کلونینگ، بیان، خالص سازی و ارزیابی آنزیم لیزواستافین نوترکیب با استفاده از سیستم کلونینگ pBAD

Background and purpose: Staphylococcus aureus is an important nosocomial pathogen which causes some diseases such as endocarditis, osteomyelitis, pneumonia, toxic shock syndrome, and food poisoning. The excessive and inappropriate use of antibiotics in the treatment of these diseases causes resistance to many antibiotics. Lysostaphin is an effective agent in the treatment of staphylococcal infections. The purpose of this study was to produce recombinant lysostaphin using pBAD cloning system. Materials and methods: In first phase, the recombinant plasmid pBAD was built using pBAD/ Thio-TOPO system (pBAD). Then, the gene expression of lysostaphin was determined by Reverse Transcriptase-test. Afterwards, the gene expression was induced by l (+) - arabinose to a final concentration of 0.2% and the expressed protein was purified by affinity- chromotagraphy using Ni-NTA agarose (Qiagen, Hilden, Germany). Finally, the enzyme activity was evaluated by Mueller Hinton agar medium and inhibition zone was measured. Results: The results of PCR and sequencing confirmed that the recombinant plasmid pBAD was created successfully. Protein expression using the l (+) - arabinose to a final concentration of 0.2% induced a very high concentration of the recombinant enzyme. Lysostaphin recombinant was found with highly specific activity against Staphylococcus aureus. Conclusion: Lysostaphin recombinant enzyme using pBAD expression system is very cost effective and highly active against Staphylococcus aureus.