关键词: 溶血性曼氏杆菌, 真核表达载体, 白细胞毒素, 致病机理

Abstract: [Objective] To amplify the leukotoxin A （LktA） genes from the A1 and A2 serotypes of ovine-derived Mannheimia haemolytica, and construct eukaryotic expression vectors for the LktA proteins of these two serotypes. [Methods] Genomic DNA extracted from A1 and A2 serotype strains was used as the templates for PCR amplification of the LktA gene with specifically designed primers. The amplified LktA genes from both serotypes were cloned into the eukaryotic expression vector Morange2-C1 to construct recombinant eukaryotic expression vectors for the LktA proteins of A1 and A2 serotypes. Recombinant plasmids were verified by double digestion with restriction enzymes EcoRⅠand KpnⅠ and sequencing. The correctly constructed vectors were transfected into 293T cells using a liposome-mediated transfection method. After 48 hours of transfection, total cellular proteins were extracted and identified by Western blotting using an anti-mOrange monoclonal antibody as the primary antibody and goat anti-mouse IgG as the secondary antibody. Additionally, subcellular localization of the target protein was observed using laser confocal microscopy. [Results] The LktA genes of the two strains were successfully amplified by PCR using genomic DNA of A1 and A2 serotype M. haemolytica as templates, with PCR products showing clears band of approximately 2 862 bp on agarose gel electrophoresis, consistent with the expected fragment size. Double digestion of the recombinant eukaryotic expression vectors with EcoRⅠand KpnⅠ produced two distinct bands, with the fragment sizes in agreement with the expected sizes of the target insert and linearized vector. Sequencing of the recombinant plasmid after double-enzyme digestion revealed 100% identity between the obtained sequences and the designed reference sequence, confirming the successful construction of the recombinant expression vector. Cell transfection and Western blotting results showed that the eukaryotic expression vectors Morange2-C1-LktA1 （A1 serotype） and Morange2-C1-LktA2 （A2 serotype） successfully expressed LktA proteins in 293T cells, with specific target bands observed at approximately 130 kDa. Confocal microscopy demonstrated that both LktA proteins were localized to the cell membrane. [Conclusion] Eukaryotic expression vectors for LktA proteins from A1 and A2 serotypes of the ovine-derived M. haemolytica were successfully constructed, and their subcellular localization was determined. These findings provide a valuable basis for further investigation into the molecular mechanisms of LktA interaction with host cell receptors.

Key words: Mannheimia haemolytica, eukaryotic expression vector, leukotoxin, pathogenic mechanism