以桿狀病毒表現犬尿胺酸3-單氧化酶並探討其在犬乳房腫瘤診斷之應用

Abstract

The kynurenine pathway is a major route for tryptophan metabolism where several metabolites have been linked to the pathogenesis of neurodegenerative disease, as well as tumor formation. Our previous studies showed that the expression level of kynurenine 3-monooxygenase (KMO), an outer mitochondrial membrane enzyme and central to the kynurenine pathway, was positively correlated with the canine mammary tumor (CMT) malignancy. So far the study of KMO involved in caner research has been ignored and lacked for species-specific molecular tool. In this study, we successful expressed the canine KMO (cKMO) protein by using baculovirus-insect cell (Sf9 cell) and characterized the sequence by software analysis. The purified recombinant proteins were used as the antigen for developing anti-KMO antibodies to select the most suitable antibody for CMT research. The comparison of KMO amino acid sequence among canine, human and mice shown that three conserved motifs folded in the interior of predicted protein structure, which might be acted as the binding site of cofactor FAD or NADPH. These structures belong to the family of NADPH-dependent flavin monooxygenase. Canine KMO gene was inserted into baculoviral genome by transposon located on the expression vector and the full-length cKMO can be detected on the mitochondria of Sf9 cells infected with these recombinant baculovirus. The best expression conditions have been optimized including 1) baculovirus passage (P3), 2) multiplicity of infection (MOI~1.5) and 3) infection time (72-96 hours). Canine KMO, a mitochondrial membrane protein, was purified using Ni-NTA affinity column under denaturing condition. A yield of 2 mg protein can be generated from 2×108 baculovirus infected Sf9 cells. Furthermore, three mice were immunized with this recombinant protein for antibody production. Hybridoma development will be conducted while the serum antibody titers of mice are elevated. In addition, the expression of KMO could be detected on the surface of CMT cell lines by using flow cytometry. We hope that the specific anti-cKMO antibodies can contribute to theranostic application of CMT in the future.