Construction And Eukaryotic Expression Of The Hybrid Antimicrobial Peptide Lf-Te Gene

Antibacterial peptides are an important antimicrobial factor for many organisms,especially insects,and are also an important part of their non-specific immune system.Hybrid antimicrobial peptides are novel antimicrobial peptides that connect parts of peptide chains of two or more antimicrobial peptides according to certain design principles by genetic engineering or chemical synthesis,which generally has features such as broad spectrum,high efficiency,non-toxic side effects,reduced hemolysis,stronger stability and activity.In this study,genetic engineering technology was used to connect the functional region of bovine lactoferrin Lfcin B and antibacterial peptide Tenecin 1 with flexible chain(ser-ser-gly-ser-gly-ser),the structure-activity relationship of the bovine lactoferrin Lfcin B and the antimicrobial peptide Tenecin 1,the selected expression vector,the selected engineering bacteria and the yeast preference codon were used for obtaining the Lf-Te sequence of the hybrid antimicrobial peptide.Primers were used to design primers,and genes were amplified by PCR amplification.Lf-Te gene was cloned into plasmid pPICZ?A to construct the recombinant expression vector pPICZ? A-Lf-Te.The recombinant plasmid was identified by plasmid size,double digestion and PCR amplification.Lf-Te gene was cloned into plasmid pPICZ?A to construct the recombinant expression vector pPICZ? A-Lf-Te.The recombinant plasmid was identified by plasmid size,double digestion,PCR amplificationand sequencing identification to verify that the target gene had been cloned into the expression vector and successfully completed construction of eukaryotic expression vector pPICZ? A-Lf-Te.The successfully constructed recombinant plasmid was linearized with restriction endonuclease Sac I and electrotultured into Pichia pastoris GS115 host strain,and positive clone transformants were screened with selection medium containing Zeocin(bleomycin).The genome was extracted by kit method,the recombinant plasmid pPICZ?-A-Lf-Te was successfully integrated into the yeast genome by PCR,which was then induced by methanol in the BMMY medium,the expressed product was digested with Tricine-SDS-PAGE electrophoresis for testing,results showed that the expressed product had a clear single band at about 7.9 kD,consistent with the expected size of the expressed product.Hybrid antimicrobial peptides expression of the optimal conditions of Lf-Te,as A2B2C2,namely the induction time of 96 h,induction temperature 30 ?,and the medium initial pH value of 5.Induced under the optimal conditions,the hybrid antimicrobial peptides Lf-Te expression protein concentration is 1.21 mg/mL.The antibacterial effect of the expressed product of hybrid antimicrobial peptide Lf-Te on Escherichia coli,Staphylococcus aureus,Salmonella enteritidis and Saccharomyces cerevisiae was detected by agar diffusion method.The results showed that the hybrid antimicrobial peptide Lf-Te had antibacterial effect on Escherichia coli,Staphylococcus aureus and Salmonella enteritidis,the minimum inhibitory concentrations were 0.120 mg / mL,0.160 mg / mL,0.200 mg / mL,respectively,it had no antibacterial effect on Beer yeast.The antimicrobial activity of the hybrid antimicrobial peptide Lf-Te was good and remained activity within 60 min after boiling.At pH 2-6,the higher the pH,the better the antibacterial activity.After pH> 6,the antibacterial activity was weakened.The activity of the hybrid antimicrobial peptide Lf-Te was affected by UV irradiation,the longer the UV irradiation time,the lower the activity of the antimicrobial peptide.The genetic stability was better.After 20 passages for culture,the target genes were not lost and the expressed products still had antibacterial activity verified by PCR.In summary,this study successfully cloned the hybrid antimicrobial peptide Lf-Te gene,constructed the eukaryotic expression vector pPICZ?A-Lf-Te,and completed its secretion expression in Pichia pastoris.The results showed that the expressed products still had good biological activity,and the antibacterial effect of the hybrid antimicrobial peptide Lf-Te was good,which provided the foundation for the further research and application of antimicrobial peptides.