Volume 25, Number 128 (9-2015)                   J Mazandaran Univ Med Sci 2015, 25(128): 18-28 | Back to browse issues page


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Mordadi A, Haji Ahmadi F, Soleimani Asl S, Arabestani M R, Szweda P. Cloning, Expression, Purification and Evaluation of Recombinant Lysostaphin using pBAD Cloning System. J Mazandaran Univ Med Sci. 2015; 25 (128) :18-28
URL: http://jmums.mazums.ac.ir/article-1-6115-en.html

Abstract:   (2877 Views)
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.
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Type of Study: Research(Original) | Subject: Microbiology

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