Construction Of Kidney CDNA Library From Sea Bass And Cloning And Expression Of Hepcidin Gene

Anti-bacterial peptide(ABP)is a kind of small sized molecular peptides encoded by a given gene,which is important for the host to defend pathogenic bacterial invaders.These peptides have a broad spectrum of antibacterial and even antitumor effect,but there are no obvious toxic effects to mammals and insects.Hepcidin is a cystein-rich antimicrobial peptide produced mainly by liver.Its nucleotide sequence and structure are conservative in vertebrates,and all cosist of three parts:signal peptide,prodomain and mature peptide.Different from other antibacterial peptides, hepcidin is also a key regulator of iron metabolism.In this study,a cDNA library of kidney was constructed from the sea bass,from which gene cloning of hepcidin was taken.Meanwhile the hepcidin recombinant pET32a-trx/hepcidin vector was constructed as a fusion protein expressed in Escherichia coli.The results are showed as follows:A cDNA library of kidney was constructed from the sea bass.After the total RNA was extracted and the mRNA isolated,the cDNA was synthesized.Through the gel electrophoresis of double-strand cDNA,the fragments of 500bp～4000bp were harvested,concentrated,ligated withλZAP Express vector and packaged in vitro.The primary cDNA library contained 7×10⁵ recombinants.The titer of amplified library was tested up to 1×10¹²pfu/mL,and the insert size of random picked phagemid was between 500bp～2000bp.PCR with specific primers of hepcidin was performed to cloning of the homologous genes from cDNA library and a fragment of hepcidin cDNA sequence was amplified.The result is the same as the reported data(GenBank AY604195.2).Therefore,the construction of cDNA library provides the basis for screening immune-related genes from sea bass.According to the mature sequence of hepcidin,we designed two 84bp complementary fragments dependent on codon biases of E.coli.After denature and anneal,a double-strand cDNA was gained.The synthetic gene was proved to be correct by the sequence analysis.Digested by NcoI/XhoI,the hepcidin recombinant pET32a-trx/hepcidin vector was constructed.The fusion protein was expressed in Escherichia coli AD494(DE3)with IPTG induction.The expressed fusion protein existed both in the dissolved state and in the form of inclusion bodies after lysis with sonication.The soluble fusion protein was purified directly by Ni-NTA column.After desalting,the hepcidin was cleaved from the fusion protein by enterokinase and used for antimicrobial assay.The expression product displayed antibacterial activity against Gram-negative E.coli,but was inactive against Gram-positive Staphylococcus aureus. Thus the recombination of hepcidin will facilitate the functional study for hepcidin in future.