Cloning, Tissue Distribution And Prokaryotic Expression Of Ghrelin Gene In Intestine Of Schizothorax Prenanti

Ghrelin is a gastric peptide, be involved in the regulation of many physiological functions including the regulation of food intake,energy balance and reproduction.To clone the gene of ghrelin in Schizothorax prenanti, thus construct prokaryotic expression vector of Schizothorax prenanti intestine ghrelin gene and obtain its expression product. According to the homology and conservation of sequences in cyprinid fish, including goldfish (AF454389), channel catfish (AB196449),common carp (AB332394),and zebrafish(EU908736)and crucian carp(HM567312), a pair of primers were designed. Base on the total RNA isolated from the intestine of healthy Schizothorax prenanti, six fish body weights were312.2±5.6g.The ghrelin cDNA was amplified by reverse transcription polymerase chain reaction(RT-PCR). The PCR fragments were inserted into plasmid pMD19-T, after cultured for overnight at temperature of37℃,positive cloning bacteria was selected and identified by PCR. The sequencing result showed that the length of ghrelin cDNA is370bp, including a completed length312bp open reading frame(ORF), which translates into a protein of103amino acid residuesIt contained a predicted signal peptide of26amino acid residues. Schizothorax prenanti ghrelin exhibited high homology with crucian carp, goldfish,common carp, zebrafish and channel catfish, homology of95.2%,93.9%,94.9%,78.8%and54.8%, respectively. Lower homology with Atlantic salmon, Rainbow trout, Japanese eel and Mozambique tilapia, the lowest one is Mozambique tilapia, homology of40.4%.Phylogenetic tree of Schizothorax prenanti and other fishes based on ghrelin is consistent with classification system.The amino acid sequence homology with crucian carp, goldfish,common carp as high as93.0%, the farthest evolution distance with Mozambique tilapia and the lowest homology of38.8%. The GenBank accession number is JQ040509.Take a pair of primers contain two restriction sites of BamH I and Xho I,a treaty251bp target gene fragment was amplified from pMD19-T-ghrelin positive cloning vector by PCR. The PCR product was cloned into the positive vector pET-32a(+) to construct pET32a(+)-n-ghrelin recombinant prokaryotic expression vector for sequencing. Then the recombinant plasmid was transformed into host cell BL21(DE3)pLysS for expression. After IPTG induction, SDS-PAGE analysis showed that the recombinant protein had successfully expressed. The molecular weight of the recombinant protein was28.9KD. The optimized expression condition of the pET-32a(+)-n-ghrelin recombinant plasmid was0.3mmol/L IPTG inducing for3hours. The protein of pET-32a (+)-n-ghrelin was fusion protein with the optimal expression condition. The expression product was observed with soluble protein and inclusion body.To measure the relative expression of ghrelin gene in different organization in Schizothorax prenanti, real-time fluorescence quantitative PCR was use to that, and (3-actin was taken as reference gene. Thirteen tissues were sampled from fish, including intestine,liver and brain. The results showed that the highest expression of ghrelin gene in intestine, followed by the liver and cholecyst, no expression were detected in gill, head kidney, kidney, pituitary, brain, eyes, skin and muscle.Through the experiment, ghrelin cDNA was firstly cloned from the intestine of Schizothorax prenanti, and fusion protein was expressed in Prokaryotic Expression Vector. Consequently, this study has established substantial bases for further study on function, eukaryotic expression of ghrelin and biological agent.