Hybrid Peptide Cecropin P1-dermaseptin S4Secretory Expression In Pichia Pastoris And Activity Detection

Purpose:Splicing two kinds of antimicrobial peptide gene fragment which have optimized, constructingexpression vector pPICZαA-cp1-ds4and efficient secretion express hybrid peptides Cecropinp1-Dermaseptin S4in Pichia pastoris X-33.Methods:1. Combine two antimicrobial peptides amino acid sequences and predicte secondary structureof hybrid peptide Cecropin P1-Dermaseptin S4use Antheprot release5.0and using theantimicrobial peptides databases (APD) hybrid peptide predicted physicochemical propertiesand the possibilities of antimicrobial peptides.2. Antibacterial peptides Dermaseptin S4gene cloning: according to the codon preference ofPichia and Dermaseptin S4and part of Cecropin P1C-end amino acid sequence in genebank,design the appropriate splicing primers and splicing together by overlap extension PCR (SOE-PCR),PCR products cloning to pMD18-T carrier vector and then transformed intoEscherichia coli DH5α and sequencing.3. Hybrid peptide gene cloning: Use pPICZ a A-cp1and pMD18T-ds4plasmid whichrespectively preserve Cecropin P1and Dermaseptin S4the right sequence as template, afterPCR amplification with their specific primers, splicing two gene fragment though SOE-PCR,the splicing fragment cloning to pMD18-T vector and then transformed into E. coli DH5αandsequencing.4. Expression vector of pPICZαA-cp1-ds4construction: recombinant plasmid pMD18T-cp1-ds4and expression vector pPICZα-A double enzyme by EcoR Ⅰ andXba Ⅰrespectively and use of T4DNA ligase for connection and then transformed into escherichia coli DH5αandsequencing.5. Construction of recombinant yeast pPICαA-cp1-ds4/X-33: The expression vectorpMD18T-cp1-ds4linearized by Sac I, using electroporation instrument EMC830low voltagemode transformed into yeast and use100mg/L Zeocin screening recombinant yeast.6. Induced recombinants expression and the preliminary activity detection: using BMGY andinduced BMMY medium with0.5%(v/v) methanol cμlture recombinants and usingTricine-sds-page testing interesting polypeptide， detecting antibacterial activity ofconcentrated supernatant which contain hybrid peptide Cecropin P1-Dermaseptin S4by agardiffusion assay.7. Purified expression products: screening mμlti-copy recombinant yeast by raising theconcentration of Zeocin, using BMGY and BMMY inducing medium cμlture and usingAKTA protein purification system purified interesting polypeptide.8. Antibacterial spectrum and the minimum inhibitory concentration determination: using agardiffusion method and micro-dilution assay heterozygous detecting antibacterial spectrum ofantimicrobial peptide Cecropin P1-Dermaseptin S4and the minimum inhibitory concentration,respectively.9. Measure the hybrid peptide Cecropin P1-Dermaseptin thermal stability S4, pH stability andthe stability of trypsin digestion by the method of calcμlating the antibacterial activity whichestablished by Hμltmark and others.10. To study the effect of hybrid peptide Cecropin P1-Dermaseptin S4to mouse myelomacells SP2/0though MTT assay.Resμlts:1. The secondary structure of hybrid peptide Cecropin P1-Dermaseptin S4was predicted byAntheprot release5.0and discovery that N-terminal and C-terminal have ability tomaintainα-helix and exist loose connection fragment between α helix structures. The hybridpeptide have possiblity to beccome one antimicrobial peptide predicted by APD.2. After splicing primers were spliced into Dermaseptin S4gene fragment(120bp)successfμlly by SOE-PCR, gene fragment was cloned into pMD18-T vector and thentransformed into E. coli DH5α and sequencing, sequencing resμlts showed that the squence is consistent with the design sequence.3. After splicing Cecropin P1and Dermaseptin S4gene by SOE-PCR successfμlly, cloned theFusion gene fragment into pMD18-T vector and then transformed into E. coli DH5α andsequencing, sequencing resμlts show that the squence is consistent with the design sequence.4. pMD18T-cp1-ds4and pPICZα-A expression vector after restriction enzyme digestion,enzyme-linked reaction, the recombinant plasmid was transformed into E. coli DH5α andsequenced, sequencing resμlt showed that the open reading frame is correct, the encodedamino acid sequence consistent with designed.5. After expression vector pPICαA-cp1-ds4linearized by Sac I,it canbe transformed intoPichia pastoris X-33successfμlly under the condition of ECM830low voltage mode (voltageof350V, pμlse time of15s, pμlse one time), positive recombinants can be screened byZeocin.6. Initial detecting the effect of concentrated supernatants to Escherichia coli andStaphylococcus aureus by agar diffusion method, adding concentrated supernatant can bothappear bacteriostatic ring.7. Mμltiple-copies recombinants can be screened though improve Zeocin centration and withthe resμlt obtaine the strain can tolerant Zeocin at the centration of400mg/L.Inducingmμltiple copies recombinant express the polypeptide and its production is approximately30mg/L, after AKTA protein purification system available a specific protein bands.8. In antibacterial spectrum measurement experiments hybrid peptide can effect on E. coliDH5α, Staphylococcus aureus, Lactobacillus acidophilus, Bacillus subtilis, ShigellaSDCDC563, Salmonella typhimurium CMCC50222with the minimum inhibitoryconcentration of approximately37.5μg/ml,75ug/ml,75ug/ml,150ug/ml,150ug/ml,150ug/ml,respectively, but invalid.for Pseudomonas aeruginosa27853.9. Hybrid peptide can also maintain a higher antibacterial activity after treate with heat, acidand trypsin digestion.10. MTT assay resμlts showed that hybrid peptide have a ability inhibit mouse myeloma cells.Conclusions:We can Secretory expression hybrid peptide Cecropin P1-Dermaseptin S4though geneticengineering successfμlly, study resμlts show that the hybrid peptide have an effect on a variety of Gram-and Gram+bacteria, the hybrid peptide has good stability after treated withthermal, pH or trypsin digestion, and also has some inhibitory effect to mouse myeloma cellsSP2/0.