Cloning and Expression of the Nucleocapsid Protein Gene from CDV and Production of the Monoclonal Antibodies

• Main Authors: Tsung-Hsun Hsieh, 謝宗訓
• Other Authors: Ming-Huei Liao
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/97888724498512691856

碩士 === 國立屏東科技大學 === 生物科技研究所 === 97 === Canine distemper (CD) causes generalized infection in dogs with prominent respiratory, gastrointestinal and central nervous system. It had a highly contagious and frequently fatal by a broad range of carnivore species such as domestic dogs and martens. CDV is a member of the genus Morbillivirus of the family Paramixoviridae. CDV is an enveloped virus with single stranded, non-segmented, and negative-sense RNA, it contained six genes in order 3’-N-P-M-F-H-L-5’. In this study, the N gene (1593 bp and 434 bp) of CDV was amplified by reverse transcription polymerase chain reaction (RT-PCR), and PCR products were confirmed by DNA sequencing. Subsequently, the N gene was subcloned into prokaryotic expression vector (pET32a) and transformed into E.coli (BL-21 (DE3) pLysS) for expression. The expressed protein was analyzed by SDS-PAGE and purified by affinity column. The purified protein was used to immunize BALB/c mice. The anti-serum of recombinant protein can recognize the N protein CDV by Western blot assay. The myeloma cell (NS-1) will fuse with spleen cell from immunized BALB/c mice to produce hybridoma cells. Antibodies secreted from hybridoma cells were screened by ELISA, Western-blot, Immuno-dot. There were 2 monoclonal antibodies prepared in this study. They can be applied to detect and diagnosis CDV in the future.