Studies On The Animal Mammary Gland Bioreactors For Human Lysozyme

Human lysozyme (hLYZ, EC3.2.1.17) hydrolyzes the glycosidic B-1, 4 linkage between N-acetyhnuramic acid and N-acetylglucosamine of the peptidoglycan polymer in the bacterial cell wall. Because of the direct exposure of polypeptidoglycan on the outside of the cell wall, hLYZ can directly lyze Gram-positive bacteria and indirectly lyze Gram-negative bacteria in the presence of slgA or complements. Furthermore, hLYZ has many other activities such as diminishing inflammation, detumescence, repairing putrescence tissues, improving blood supply in local tissues, reducing pus and anti-virus.hLYZ can be purified from human breast milk, neutrophils, placenta and urine of hemodialysis patients. Because of the limited supply and concerns over the potential risk of transmitting pathogens to the nursing infants, development of safer recombinant hLYZ is needed. Expression of rhLYZ in prokaryotic bacteria or yeast by utilizing gene-engineering techniques has some difficulties due to their lack of appropriate post-translational modifications and the potential lysis of the host cells. Animal mammary gland bioreactor is a novel alternative strategy for production of recombinant proteins with many advantages including high production capacities, easy purification, faithful translational modifications and authentic biological activities of the expressed products.To develop animal mammary gland bioreactors expressing hLYZ, 1.5 kb dscDNA for hLYZ was amplified by PCR from pooled first-stranded cDNAs from human mammary gland and subcloned into pGEM-T vector. Sequence analysis showed that the PCR-amplified cDNA was identical to that cloned from human placenta, macrophages and colon. The cDNA included an open reading frame of 447 bps and a pair of Alu elements in reverse orientation at the 3' -noncoding region. The open reading frameencoded a polypeptide of 130 amino acids, the first 18 amino acids of which were predicted to be signal pepeptide. The mature enzyme had a predicted molecular weight of 14.7 kDa and pI of 9.02. The deduced amino acid sequence of the cDNA was also identical to that cloned from human placenta, macrophages and colon and differed by 1 or 5 amino acids from that cloned from histiocytes or Chinese placenta.The cDNA was subcloned into eukaryotic expression vector pcDNA3 and transfected into COS-1 cells following mixing with 25 kDa branched polyethylenimine(PEI). Expression of hLYZ cDNA was revealed by indirect immunofluorecence using hLYZ-specific antibody. The hLYZ cDNA was then subcloned into mammary gland-specific vectors p205C3, pBJ41 and pBCl. After mixed with 25 kDa PEI, the three recombinant expression vectors p205C3LYZ, pBJLYZ and pBCLYZ were injected into lactating mice via tail vein route. Micrococcal lysis assay of the collected milk samples showed that rhLYZ was efficiently expressed in the mammary glands and secreted into the milk with maximal concentrations of 87, 69 and 60 mg/L, respectively, for the three recombinant vectors. The expression of the three vectors was restricted to mammary glands with some degrees of ectopic expression in spleen, intestines and/or kidney among 12 different tissues tested by dot blotting and micrococcal lysis assay. These data indicate that the self-constructed mammary gland-specific vector p205C3 can replace the other two vectors obtained from abroad for development of animal mammary gland bioreactors.The recombinant vector p205C3LYZ was used to generate transgenic mice by microinjection. A total of 136 F0 mice were obtained, of which 7 (2 5 ) and 4 (1 3 ) were showed to be transgenic by PCR and Southern blotting, respectively. Based on the micrococcal lysis assay of the milk samples, the only one female founder did not express the gene of interest. The three male founders were mated with normal mice and four female offspring of the only one male founder expressed hLYZ activities at levels of 5-200 mg/L. From the F1 generation on, the transgenic mice were mated with their sisters or brothers and a mouse colony was established with stable transmission and expre...