The Influence Of AAAV Sequences On Oviduct-Specific Expression Of Human Lysozyme Gene

Adeno-associated virus (AAV) is a good vector for gene therapy, human AAV in particular, which can site-specifically integrate its genome or foreign genes into human genome. Avian adeno-associated virus (AAAV) has a similar persistent infection and genome structure to AAAV. To explore the feasibility of using AAAV sequences to promote oviduct-specific expression of human lysozyme (hLYZ) gene, we inserted the oviduct-specific expression cassette between the AAAV ITRs with a CMV promoter controlled Rep-expression cassette in the same vector. The resultant vector, called pRep2ITR-OV4-LYZ, was used for expression study with oviduct-specific expression vector pOV4-LYZ as the control. Egg-laying hens were injected via wing vein route with vector pRep2ITR-OV4-LYZ or control vector pOV4-LYZ with the same dose of 1014 copies. After twine injection, egg whites were collected for enzymatic assay. The results showed that a higher and longer hLYZ expression was observed in egg whites of pRep2ITR-OV4-LYZ-injected hens than that in the control vector-injected birds. Further studies showed that three successive vector injection had a higher and longer hLYZ expression than twice injection. These data indicate that AAAV ITRs and Rep gene can promote transfection and/or integration of oviduct-specific expression vector, but can not significantly promote foreign gene expression.To explore the feasibility of using oviduct-expressed hLYZ for bacterial diseases in fish and dairy cows, hLYZ was purified from egg whites of pRep2ITR-OV4-LYZ injected hens by using direct crystallization method, which showed a relatively high purity by SDS-PAGE. In vitro bacterial growth inhibition tests showed that the purified hLYZ could significantly inhibit pathogenic bacteria for cow mastitis, but not for fish bacteria. Egg white powder containing hLYZ was prepared by using spray drying without significant loss of enzymatic activity.