[Cloning, prokaryotic expression of cattle Ghrelin gene and biological activity detection of the expressed protein].
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.
College of Animal Science and Technology, Laboratory of Molecular Biology for Agriculture, Northwest A & F University, Yangling 712100, China.
Ailing
A
Zhang
Li
L
Zhang
Hong
H
Chen
Liangzhi
L
Zhang
Xianyong
X
Lan
Chunlei
C
Zhang
Cunfang
C
Zhang
Zeyi
Z
Zhu
D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D054439:Ghrelin / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D054439:Ghrelin; D011993:Recombinant Fusion Proteins