Genetic Engineering Preparation For Channel Catfish(Ictalurus Punctatus) Hepcidin Antimicrobial Peptide

China as the world aquaculture producer, is the only one aquacultureproduction is higher than the national fishing yield in the world. However,a key problem to be solved urgently, result from germplasm, disease andenvironment is becoming more and more serious. Bacterial infection andbacterial diseases are on the rise, in order to solve these problems led to theabuse of antibiotics, which results in many drug-resistant bacteria and drug residue problems. Genetic engineering of antibacterial peptide as greenfeed additive, which can enhance the aquaculture biological resistance, butalso can improve the quality of aquatic products.Antimicrobial peptides is an important part of animal immune defensesystem. Antimicrobial peptides have a broad spectrum of antimicrobialactivity, but also has high antifungal, antiviral and antitumor activity.Therefore, antimicrobial peptides have a wide application prospect inmedicine and agriculture. This study relates to the hepatic expression ofantibacterial peptide hepcidin, originally isolated from human blood. Thereare reports that hepcidin against gram positive and negative bacteriashowed antibacterial activity, so it could be concluded that hepcidin can beused as a kind of green feed additive replace antimicrobial peptidesapplication in aquaculture. This study selected channel catfish as researchobject, to the preparation of antibacterial peptide using recombinant DNAtechnology.This research is mainly divided into five parts, the first part is focuseson cDNA cloning of prepro-peptide and mature peptide gene of the channelcatfish hepcidin. The total RNA was extracted from the liver of the channelcatfish. Using a pair of specific primer, which was designed with referenceto sequences of channel catfish hepcidin gene, a cDNA fragment of291bpwas amplified from the liver of channel catfish by RT-PCR. Two cDNAfragment of219bp and78bp was amplified from the hepcidin by nestedPCR respectively. The target fragment was sequenced after being clonedinto T vector. The open reading frame of the hepcidin encoded a peptideconsisting of96amino acid residues, which contained a signal peptide of24residues and a mature peptide of25residues. It was noted that when thechannel catfish hepcidin gene was compared with those not only from fish,but also from mammalian, eight conserved cysteine residues occurred atC-terminal regions of these hepcidin, suggesting that these eight residuespossibly related to antimicrobial activity of hepcidin.The second part is focuses on the construction of recombinant expression plasmid―pET32a-pCH‖and―pET32a-mCH‖. The verifiedfragment by sequencing was again amplified by using a pair of primercarrying EcoR I and Hind III site to create restriction sites. The sequencingresult for this newly amplified fragment indicated that the restriction siteswere successfully created. Subsequently, this fragment with the restrictionsites was suffered from digesting, and successfully linked into theexpression vector pET32a carrying the same restriction sites.The third part is focuses pCH and mCH fusion expression inEscherichia coli and purification of expressed product. The recombinantexpression plasmid pET32a-pCH and pET32a-mCH were transformed intohost bacteria E.coli BL21(DE3), at25℃by adding revulsant IPTG, at thelate log phase of E.coli BL21(DE3). The Tricine-SDS-PAGE analysisshowed that: the fusion protein TrxA-pCH and TrxA-mCH was highlyexpressed. In order to obtain fusion protein of high purity, chromatographyon total protein solution after ultrasonic using IMAC Ni-IDA agarose gelcolumn, were eluted using gradient of imidazole, the Tricine-SDS-PAGEanalysis proved that the high purity of TrxA-pCH and TrxA-mCH fusionprotein is obtained. The high concentration of TrxA-mCH fusion protein byenterokinase digestion treatment. After restriction enzyme digestionmixture is then subjected to ultrafiltration separation. Tricine-SDS-PAGEgel containing target protein mCH were analyzed by MALDI-TOF-TOF,were proved to acquired high purity target protein mCH.The fourth part is focuses on the antibacterial activity of mCH maturepeptide. Antibacterial activity was identified by agarose diffusion method,with50μ g/mL of ampicillin as positive control, with sterile ddH2O asblank control. The positive control had a significant inhibition zone, andblank control did not produce inhibition zone, illustrate the bacteriostaticexperiment was effective. mCH has inhibitory effect on Gram-positive andGram-negative bacteria.The fifth part is focuses on hepcidin mature peptide gene mCH-mTHcDNA cloning and eukaryotic expression vector construction. According to the existing channel catfish hepcidin mature peptide gene mCH and Niletilapia hepcidin mature peptide gene mTH as template, primer whichcontaining cross complementary sequence was designed, a cDNA fragmentof156bp was amplified by SOE-PCR. The verified fragment by sequencingwas again amplified by using a pair of primer carrying Xho I and Xba I siteto create restriction sites. The sequencing result for this newly amplifiedfragment indicated that the restriction sites were successfully created.Subsequently, this fragment with the restriction sites was suffered fromdigesting, and successfully linked into the expression vector pGAPZ α Acarrying the same restriction sites.