattle Ghrelin gene was amplified from abomasum fundic gland mRNA of Qinchuan Cattle by RT-PCR. PCR product was cloned into the T vector pGM-T to construct pGh-T1 for sequencing. Then the cDNA was subcloned into the prokaryotic expressing plasmid vector pET32a (+) and transformed into host Escherichia coli strain BL21 (DE3) for expression. The expression of pGh-32 mature Ghrelin protein was induced by IPTG and was identified by SDS-PAGE. The expression product was observed with soluble protein and inclusion body. Western blotting showed that the recombinant protein was recognized by his-antibody specifically. The protein was purified by Ni-NTA column and was used to inject rabbits to obtain polyclona antibody. ELISA result showed that the antibody titer was 1:12 800. The immunohistochemistry test between the hypothalamus arcuate nucleus and the antibody showed that fusion protein had biological activity. This will provide a basis for further study on the biological function of Ghrelin protein to growth and development and fat deposition of cattle.
Texto completo:
1
Base de datos:
WPRIM
Asunto principal:
Proteínas Recombinantes de Fusión
/
Clonación Molecular
/
Escherichia coli
/
Ghrelina
/
Genética
/
Metabolismo
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
Idioma:
Zh
Revista:
Chinese Journal of Biotechnology
Año:
2009
Tipo del documento:
Article