Clone Of The Hepcidin Gene In Yak And Its Prokaryotic Expression

Hepcidin is a special antimicrobial peptide, rich in cysteine, firstly found from human blood in 2000, not only it has remarkably antimicrobial activity, but also is considered a key regulator of iron metabolism. It was greatly noticed because of its important value of medical applications. This Gaoyuan Yak was takan as the candidates for the reasch. A pair of primers was designed and synthesized according to the bovine Hepcidin gene sequence in Genbank (Accession Number:XM-589792.3). Coding sequence of Hepcidin gene was amplified by RT-PCR technology from total RNA of yak's liver, the product was cloned to pGEM-T easy vector and transfer into competent DH5α-cells then got the positive recombinant; tested the sequence and analysed with molecular biology software. Phylogenetic tree of Hepcidin protein was constructed. The coding region of Yak's Hepcidin gene was cloned to PGEX-4T-1 vector to construct an expression plasmid PGEX-4T/Hepcidin. After inducation with IPTG, it groped the best expression conditions of the fusion protein PGEX-4T/Hepcidin.The results showed that there was a 322 bp cDNA sequence of Hepcidin gene cloned, including 289 bp open reading frames (Genbank: EU863791), encoding 82 amino acids which is contained 22 amino acids coded signal peptide. Forecast for protein molecular weight and isoelectric points, which is respectively 9.24 and 8.88 kD. Sequence alignment indicated that there were one mutations in the coding region between yak and bovine, but don't cause amino acid mutation in Hepcidin protein. Hepcidin protein has high conservation in different species; phylogenetic tree conformed to the laws of species evolution. SDS-PAGE electrophoresis showed that the band with 35 kD of molecular weight was consistent as expected. The result indicated that the successful expression of Hepcidin antimicrobial peptide gene in Escherichia.coli DH5α.