Characterization, Expression, And Prokaryotic Recombination Of Hepcidin From Cobia (Rachycentron Canadum)

Basing on the techniques of homology cloning, RACE-PCR, real time PCR,the fullcDNA of Hepcidin gene from cobia (Rachycentron canadum) was cloned. The mRNAexpression of hepcidin in tissues of normal fishes and fishes injected with LPS and Vibriocarchariae via i.p. respectively, were detected by QRT-PCR. The prokaryotic recombinationvector was constructed and the expression of recombined hepcidin were analyzed. The mainresults are showed as follows:The full length cDNA of hepcidin comprises of 714 bp with a 5'untranslatedregion(UTR) of 213 bp, a 3'UTR of 228 bp and an open reading frame (ORF) of 273 bp,encoding a polypeptide of 90 amino acid residues, including signal peptide, prodomain andmature peptide regions. The predicted molecular weight was 10.03 kDa and theoreticalisoelectric point was 7.54. phylogenetic tree based on hepcidin amino acid sequence wasconstructed by amino acid alignment, and results showed that the relationship of hepcidinbetween cobia and finless eel (Monopterus albus) was closest. The putative protein showedhomology varying from 85.6% to 24.4% within some known hepcidin amino acids of fishesand mammals.QRT-PCR PCR indicated cobia hepcidin gene expressed in all detected tissues withvariable quantity. High expression was detected in the liver, head kidney and spleen, andmoderate expression was detected in the intestine, gill, heart and skin, while low expressionwas in blood, brain and stomach. Expression of hepcidin in the liver, head kidney and spleenincreased after LPS and V. carchariae stimulated. The result show that the exogenous effectfactor can induced expression of hepcidin in immune organs of cobia, to kill the pathogens.The recombinant expression vector of pTYB11-Hepcidin was successfully constructedand identified, and then transformed into E.coli ER2566. The expression of recombinedhepcidin induced by IPTG were assessed. The products of the prokaryotic expression weretested by SDS-PAGE. The results showed that the recombinant pTYB11-Hepcidin protein was66.03 kDa, which was in the forms of inclusion bodies. It provides a good experimentalmaterial for further study of the recombinant protein immune function.