Environmental Release Of Recombinant Plasmid P215C3LYZ For Treating Bovine Mastitis

Mastitis is one of the most common, hard to control diseases in dairy cows. Our previous studies have shown that the recombinant plasmid p215C3LYZ can express recombinant human lysozyme in cow mammary glands, which can replace antibiotics for treating bovine mastitis. To evaluate the biosafety, the plasmid was used in cattle farms as open system during this environmental release experiment. KanR gene was subcloned into prokaryotic expression vector pQE-31. After transformation and cultivation on Kan-containing agar plates, Kan-resistant E.coli colonies were obtained and an expected recombinant protein was expressed after IPTG induction. Specific antiserum was obtained by immunization of mice. The vector pQE-Kan and vector p215C3LYZ were transfected into COS-1 cells, DH5a E.coli was inoculated into the supernatants and the OD600 values were detected after 24h cultivation. The results showed that growth of the indicator bacterium was inhibited significantly in the presence of Kan, indicating that the Kan resistant protein was not expressed in the supernatants of the vector-transfected cells. Western blotting of the cell lysates confirmed that Kan resistant protein was not expressed in the vector-transfected cells. No Kan resistant protein was detected in concentrated milk samples from cows after intramammary injection with vector p215C3LYZ. The samples of blood, milk, saliva, feces, urine, of the experimental cows, and air,cushion samples from the cattle house were also submitted to PCR detection, which showed only the milk samples one day after plasmid injection was positive. These experimental data demonstrate that the plasmid p215C3LYZ can't be released to the environment and can't express the Kan resistant protein, providing additional safety evidence using the p215C3LYZ vector for treating bovine mastitis.