Cloning And Fusion Expression Of Antibactorial Peptide LactoferricinB In Escherichia Coli

Background & Objective LactofenicinB is a 25-residue antimicrobial peptide release by pepsin cleavage of lactoferrin, an 80kDa iron-binding glycoprotein with many immunologically important functions. Several studies have shown that lactoferricin B has a broad-spectrum activity against various gram-positive and gram-negative bacterial. In addition the peptide has been shown to have antifungal, antiviral activity. Moreover, it is known to bind lipopolysaccharides, thus protecting organisms from the harmful effects of sepsis. Moreover, the peptide is apable of stimulating the adaptive immune respones and has anti-inflammatory properties. All of the beneficial physiological activities make it an obvious choice for detailed molecular studies. In this reseach, we want to construct the prokaryotic expression plasmid of lactoferricinB gene by recombinant technology and express fusion protein for identifying antibacterial potency. Methods Based on the amino acid sequence of lactoferricinB, We designed the cDNA sequence and obtained lactoferricinB gene through ologodeoxynucletides synthesis. Then lactoferricinB gene was inserted into the prokaryotic expression vector pGEX1 λ T and recombinant plasmid pGEX1 λ T-LfcinB was transformed into E.coli JM109. The positive clonewere identified by restriction enzyme analysis and PCR. Fusion protein was expressed after the host bacterium was induced with IPTG. Assaying the expression product with SDS-PAGE. Fusion protein was purified with GSTrap FF affinity chromatography and cleaved by thrombin. Then testing antibacterial activity.Result The evidences of enzyme digestion. PCR and sequence analysis confirmed that lactoferricin B gene has been correctly inserted into pGEX-1 A. T vector. The recombinant expression plasmid pGEXl X T-LfcinB successfully expressed GST/LfcinB fusion protein in E.coli JM109. But the expression product has no antibacterial activity to most of the tested pathogenic bacteria after purification and enzyme cleavage. Conclusion we succeeded in constructing the recombinant plasmid expressing GST/LfcinB fusion protein. The construction of prokaryotic expression plasmid of lactoferricin B gene established a solid basis for further studying the antibacterial potency .