The Expression And Identification Of Human Beta-Defensin-4

ObjectiveWith a large number improper use of traditional antibiotics, the problem of drugresistance becomes more and more serious. Thus, researching and developping theefficient and non-toxic alternatives to existing antibiotics are a hot issue today.Antimicrobial peptides (AMPs) are produced in vivo by the induction of a class ofbiologically active small molecule peptide. It is an important part of the host’simmune defense system, with broad-spectrum anti-bacterial, anti-fungal parasites,anti-enveloped viruses, anti-cancer cells and other biological functions. Meanwhile,AMPs have a unique antimicrobial mechanism and not easily let the bacteria producethe drug resistance. So it shows huge potential and broad prospect for application inan alternative to traditional antibiotics.Human defensins are the important endogenous AMPs of the human body whichbelong to the AMPs family. It is an important part of the immune defense system.Among them, the human β-defensin-4(hBD-4) was discovered in2001by Jose-Ramon Human β-defensin, with broad-spectrum antimicrobial activity,especially has a powerful killing activity to S. carnosus and P. aeruginosa which haveserious resistance, so it is a very promising new antibiotic developed which can kill avariety of drug-resistant bacterial such as Staphylococcus and P. aeruginosa.Undoubtedly, the best choice of mass-produced hBD-4is genetic engineeringtechnique due to less natural production and tedious extraction step, as well asexpensive chemical synthesis. Therefore, this study will artificially synthesize thehBD-4oligonucleotide fragments according to the hBD-4mature peptide geneinformation from the GenBank database, and bioinformatic analysis of its structure,build the prokaryotic fusion expression system with pET system which is mostpowerful expression system to the recombinant proteins, then opmimize theexpression conditions. It will establish the foundation to realize the large-scaleproduction of hBD-4and the other further research.Methods1. Adopting the method of PCR-based gene SOEing synthesis to artificiallysynthesize hBD-4according to the hBD-4mature peptide gene informationfrom the GenBank database. Connecting the hBD-4gene fraction and pET-28avector and constructing pET-28a-hBD-4pronucleus expression vector. Andtransforming the pET-28a-hBD-4to E. coli Competent Cell JM109, then thepositive clone will be identified by collect the plasmid, agarose gel electrophoresisrecovery and purification, sequencing.2. Using DNAClub to analyze the restriction endonuclease site of hBD-4nucleotidesequence, using Expasy analysis tools (ProtParam, InterProScan, SOPMA,Protscale) and Cn3D4.1procedure to analyze the amino acid sequence andpredict the structure.3. Transforming the positive clone vector to E. coli BL21(DE3) competent cell.Detecting the positive transformed strain by PCR.4. Using SDS-PAGE and Western Blot to testify the expression of the hBD-4. 5. Screening the best expression condions of fusion protein, namely the optimalinduction time, the optimal induction temperature as well as the best IPTGinduction concentration.6. Detecting the expression form of recombinant protein, and purifing byImmobilized metal ion chromatography (IMAC) after the large expression, andthen determining hBD-4final yield by the BCA method.7. Determining the antibacterial activity to S. aureus, E. coli, Shigella, P. vulgarisand Salmonella by agarose hole diffusion method. Utilizing microdilution methodto determine the hBD-4MIC to S. aureus and E. coli, and measuring the hBD-4impaction to growth curve of S. aureus and E. coli.8. Using agarose hole diffusion method to detect the antibacterial activity of hBD-4to S. aureus and E. coli after boiling, repeated freeze-thaw and pH treatment.Results1. Successfully constructed the pET-28a-hBD-4in E. coli BL21(DE3) pronucleusexpression system.2. hBD-4exists only Alu I and Fok I restriction endonuclease sites, the theoreticalmolecular weight (NW) is5988.9Da, isoelectric point (pI) is9.27, and it is aamphiphilic α-helical and β-sheet peptide.3. Determined the optimal induction of expression conditions: inducing6h with the1mM IPTG at30℃. With this inducing condition, the target protein expressed insoluble form and a very small amount of inclusion bodies’ form.4. Obtained pure recombinant protein, the final yield is316.33μg/ml.5. Purified hBD-4has a significantly antibacterial activity to S. aureus and E. coli,but not to Shigella, P. vulgaris and Salmonella. The minimum inhibitoryconcentration (MIC) to S. aureus and E. coli is8~16μg/ml and8μg/mlrespectively, and the antibacterial activity to S. aureus and E. coli is in adose-dependent manner.6. The thermal stability experiment showed hBD-4in boiling water for5min, the activity is not affected, the activity was slightly lower for10-20min and theactivity disappeared after30min. Freeze-thaw test showed after repeated freezingand thawing at six times, the antibacterial activity had not been significantlyaffected. PH stability results showed the activity of hBD-4is substantiallystabilized in the pH6.0-9.0. When the pH value is less than6.0or greater than9.0,the activity is gradually reduced, and hBD-4is more stable when in the alkalinecondition than in the acidic condition.ConclusionThis study successfully constructed prokaryotic expression vector ofpET-28a-hBD-4, and obtained a high yield of recombinant hBD-4protein with goodbiological activity and vitro stability after the recombinant plasmid being transferredto BL21(DE3). The research laid the foundation not only to achieve mass productionof hBD-4but also to create the condition for hBD-4post-R&D and clinicalapplication.