Cloning and Expression of Hemagglutinin Protein from Canine Distemper Virus and Production of the Monoclonal Antibodies

• Main Authors: Chia-Yu Wang, 王嘉裕
• Other Authors: Ming-Hui Liao
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/41072841154757498702

碩士 === 國立屏東科技大學 === 生物科技研究所 === 97 === (Canine Distemper Virus, CDV) cause dogs to suffer from dog's acute communicable diseases. it is infective to highly keep in touch, high incidence of disease and high death rate and causing the etiology mainly. CDV is a member of the genus Morbillivirus of the family Paramixoviridae. CDV is an enveloped virus with single stranded, linear, non-segmented, and negative-sense RNA, it contained six genes in order 3’-N-P-M-F-H-L-5’. In this study, H protein has higher antigenicity in comparison with amino acid sequence of other CDV. The H gene fragment of CDV was amplified using reverse transcription polymerase chain reaction (RT-PCR) and PCR products were confirmed by DNA sequencing. Subsequently, The H 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 then immunize BALB/c mice. The anti-serum of purified H protein was needed to recognize the H protein of CDV by Western blot. The myeloma cells (NS-1) will fuse with spleen cells from immune BALB/c mice to produce hybridoma cells. The expressed H protein will be coated as antigen. Antibodies secreted from hybridoma cells were screened by ELISA, Western-blot, and Immuno-dot. There are 7 monoclonal antibodies prepared in this study, including 1 anti-pET32a-Tag and 6 anti-H protein antibodies. Immuno-dot and Western-blot analyses suggested that these 7 monoclonal antibodies recognized a linear epitope. The 6 monoclonal antibodies which anti expressed H protein can recognize the H protein of virus. These monoclonal antibodies can be used in further research to develop CDV testing reagent.