The Cloning and Expression of Hemagglutinin Gene of Canine Distemper Virus

• Main Authors: Hsien-Hua Hsieh, 謝嫻樺
• Other Authors: Min-Liang Wong
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/78104506721410620226

碩士 === 國立中興大學 === 獸醫學系 === 92 === Canine distemper (CD), is caused by canine distemper virus (CDV) and can induce systemic and neurologic signs. CD is typically an acute-subacute disease, which is very contagious, highly incident, and usually fatal. The extensive use of live attenuated CDV vaccines has reduced the incidence of CD in dogs. CDV is classified in the Morbillivirus genus of the family Paramyxoviridae. Virus particle is surrounded by a lipoprotein envelope; its genome is composed of single-stranded non-segmented RNA of negative polarity. The viral envelope protein, hemagglutinin (H), mediates virus entry and can induce immune response. In this work, we cloned the gene of H protein and expressed H protein in bacteria. We used RT-PCR to amplify H gene, and confirmed its identity by DNA sequencing (GenBank No. AY378091, CDTaichung). Then, the cDNA of H gene was cloned into expression vector pET32a. The plasmids were transformed into the E. coli. strain BL21(DE3)LysS and the expression of recombinant protein was induced by IPTG. Our results showed that the full-length H was not detected by Coomassie blue staining but detectable by Western blotting. Moreover, we created various truncated constructs to express shorter H proteins in E. coli., and three of them were purified by affinity chromatography. In addition, we cloned the H gene into a mammalian expression vector pcDNA3.1(-) and examined the effect of H protein on the promoter of hsp70 gene by CAT reporter gene assay. The results indicated, the promoter of hsp70 was inhibited.