Cloning And Expression Of Cathelicidins Bac5 From Dairy Cattle And The Antimicrobial Activity Analysis

In recent years, the drug resistance problem is becoming more and more serious due to the long-time abuse of antibiotics, which has increased the difficulty of prevention and treatment of the animal diseases. Meamwhile, the antibiotic residues in animal product have a great influence on the security of animal derived food. Therefore it is urgent to research and develop some kind of new environment-protection antibiotics. Antimicrobial peptide widely distributes in plants and animals, and plays an important role in the innate and acquired immunity. Moreover it has a powerful antimicrobial effect on the resistant strains. So it has got much attention. Bovine Bac5 with extended helical structure is a kind of potent cationic antimicrobial peptide, which is classified as the Cathelicidins and plays an important role in preventing bovines from invasion of foreign pathogens. The traditional technique which extracts Bac5 from the bovine tissues has the shortage of complex operation, high cost, low yeild and unavailability of large-scale production. Application of genetic engineering technology to produce Bac5 recombinant protein is expected to solve this problem and has become the new direction to develop new enviroment-protection antimicrobial agents.In the present study, based on the bovine Bac5 gene sequences reported in the GeneBank datebase, a pair of specific primers was designed and synthesized, then the Bac5 gene fragement was amplified by RT-PCR from the total RNA of Holstein bull bone marrow. After the digestion of EcoRⅠand XhoⅠ, the target gene fragement was subcloned into the prokaryotic expression vector pET28a to construct the recombinant expression plasmid pET28a-Bac5, the recombinants were transformed into E.coli DH5α. Screening the positive clone strains in the LB culture medium with kanamycin(50μg/ mL), identified by PCR amplification, double restriction enzyme digestion and sequencing, the recombinant expression plasmid pET28a-Bac5 was transformed into E.coli Rosetta. Screening the positive clones, and identified by PCR and sequencing again, the positive E.coli Rosetta liquid was induced by IPTG for expression, and the expressed product was analyzed by SDS-PAGE. Then the antimicrobial activity of expressed recombinant protein(Bac5) was identified by antimicrobial tests with 2 standard strains(CVCC1450 and CVCC545) and 3 main strains separated from mastitis milk(escherichia coli, staphylococcus aureus and streptococcus) . The results suggest that Bac5 gene fragement without signal peptide was successfully amplified from bovine bone marrow, and the recombinant expression plasmid pET28a-Bac5 was also successfully constructed. Sequencing result shows that the inserted target gene fragement is 414bp and encoding 137 amino acid residue, which is 100% homologous compared with the bovine Bac5 gene sequences reported in the GeneBank datebase(L02650.1), and the open reading frame is correct. E.coli Rosetta containing recombinant expression plasmid pET28a-Bac5 expressed the Bac5 recombinant protein with the molecular weight of about 19 KD, which is consistent with theoretical prediction. The results from antimcrobial test illustrate that the three main strains separated from mastitis milk have some drug resistance to Penicillin and Streptomycin, but is sensitive to the Bac5 recombinant protein. In addition, Bac5 recombinant protein showed strong antibacterial activity to the two standard strains.The present study lays the foundation for the clinical treatment of mastitis and the research and development of novel and environment-protection antibacterial preparations.