Generation Of Chicken Oviduct Transient Bioreactor Expressing Human Lysozyme

Lysozyme (LYZ) can directly hydrolyze the glycosidic β-1,4 linkage of G~+ bacteria and thus has widespread uses in clinic and food industry. Presently available LYZ is mainly purified from chicken egg white, which may have sideeffects for human injection. The source of natural human lysozyme (hLYZ,) is highly limited with potential risk of pathogen contamination. Transgenic bioreactors have many advantages, including high productivity, easy purifucation, faithful post-translational modifications and authentic biological activities, but many technique bottlenecks remain to be overcomed.To generate chicken oviduct transient expression system expressing hLYZ, the correct hLYZ cDNA was subcloned to oviduct-specific espression vectors pOVl and pOV2 and the recombinant vector pOV1LYZ or pOV2LYZ was injected into laying hens via wing vein route following mixing with 25kDa polyethyleneimine. RBB-R-labeled colorimetric assay of egg white of the vector-injected hens showed that the expression level of pOV2LYZ was higher than that of pOV1LYZ and thus was selected for further expression studies. 16 laying hens were divided into 4 groups and each hen was injected with 2, 3 or 4 mg of pOV2LYZ mixed with PEL Following once or twice injection of each hen with 2 mg of pOV2LYZ, secretion of hLYZ into egg white reached a maximum level of 750μg/ml, which lasted for more than 7 days. Although expression levels of 3- and 4-mg injection groups were higher than that of 2-mg injection group, overt sideeffect such as lowed egg production was observed.When activity of the recombinant enzyme dropped to its prior-injection level, the hens were reinjected with same dose of pOV2 vector using same method and evenhigher and longer expression was obtained. Westerning blotting of egg white from vector-injected hens showed that hLYZ was able to be recognized by hLYZ-specific antibody with a expected molecular weight of 14.7 kDa. The results of characterization showed that the rhLYZ expressed in egg white had a similar thermo-stability, pH profile metal ion sensitivity and microbialcidal effect to that of natural LYZ from human milk. These results indicate that an oviduct transcient expression method has been established, with which relatively high levels of active rhLYZ can be produced.