Expression Of Chicken AvBD2Mature Peptide In Pichia Pastoris And Analysis Of Antimicrobial Activity Of The Recombinant Protein

The chicken antimicrobial peptides which were generated when externalpathogenic microorganisms invaded chicken were positively charged small moleculeactive peptides. As one of the antimicrobial peptides, avian beta-defensin （AVBD） isimportant. Because of its anti-bacterial broad spectrumhigh efficiency, particularmechanism and pathogenic microorganism is difficult to produce resistivity,antimicrobial peptides is likely to become sources of new antibiotic drugs incircumstances which antibiotic resistance is becoming more serious. Therefore thechicken beta defensin became one of the hotspots in current research. With avian betadefensin2（AvBD2） as the research object，synthetic AvBD2mature peptide gene wassuccessfully cloned into the pPIC9K carrier using genetic engineering technology.The pPIC9K-AvBD2reorganization expression vector was built, then the supernatantof the AvBD2target protein was obtained after the recombinant Pichia pastoris wasinduced by methanol, at last, a preliminary research on the antimicrobial activity ofthe supernatant products was conducted. The specific contents and results are asfollows.1Synthesis ofAvBD2mature peptide geneAccording to the NCBI reported avian β-defensin-2（AvBD2accession number:NM₂04992） cDNA sequence, the AvBD2mature peptide gene was synthetized bythe TaKaRa Company after adding the appropriate restriction sites EcoRI and NotI atthe both ends of AvBD2mature peptide gene. Mature peptide gene was cloned intothe pMD18-T-simple vector and successfully constructed the cloning vector of thepMD18-T-simple-AvBD2.2Expression of AvBD2mature peptide in pichia pastoris and its in vitro antibacterialtestAfter pMD18-T-simple-AvBD2clone vector was digested by double digestionreaction. AvBD2mature gene fragment was cloned into the pPIC9K vector andpPIC9K-AvBD2expression vector was built. Then positive strains which identifiedby PCR were extracted and the sequencing results by BLAST comparison showedthat the AvBD2mature peptide gene in constructed expression vector was consistentwith that published in GenBank, and the homology reached100%. Then the obtainedrecombinant secretory expression plasmid pPIC9K-AvBD2was linearized by SacIenzyme and was electrotransformed into the Pichia pastoris GS115. After the positive clones were screened and induced with methanol, the expression supernatant wasdetected by Tricine-SDS-PAGE and bacteria experimental. The results showed thatAvBD2mature peptide gene was successfully integrated into the the genome ofGS115, the yeast strains which steadily expressed AvBD2mature peptide wereobtained. The Tricine-SDS-PAGE detection manifested the molecular weight ofAvBD2protein is about5.8kD and the expression supernatant can inhibit a variety ofbacterial growth including Escherichia coli （CMCC44102）, Avian pathogenic E.coli（CVCC1565）, Salmonella （S2）, Pseudomonas aeruginosa （ATCC27853）,Staphylococcus aureus （ATCC25923）, Enterobacter aerogene （CMCC45103）.3Study of expression supernatant of AvBD2mature peptide to the effect onprevention of chickens infected colibacillosisWith Roman laying hens as object, the preventive effect on chickens infectedwith pathogen was researched. This experiment selected the avian pathogenicEscherichia coli CVCC1565to infect chicken after ascertaining the best bacterialattacking dosage. The expression supernatant with different concentrations werecontinuously intraperitoneal injected into11days old chicks for5days,0.2mL forevery day and every chick. Then1×109cfu/mL CVCC1565was intraperitonealinjected into21days old chicks,0.5mL for every chick. After48hours, not all of thechicks in PBS group was died, then1×108cfu/mL CVCC1565was intraperitonealinjected into survived chicks,0.5mL for ever chick. After the observation of48h, allof the chicks in PBS group were died, then infecting was stopped. The results ofinfection experiment were that all of the chicks in the groups which were injecteddifferent concentrations of the expression supernatant were survival, and part of thechicks in the group which were injected expression supernatant of empty yeast werenot dead, however, all of the chicks in the PBS control group were died. It showedthat the prevention effct of expressed supernate reached high level.In conclusion, the expression vector pPIC9K-AvBD2was successfullyconstructed and transformed into Pichia pastoris GS115by electroporation. Theninduced by methanol and the5.8kD chicken AvBD2protein was detected byTricine-SDS-PAGE. The expressed supernate showed an inhibiting effect on thegrowth of many bacterias including Escherichia coli （CMCC44102）, Avianpathogenic E.coli （CVCC1565）, Salmonella （S2）, Pseudomonas aeruginosa（ATCC27853）, Staphylococcus aureus （ATCC25923）, Enterobacter aerogene（CMCC45103）. In the experiment of chicken infected by E.Coli CVCC1565, compared to injection of empty yeast expression supernatant group and PBS controlgroup, the mortality of AvBD2of different concentration expression supernatantgroup was significantly reduced （P<0.01）. The prevention effect of AvBD2expressedsupernate reached high level.