Cloning of Feline Herpesvirus 1 Uracil-DNA Glycosylase Gene and Its Expression in Escherichia coli

• Main Authors: Yueh-Min CHU, 瞿玥岷
• Other Authors: Yang-Tsung CHUNG
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/71280138472339150841

碩士 === 國立中興大學 === 獸醫學系 === 92 === There are no clinical diagnosis about the diseases infection both animals and human in the veterinary hospital or in the pet animals clinic in Taiwan. For seeking rapid and sensitivity diagnosis analysis, we used polymerase chain reaction (PCR) to detect the cats having upper respiratory tract diseases of feline herpesvirus 1 (FHV-1). We collected 227 samples of conjunctional and throat secretions from the veterinary hospitals then proceeded PCR. The results showed that we could detect FHV-1. But the sensitivity of primers which we used to detect FHV-1 not very well, so we re-designed FHV-1 uracil-DNA glycosylase (UDGase) primers to detect FHV-1. UDGase hydrolyzes uracil-glycosidic bonds in single- or double-stranded DNA excising uracil and creating alkaline sensitive abasic sites in the DNA. In recent years, this enzyme has been widely used in recombinant DNA technology and become an important tool of DNA manipulation. The results showed that the sensitivity of UDGase primers are better than previous primers, and compared to FHV-1 UDGase of American strain, the identity reach to 99%. Then we cut the UDGase fragment from pBlueBac4.5/His TOPO into E. coli expressing vector to expressed protein. Subsequent enzymatic assay verified that the purified protein does have UDGase activity.