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

Expression of Canine Kynurenine 3-monooxygenase by Baculovirus for the Diagnostic Application in Canine Mammary Tumor

陳薈宇(Hui-Yu Chen)

指導教授 ： 林辰栖

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10668

摘要

無資料

關鍵字

犬尿胺酸 3-單氧化? ； 犬尿胺酸代謝途徑 ； 桿狀病毒表現系統 ； 粒線體膜蛋白 ； 犬乳房腫瘤

並列摘要

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.

並列關鍵字

kynurenine 3-monooxygenase ； kynurenine pathway ； baculovirus expression system ； mitochondrial membrane protein ； canine mammary tumor

參考文獻

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