Abstract
Human lysozyme is an important natural non-specific immune protein that is highly expressed in breast milk and participates in the immune response of infants against bacterial and viral infections. Considering the medicinal value and market demand for human lysozyme, an animal model for large-scale production of recombinant human lysozyme (rhLZ) is needed. In this study, we generated transgenic cloned cows with the marker-free vector pBAC-hLF-hLZ, which was shown to efficiently express rhLZ in cow milk. Seven transgenic cloned cows, identified by polymerase chain reaction, Southern blot, and western blot analyses, produced rhLZ in milk at concentrations of up to 3149.19 ± 24.80 mg/L. The purified rhLZ had a similar molecular weight and enzymatic activity as wild-type human lysozyme possessed the same C-terminal and N-terminal amino acid sequences. The preliminary results from the milk yield and milk compositions from a naturally lactating transgenic cloned cow 0906 were also tested. These results provide a solid foundation for the large-scale production of rhLZ in the future.
Highlights
With different rhLZ constructs[6,20], the low expression of rhLZ in the mammary gland of those cows has limited its commercial application
The results of our previous studies showed that the vector pBAC-hLF-hLZ-Neo can efficiently express rhLZ both in mice[22] and pigs[23]
Human lysozyme is abundant in breast milk and plays a major role in the innate immune system of infants
Summary
With different rhLZ constructs[6,20], the low expression of rhLZ in the mammary gland of those cows has limited its commercial application. The results of our previous studies showed that the vector pBAC-hLF-hLZ-Neo can efficiently express rhLZ both in mice[22] and pigs[23]. In consideration of bio-safety issues, we modified the vector pBAC-hLF-hLZ-Neo and removed the marker gene neomycin in vitro. Using this marker-free vector, we generated seven transgenic cloned cows that produced rhLZ in milk at concentrations of up to 3149.19 ± 24.80 mg/L. These transgenic cows produced high concentrations of rhLZ in milk, thereby laying a solid foundation to produce rhLZ via the mammary gland bioreactor system and to improve milk quality and resistance to mastitis
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