- 期刊
豬繁殖與呼吸道症候群病毒之結構蛋白分析與其單源抗體之研製
Characterization of the Structural Proteins of Porcine Reproductive and Respiratory Syndrome Virus and Production of Monolonal Antibodies against the Virus
摘要
豬繁殖與呼吸道症候群病毒（porcine reproductive and respiratory syndrome virus, PRRSV）台灣分離株FI，純化後以電子顯微鏡負染色法觀察之，發現病毒顆粒呈多形性，以圓形為主，經由免疫金染色（immunogold）電子顯微鏡的觀察，確認為PRRSV。而病毒蛋白的結構分析係以純化之病毒直接進行SDSPAGE（sodium dodecyl sulfate-polyacrylamide gel electrophoresis），及以西方草轉漬法（Western blotting）分析。結果可觀察到15, 17, 19, 29與42 KDa等病毒蛋白。單源抗體之製備，乃以純化PRRSV免疫BALB/c鼷鼠，取其脾臟細胞與小白鼠骨髓瘤細胞NS-1進行融合，以酵索連接吸附法（ELISA）及間接螢光染色法（IFA）篩選具有分泌特異性抗體之敵合瘤，經過二次的極限稀釋，再以西方轉漬法與放射線免疫沈澱法進行定性分析，最後篩選出一株抗64 KDa病毒蛋白及二株抗15 KDa核蛋白之單源抗體，這些單源抗體皆不具中和病毒的能力，抗64 KDa病毒蛋白之單源抗體為IgG2b，輕鍵為κ鍵，不具有血球凝集抑制的功能，而抗15 KDa核蛋白之單源抗體則具有血球凝集抑制的能力。在抗原性分析方面，以各國分離株之病毒與各不同來源之單源抗體進行交叉間接免疫螢光染色，結果發現台灣分離株與美國分離株之抗原性較相近，而與英國分離株之抗原性差異較大。
並列摘要
Porcine reproductive and respiratory syndrome virus (PRRSV) of Taiwanese isolate FI was purified by sucrose gradient centrifugation. Pleomorphic, mostly spherical, virus particles with the sizes of 50-60 nm were observed in all fractions of the gradient by the transmission electron microscopy with negative stain. It was also confirmed by the protein A-gold immunolabelling. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) with Coomaasie Blue R250 staining and Western blotting were used to analyze the viral proteins. The presentation of protein bands was variable due to different methods, reducing or nonreducing conditions for analysis. The bands of the 29 KDa and the 42 KDa envelope proteins were most predominant. 15, 17, and 19 KDa of proteins could be observed as well. Monoclonal antibodies against PRRSV were produced by fusing NS-1 myeloma with the splenocytes of BALB/c mice which were immunized with purified PRRSV. The hybridomas were selected by the medium with HAT (hypoxanthine, aminopterin, thymidine). Then enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFA) were used to screen the cultural supernatant of hybridomas. The hybridomas with the specific antibody-activities were further cloned twice by limiting dilution. Monoclonal antibodies were then characterized by Western blotting, radioimmunoprecipitation, and isotyping. After this, three monoclonal antibodies were obtained. Two of the three monoclonal antibodies with the ability of hemagglutination-inhibition precipitated the 15 KDa viral nuleocapsid protein by radioimmunoprecipitation. The other one without the ability of hemagglutination-inhibition recognized the 64 KDa viral protein by Western blotting and belonged to IgG2b with K light chain. None of them could neutralize the viruses. Furthermore, the monoclonal antibodies to the American, British, and Taiwanese isolates were reacted with eleven different PRRSV isolates by IFA. The results indicated that the six Taiwanese isolates of PRRSV were antigenically closely related to American isolates, but distantly related to European isolates.
延伸閱讀
- Peng, J. Y. (2018). 豬流行性下痢病毒之病變、診斷試劑開發及抗病毒飼料添加物研究 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU201801238
- 鍾文彬、鄭倉瑋、林敏雯、楊平政（1995）。應用豬隻周邊血液單核球檢測豬繁殖與呼吸道症候群病毒。中華民國獸醫學會雜誌，21(5)，308-315。https://doi.org/10.7009/JTCSVS.199510.0308
- Kaewprom, K. (2020). 台灣豬生殖與呼吸道病毒序列分析及抗病毒中藥之篩選 [doctoral dissertation, National Pingtung University of Science and Technology]. Airiti Library. https://doi.org/10.6346/NPUST202100020
- 邱宇恩（2018）。Investigation of the Stability and Proteasome Degradation Efficiency of Influenza A Virus PB1-F2 Proteins from Different Viral Strains in Human Cell Lines〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU201803869
- 黃淑敏、莊秀琪、連毅、鍾文彬（2002）。Distribution and Quantitation of Porcine Reproductive and Respiratory Syndrome Virus in Naturally Infected Pigs。臺灣獸醫學雜誌，28(2)，125-133。https://doi.org/10.7009/TVJ.200206.0125