Expression Of Recombinant Lactoperoxidase In The Mammary Gland Of Transgenic Mice Generated With BAC Vectors

As a promising method for production of valuable proteins, mammary gland bioreactor has a wide range of applications. The promoters of milk protein encoding genes including casein, whey acid protein, lactalbumin, lactoglobulin and lactoferrin (LF) are usually used to regulate tissue specific expression of foreign genes in the mammary gland. Utilization of bacterial aritificial chromosome (BAC) in generating transgenic animals has several advantages including integrity of regulatory elements, less positional effect, expression level in a copy dependent manner. In this study we verified the capacity of BAC as efficient expressing vector to regulate the expression of foreign genes. Bovine lactoperoxidase (bLPO) gene was introduced into milk protein-gene-containing BAC vectors and expressed in the mouse mammary gland because of its potential value. LPO combined with hydrogen peroxide and thiocyanide are called lactoperoxidase system (LPS), which is a natural bactericide. LPS has highly potential value because of its multiple biological functions including antifungal, antivirus, anti-free radials and immuno-stimulating activties.Expression efficiencies of three BAC vectors with bLPO genomic sequence were compared in transgenic mice. Three BAC vectors contain gene promoters of bLPO, αsl casein and lactoferrin, respectively. The insertion and expression of bLPO gene in transgenic mice were determined. And the results showed that bLPO promoter failed to drive bLPO expression at the protein level in the milk although a low level of mRNA could be detected. Both the αsl casein and lactoferrin gene promoters successfully drove bLPO expression in transgenic mouse milk. Although the highest level of LPO was observed in one LF BAC transgenic line, the average level of bLPO in LF BAC transgenic mice was lower than that in αsl casein BAC transgenic mice. Western blotting and deglycosylation analysis indicated that recombinant LPO (rLPO) in transgenic milk displayed a similar glycosylation pattern to native bLPO, but with a slight difference in molecular mass. The range of rLPO concentration in αsl casein transgenic mice was from 0.118 μg/μL to 0.216 μg/μL, while the level of ranged from 0.041 to 0.364 μg/μL in lactoferrin transgenic mice. In both types of transgenic mice, the expression level of rbLPO was higher than the LPO level in bovine milk (～0.03 μg/μL). ABTS assay results showed that rbLPO enzyme activity (U/mL) in 1μL transgenic mouse milk was from 0.0198 to 0.0261, about 56.1% ～73.9% activity of 1μg strandard bLPO. Activity of rLPO is concentration dependent. Antibacterial activity of rbLPO was verified by bacterial inhibition ring. Both transgenic mouse milk could inhibit growth of Escherichia coli, Salmonella enteritidis and Staphylococcus aureus as evidenced by the inhibiton ring.The results in this study indicated that rbLPO was successfully expressed in transgenic mice and the highest concentration of rbLPO in transgenic milk was 0.364 μg/μL. Both αsl-casein gene and lactoferrin gene BACs could be used as efficient vectors to express foreign genes. And these two BACs could be applied in generation of transgenic models, study of foreign protein function and large-scale production of foreign proteins.