Expression Of Turbot Hepcidin Gene In Pichia Pastoris

Turbot, a rare and economic fish, was brought from Europe and initially formindustrialization breed with broad outlook for its abundat nutrition delicious flavourand highly economic value. But now in the prevention and cure area of turbot disease,culturists mainly use plenty of antibiotic and other heavy metal compound medicament,whichresult in turbot was endurable to the medicine or medicin rudimental and heavy metal rudimentalwas excess the regulated standard.It is eager and urgent to find other new antibiosis substance toreplace the antibiotic or other heavy metal compound medicament.Antimicrobial peptides (AMPs) are regarded as important componentsof the hostinnate immune system and play crucial roles in host defense against microbialinvasion. Gene-encoded, ribosomally synthesized antimicrobial peptides are widelydistributed in nature and they display strong antimicrobial activity against a broadrange of microbes including Gram positive and Gram negative bacteria, fungi,protozoa and viruses. Several AMPs exhibit antiparasitic and anticancer properties.In order to explore the potential purpose of AMPs and lay emphasis on therecombinant strain as additive feedstuff of fishery, we sought to make use of the DNArecombination technique to consutructed the turbot hepcidin gene mature peptide intopGAPZB expression vector, and transformed the recombint pGAPZB-TH vector intoP. pastoris by electroporation method, then analysised the expression protein byTricine SDS-PAGE and Western-blotting, finally we identified the maximumexpression timeThe current study mainly focus on:1. construction of the intracellularly expression vector: extracted the total RNA fromthe turbot liver and reverse-transcripted into cDNA, primers were designed toamplificate the hepcidin mature peptide sequence according to the turbot hepcidinsequence, add the thrombin sequence behind the mature peptide sequence for thedownstream purification and mutate some code in the front of the mature peptidesequence for the love of Pichia pastoris, subcloned the turbot hepcidin mature peptidesequence into pGAPZB vector. 2.Transformation of Pichia pastoris and selection of transformants: The expressionPlasmids pGAPZB-TH and pGAPZB were linearized by SalI and transformed into thecompetent X-33 cells by electroporation. All Zeocin-resistant colonies in YPDSmedium, the recombinants were extracted genomic DNA followed identified by PCRusing PGAPF and 3-AOX1 primers. And finally three recombinant strains and threenegative control strains were found.3. Identification of the recombinant strains by Tricine SDS-PAGE andWestern-blotting: recombinant proteins expressed in the recombinant strains andnegative control strains and the X-33 strains were extracted by snail enzymemathod.the expressed protein was found in the recombinant strains but not in the othertwo strains. Western blotting demonstrated that the recombinant protein couldspecially contact with anti-6his antibody. The results suggested that hepcidin gene wassuccessfully expressed intracellularly in Pichia pastoris.4. Identification of the maximum expression time of recombinant strain: The positivelytransformed yeast cells were cultured in YPD and ten microliters of the expressionmedium was taken at 24h time shelf and analyzed by Tricine SDS-PAGE aftermeasured the OD600.the result demonstrated that the maximum expression time ofthe recombinant protein was at 96 hours.