[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.
Zhang
Ailing
A
College of Animal Science and Technology, Laboratory of Molecular Biology for Agriculture, Northwest A & F University, Yangling 712100, China.
Zhang
Li
L
Chen
Hong
H
Zhang
Liangzhi
L
Lan
Xianyong
X
Zhang
Chunlei
C
Zhang
Cunfang
C
Zhu
Zeyi
Z
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