<div>Background: Mastitis caused by Staphylococcus aureus remains a significant challenge in dairy livestock due to the emergence of multidrug-resistant strains and the limited effectiveness of conventional therapeutic approaches. Lysostaphin, a staphylolytic enzyme with high specificity against Staphylococcus species, has emerged as a promising alternative antimicrobial agent. In this study, we aimed to establish a preliminary mammalian expression strategy for production of biologically active recombinant lysostaphin in buffalo fibroblast cells. 
 
Methods: A modified lysostaphin gene, designed to eliminate predicted N-glycosylation sites, was synthesized and initially cloned into the pUC57 vector followed by subcloning into the pcDNA3.1/CT-GFP-TOPO expression vector. Recombinant constructs were confirmed by colony PCR and Sanger sequencing before transfection into buffalo fibroblast cells. Recombinant expression was qualitatively assessed using GFP fluorescence and antibacterial activity was evaluated using a qualitative plate inhibition assay against Staphylococcus aureus. 
 
Result: The lysostaphin gene (~798 bp) was successfully amplified and cloned into the mammalian expression vector. Screening and sequencing confirmed the correct insertion and orientation without mutations. Transfected buffalo fibroblast cells exhibited GFP fluorescence, indicating successful expression of the recombinant construct. Lysates from transfected cells produced visible zones of inhibition against S. aureus, indicating antibacterial activity of the expressed lysostaphin. 
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A Simplified and Robust Approach for Recombinant Lysostaphin Production in Buffalo Fibroblast Cells