人溶菌酶原核表达载体的构建及其对奶牛乳房炎常见致病菌的抗菌活性OA

To develop a novel biotherapeutic agent with potent antibacterial activity against bovine mastitis pathogens and a low risk of inducing antimicrobial resistance,a recombinant prokaryotic expression plasmid was constructed by inserting the human lysozyme(hLYZ)gene fragment into the pGEX-4T-1 expression vector using recombinant DNA technology.The recombinant plasmid was transformed into an Escherichia coli expression system,and protein expression was induced with isopropyl β-D-1-thiogalactopyranoside(IPTG).Inclusion bodies were purified from the precipitated proteins to obtain highly purified target protein,which was subsequently used to immunize rabbits for the production of specific polyclonal antibodies.Antibody titers were determined by enzyme-linked immunosorbent assay(ELISA),and antibody specificity was verified by Western blotting(WB).The antibacterial activities of the recombinant protein against Staphylococcus aureus,Escherichia coli,and Streptococcus agalactiae were evaluated in vitro.The results showed that the recombinant prokaryotic plasmid pGEX-4T-1-hLYZ was successfully constructed.After transformation into E.coli BL21 competent cells,the highest expression level of hLYZ protein was achieved by induction with 0.5 mmol/L IPTG at 16℃for 12 h.Rabbit polyclonal antibodies against hLYZ were successfully generated using purified hLYZ-GST fusion protein as the immunogen.ELISA results demonstrated that the antibody titer reached at least 1∶102 400.WB analysis revealed a specific immunoreactive band at approximately 45 kDa,indicating good specificity of the prepared rabbit anti-hLYZ polyclonal antibody.In vitro antibacterial assays showed that the recombinant hLYZ protein exhibited significant inhibitory activity against S.aureus,E.coli,and S.agalactiae.These findings indicate that the pGEX-4T-1-hLYZ prokaryotic expression vector constructed through recombinant DNA technology can efficiently express biologically active recombinant hLYZ protein with antibacterial properties,providing a basis for the future development of biological agents for the prevention and control of bovine mastitis.