- 學位論文
犬瘟熱病毒核鞘蛋白基因之分子選殖表現與單株抗體之製備
Cloning and Expression of the Nucleocapsid Protein Gene from CDV and Production of the Monoclonal Antibodies
摘要
犬瘟熱(Canine distemper, CD)為一種經常造成犬隻呼吸道、消化道症狀及引起全身性或中樞神經系統障礙的疾病，感染對象以犬、貂等食肉目動物為主。在分類上犬瘟熱病毒屬於副黏液病毒科(family Paramyxoviridae)，麻疹病毒屬(genus Morbillivirus)，為具封套，負向單股的RNA病毒。主要可以轉譯出6種結構蛋白分別為N、P、M、F、H、L。本實驗設計二對引子對，以RT-PCR增幅出核鞘蛋白(Nucleocapsid protein, N)全長1593 bp與片段434 bp基因，經定序後與NCBI資料庫中的序列進行比對。再將基因構築至pET32a表現戴體，並利用原核表達系統表現重組N蛋白，接著以膠體電泳SDS-PAGE分析所表現的蛋白質，再將此重組蛋白利用親合性管柱進行純化，之後免疫BALB/c鼷鼠，取其血清進行西方轉漬法以證明其抗原性。隨後將免疫的BALB/c鼷鼠之脾細胞與骨髓瘤細胞(NS-1)進行融合，以製備N蛋白的單株抗體。結果經ELISA、西方轉漬法、免疫墨點法等分析確認後，最後共篩得2株抗體，未來此抗體將可應用於犬瘟熱檢測與診斷上。
並列摘要
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.
參考文獻
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