Cloning And Expression Ghrelin Genein Crucian Carp (Carassius Auratus)

Accroding to the homology and conservation of sequences of goldfish (AF454389) common carp (AB332394), channel catfish (AB196449) and zebrafish (EU908736) from the NCBI databases, the primers were designed by PREMER5.0 softeware. A treaty 490bp target gene fragment was amplified from total RNA of Crucian carp intestine tract by RT-PCR. The target gene was inserted into the pMD-19 vector. The result of the sequencing indicated that the sequence contained the complete coding sequence of ghrelin protein, which was 312 bp, encoded 103 amino acids. The molecular weight of crucian carp ghrelin was 11519.48 Da, isoelectric point was 5.20, estimated half-life was about 1.20 hours, and instability index was 31.29. Grand average of hydropathicity of ghrelin protein was 0.727. while the hydrophobicity was between-2.900 and 2.856. The protein had one signal peptide between 1 and 26 sites; there were 44a-Helix,8β-Sheet and 43 coils in crucian carp ghrelin. The phylogenetic tree showed that the evolution distance of crucian carp ghrelin gene was the most homogeneous to goldfish ghrelin gene.The experiment found that the expression of ghrelin gene mRNA was detected in intestinal tract, liver, kidney, pituitary gland, muscle and spleen of crucian carp by fluorescence quantitative techniques, which is the highest expression in intestinal tract. Meanwhile, we also found the expression of ghrelin gene mRNA was not decteded in skin, heart and gills of crucian carp.To obtain the region of the complete ORF was amplified from the the recombinant plasmid of pMD19-ghrelin, we designed the preimers with restriction enzyme of BamH I and Xho I by the primer5.0 software. The target fragment was was inserted into bacterial expression vector of pET-32α(+), and then recombinant prokaryotic expression vector in pET-32α-ghrelin was constructed successfully. The recombinant plasmid of pET-32α-ghrelin was transformed into host cell and was induced by IPTG at 37℃. The result was identified by electrophorese of SDS-PAGE, it indicated that the expression of ghrelin fusion protein was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG). Its molecular weight was about 27.0 KDa as expected. The optimized expression condition of the pET32a(+)-n-ghrelin recombinant plasmid, was determined to be induced by 0.3mmol/L IPTG (OD600=0.5) for 3 hours. The protein of pET32a (+)-n-ghrelin was fusion protein with the optimal exression condition.In our study, the ghrelin was firstly cloned from the intestine tract of the Crucian carp. We successfully constructed the prokaryotic expression vector of the pET32α(+)-ghrelin, and the molecular weight of fusion protein was 27.0KD. In the future, the current study provides useful experimental material for further functional analysis of ghrelin in crucian carp.