Cloning, prokaryotic expression of cattle Ghrelin gene and biological activity detection of the expressed protein.
- Author:
Ailing ZHANG
1
;
Li ZHANG
;
Hong CHEN
;
Liangzhi ZHANG
;
Xianyong LAN
;
Chunlei ZHANG
;
Cunfang ZHANG
;
Zeyi ZHU
Author Information
1. College of Animal Science and Technology, Laboratory of Molecular Biology for Agriculture, Northwest A & F University, Yangling 712100, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cattle;
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Ghrelin;
genetics;
metabolism;
Recombinant Fusion Proteins;
genetics;
metabolism
- From:
Chinese Journal of Biotechnology
2009;25(1):23-28
- CountryChina
- Language:Chinese
-
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.
