Expression Of Thanatin And Ietalurus Punetaus β-defensin Gene Based On Pichia Pastoris And Its Antimicrobial Activity

Antibiotics have saved countless lives,but its abuse caused a series of "bacterial resistance","super resistant bacteria","DNA pollution" and other problems have been very serious,which has posed a huge threat to global biosafety.Combined with the "Antibiotic reduce" and "Antibiotic prohibition" policy,It is imperative to find antibiotic alternatives and study new anti-infectious drugs.Antimicrobial peptides（AMPs）are important active molecules of biological natural immunity,It is easy to degrade,acid,alkali and high temperature,not easy to make bacteria resistant,and is expected to become an alternative to antibiotics.Thanatin originated from thorn（Podisus maciventris）,thanatin is a small molecule antimicrobial peptide containing 21 amino acids isolated from blood lymph,with broad-spectrum antibacterial activity,but with low resistance to S.aureus.Ietalurus punetaus is a lower fish,and benthic life,complex living environment,susceptible to pathogenic microorganisms,β-defensin plays an important immune role.In this study,Thanatin gene and the Ietalurus punetaus β-defensin gene（named defbl）were selected and optimized by Pichia preferred codon,Sal I and Eco R I,6×His tag,stop codons were added to the gene sequence,The single genome and the co-expression group（named TD）which linked with T2 A cleaved peptide were construct into the p MD19-T vector,Later,the target gene were subcloned into the yeast expression vector pPICZαA Vector（Zeocin TM）,By Zeocin resistance screening,PCR,double digestion validation and sequencing validation,The recombinant eukaryotic expression vectors pPICZɑA-thanatin,pPICZɑA-defbl,and pPICZɑA-TD were successfully constructed.The recombinant vector were linearized with Sac I and electrotransfered into the P.pastoris GS115 cells,and The recombinant high-anti-positive transformants GS115-pPICZɑA-thanatin,GS115-pPICZɑA-defbl and GS115-pPICZɑA-TD（Mut+）which tolerated 1 000 μg/m L Zeocin were selected by Zeocin and PCR.0.5% methanol was used to induce the expression of target proteins and the collected proteins from methanol induction for 0 h,12 h,24 h,36 h,48 h,60 h,72 h and 96 h for Western blotting,The results showed that the target protein was expressed in the medium supernatant with 0.5% methanol induction,there were specific bands appeared at 2.3 k Da and 7.8 k Da in the thanatin and dbfel groups respectively,and the two bands appeared were both inappeared in the TD group,the protein sizes were all consistent as expected.After purification,the protein concentrations of thanatin,dbfel and TD were 600 μg/m L,300 μg/m L and 700 μg/m L,the optimal expression time was 72 h for thanatin,96 h for defbl and 96 h for TD.Expanding culture collection of purified each recombinant protein for in vitro antibacterial experiment and hemolytic test,the results showed: 1.All recombinant antimicrobial peptide proteins showed antibacterial activity against the eight indicated strains used in the test,Among the Gram-negative bacteria: E.coli K88,Balcillus proteus mirabilis,Klebsiella pneumoniae ATCC700603,Pasteurella multocida and the Gram+positive bacteria: Staphylococcus epidermidis ATCC 12228,Staphylococcus aureus CMCC（B）26001,streptococcus dysgalactiae,Streptococcus agalactiae.Group thanatin recombinant proteins have better inhibited to Pasteurella multocida,E.coli K88,Klebsiella pneumoniae ATCC700603 than Staphylococcus aureus CMCC（B）26001,streptococcus dysgalactiae,Streptococcus agalactiae,the antibacterial rate was all between80% and 95%,The antibacterial rate of the defbl group was above 90%,The recombinant proteins in the TD group inhibited best against Klebsiella pneumoniae ATCC700603 with MIC 25 μg/m L,which significantly outperformed the thanatin and defbl groups,The inhibitory effect of S.aureus was significantly better than the thanatin group and better than the single genome for all strains.2.The hemolysis rate of each recombinant protein at 300μg/m L concentration was less than 5%,indicating that the recombinant antimicrobial peptide obtained in this test did not possess erythrocyte hemolysis.The purified recombinant proteins150 μg/m L were acting on HEK293 cells for 12 h,24 h and 36 h,CCK-8 analysis of cells showed that: Each recombinant protein acts on HEK293 cells within 36 h had no cytotoxic effect on HEK293 cells,DAPI staining at 36 h also indicated that none of each recombinant protein caused apoptosis.In conclusion,this test successfully constructed a recombinant expression vector containing thanatin gene,Ietalurus punetaus β-defensin gene and both co-genes,By the yeast expression system the antimicrobial proteins were got successfully,The recombinant proteins showed good antibacterial activity against the tested strains,had no red blood cell hemolysis,no cytotoxic effect,not causing apoptosis,had a good safety profile treatment of bacterial diseases.In contrast,the TD group had better antibacterial effect and more research value.This experiment provides a theoretical and experimental basis for the subsequent research on the function and application of the protein,and provides new ideas for the prevention and treatment of bacterial diseases.