AAAV Sequence-Enhanced Expression, Characterization And Quantitative Detection Of Recombinant Human Tissue Kallikrein In Hen Oviduct

Adeno-associated virus (AAV) is a promising vector for gene therapy. 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 Human Tissue Kallikrein (rhK1) gene, we inserted the oviduct-specific expression cassette between the AAAV ITRs and included the CMV promoter-controlled Rep-expression cassette in the same vector, resulting in recombinant vector pRep2ITR-OV4-KLK1. The AAAV sequence-containing vector, as well as the control vector pOV4K, was injected into egg-laying hens via wing vein route after mixing with polyethyleneimine (PEI) at N/P=5. After twine injection, egg whites were collected for enzymatic assay. The results showed that a higher and longer hK1 expression was observed in egg whites of pRep2ITR-OV4-KLK1-injected hens than that in the control vector-injected birds. These data indicate that AAAV ITRs and/or Rep gene can promote KLK1 gene expression in an oviduct-specific manner without evidence for persistent expression.To characterize the rhK1, egg white of the vector-injected laying hens submitted to Western blotting analysis using an antibody specific for human tissue kallikrein. The results showed that a specific protein band of 37kDa was detected, which corresponded to the mature enzyme. Further biochemical studies showed that the rhK1 had a similar thermo-stability, optimal pH and sensitivity to different ions to the natural enzymes from human and porcine tissues.To establish a quantitative detection method for rhK1, by using affinity-purifed monoclonal antibody against hK1 as the coating antibody and the E.coli-expressed GST-KLK1 fusion protein as the known antigen, a sandwich ELISA was established. Under the optimized conditions, the ELISA reading values had intra- and inter-plate linear relationship with the antigen concentrations ranging from 16μg/ml to 125μg/ml. The egg whites of the vector–injected hens were submitted to the ELISA detection and the hK1 of 332, 476 and 248μg/ml was detected at days 3, 6 and 9 after vector injection, respectively.