Fusion Expression Of DCD-1L In Escherichia Coli And Its Antimicrobial Activity Analysis

Objective:Dermcidin (DCD) is German scientist Schittek B first found as a new gene expressed specifically in the body’s sweat glands. Because it is difficult to obtain purified antibacterial peptide from biotic tissues in large quantity, and the cost of chemical synthesis method is high,by using the gene engineering method to prepare antimicrobial peptide has become a hot spot of scientific research in recent years. The study is to construct prokaryotic expression vector of pET-32a-DCD-1L by genetic engineering method, proteins was expressed in E.coil transetta(DE3) and to identify the antimicrobial activity, it will be the base of its antibacterial mechanism and application research. MethodsiThe two gene oligonucleotide fragments of DCD-1L were designed and synthesized based on the amino acid sequence of DCD-1L and the codon bias of E.coli,we use the two gene fragments synthetic DCD-1L through the method of overlap extension PCR.Then,through restriction enzymes digestion, connection and transformation achieve the recombinant expression vector pET-32a-DCD-1L,identify it by undergo PCR, enzyme cleavage, and sequencing, transformed right pET-32a-DCD-1L into E.coli transetta(DE3) cell to express DCD-1L.Have the induced by IPTG of final concentration is0.5mmo1/L, DCD-1L protein were obtained by spalling cells, and the indication of the existence of objective protein is discovered by SDS-PAGE experiment.Use the inhibition zone test to test for inhibition of anti-bacterial activity.ResuIts:The results showed that pET-32a-DCD-1L gene expression vector was established correctly after resrtiction enzyme analysis and DNA sequencing. SDS-PAGE analysis showed that the molecular weight of28kDa,and the inhibition zone test indicates that DCD-1L has obvious inhibiting effects on B.licheniformis, S.aureus, P.aeruginosa and S.pneumoniae.Conclusion:The expression vector pET-32a-DCD-1L was successfully constructed and expressed in E.coli transetta(DE3), the expression product DCD-1L has obvious inhibiting effects on Gram-positive and Gram-negative bacterium. This research establishes the foundation for the recombinant expression of antimicrobial peptides and clinical application research. This research establishes the foundation for the recombinant expression of antimicrobial peptides and clinical application research.