From Gene Design To Activity Characterization, A New Antibacterial Peptide: LfampinB/LfcinB

Lactoferrin was first isolated from bovine milk, and it has many biological functions, including antibiosis, antivirus, anti-inflammatory, immunological regulation etc. Bovine lactoferrampin (LfampinB) and bovine lactoferrcin (LfcinB) are two antimicrobial fragments, situated in close proximity in the N1-domain of bovine lactoferrin (bLF). They are released by proteolytic digestion of bLF, and their antibacterial activities are more powerful than bLF. In recent years, many studies of LFchimera which contains LfampinB and LfcinB conducted that its antibacterial activity is much stronger than its constituent. LFcimera as an antibiotic peptide, it will display an extensive research perspective in substituting traditional antibiotics.By comparing the prokaryotic and eukaryotic expression, the author constructed the high-level expression vector LfampinB/LfcinB and wished to product antibactericidal peptide for controling diseases. According to the LfampinB and LfcinB structural features and antibacterial mechanism, the recombinant LfampinB/LfcinB gene was designed and synthesized. To obtain the prokaryotic expression vector pME290-SDLfa/Lfc, the LfampinB gene was linked to the pPL-SD plasmid, and then cloned on the expression vector pME290. With the multiple clone site of excretive expression vector pPIC9K in Pichia pastoris, the LfampinB/LfcinB recombinant gene was cloned on the expression vector pPIC9K, the eukaryotic expression vector pPIC9K-Lfa/Lfc was constructed after the identification of restriction enzymes, PCR and sequencing.The pME290-SDLfa/Lfc was transformed into the competent cells of pseudomonas aeruginosa for expression, the supernatant was analyzed by SDS-PAGE after freezing concentration, and the result showed that the LfampinB/LfcinB fusion protein was expressed with molecular weight of 16kDa approximately. The pPIC9K-Lfa/Lfc was transformed into Pichia pastoris GS115 strain, and gained the high-copy methanol utilization plus (Mut+) phenotype clone. Under the induction of 0.5~1.0% methanol for 72 hours, the LfampinB/LfcinB fusion protein was highly expressed into supernatant. With the analysis of SDS-PAGE and Gel scanning, the molecular weight of LfampinB/LfcinB was 10kDa and its yield reached 0.23mg/mL.The two kinds of LfampinB/LfcinB fusion protein were detected for antibacterial activity, the result showed that the LfampinB/LfcinB fusion protein which was expressed by pseudomonas aeruginosa had few antibacterial activity, but the LfampinB/LfcinB fusion protein which was expressed by Pichia pastoris GS115 showed much higher antibacterial activity to 10 species of six genus including Escherichia coli C600λ, Fusobacterium necrophorum, Bacillus welchii, Pseudomonas aeruginosa, salmonella typhimurium,Salmonella pullorum, Salmonella choleraesuis, Staphylococcus aureus, Streptococcus agalactiae and Streptococcus dysgalactia.The LfampinB/LfcinB fusion protein may be exploited to be a safe, effective and broad-spectrum antibacterial drug.