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題名 | 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 Monoclonal Antibodies Against the Virus |
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作者 | 阮馨瑢; 賴秀穗; | 書刊名 | 中華民國獸醫學會雜誌 |
卷期 | 24:3 1998.09[民87.09] |
頁次 | 頁145-155 |
分類號 | 437.657 |
關鍵詞 | 豬繁殖與呼吸道症候群病毒; 單源抗體; 抗原性; PRRSV; Monoclonal antibody; Antigenecity; |
語文 | 中文(Chinese) |
中文摘要 | 豬繁殖與呼吸道症候群病毒( porcine reproductive and respiratory syndrome virus, PRRSV )臺灣分離株 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, 以酵素 連接吸咐法( ELISA )及間接螢光染色法( IFA )篩選具有分泌特異性抗體之融合瘤,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 k 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,結果發現臺灣分離株與 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。 |
英文摘要 | 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 electon microscopy with negative stain. It was also confirmed by the protein A-gold immunolabelling. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) with Coomassie 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 nuleocapsd 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 chanin. 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. |
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