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題名 | 豬生殖與呼吸綜合症病毒「gp5重組蛋白」的表現與單株抗體之製備=Expression of the Gp5 Recombinant Protein of Porcine Reproductive and Respiratory Syndrome Virus and Preparation of Monoclonal Antibodies |
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作者 | 陳金滄; 賴秀穗; Chen, Jing-tsang; Lai, Shiow-suey; |
期刊 | 中華民國獸醫學會雜誌 |
出版日期 | 19990900 |
卷期 | 25:3 1999.09[民88.09] |
頁次 | 頁171-182 |
分類號 | 437.657 |
語文 | chi |
關鍵詞 | 豬生殖與呼吸綜合症病毒; gp5醣蛋白; 單株抗體; PRRSV; Glycoprotein5; Monoclonal antibodies; |
中文摘要 | 經反轉錄聚合�t鏈反應(reverse transcription-polymerase chain reaction; RT-PCR)將豬生殖與呼吸綜合症病毒(porcine reproductive and respiratory syndrome virus; PRRSV)Fl臺灣分離株之gp5醣蛋白基因加以選殖並增幅,且將選殖所得之互補DNA(cDNA)片段植入pGEX-2T表現載體,並於大腸桿菌內進行大量表現,經誘發表現後,得到一分子量45 kDa之GST-gp5重組融合蛋白,以此重組蛋白進行製備單株抗體,共得到6株單株抗體,其中5株屬於lgG���f,1株屬於lgG���k,其輕鍵皆為�R鍵。經西方免疫轉漬試驗分析該單株抗體之特異性,結果皆可與分子量25 kDa之PRRSV病毒封套gp5醣蛋白反應;但皆不具中和PRRS病毒的能力。此外,利用該單株抗體進行間接免疫螢光試驗分析不同PRRS病毒分離株發現,該單株抗體可與臺灣分離株和美國分離株病毒反應,而無法與英國分離株反應,此顯示臺灣分離株與美國分離株之gp5醣蛋白的抗原性較為接近,而與英國分離株之抗原性則有明顯之差異。 |
英文摘要 | The gp5 (glycoprotein5) gene fragment of a Taiwan isolate (Fl strain of porcine reproductive and respiratory syndrome virus (PRRSV) was amplified by reverse transcription-polymerase chain reaction. The resulting cDNA was cloned into the expression vector pGEX-2T to produce the recombinant protein in prokaryotic cells (Escherichia coli). After induction, the 45 kDa GST-gp5 (glutathione-s-transferase-gp5) fusion protein was expressed. The fusion protein was used to prepare monoclonal antibodies (MAbs). Six hybridoma cell lines secreting MAbs against the 25 kDa viral envelope protein (gp5) were obtained. Five of six MAbs belong to the lgG���f isotype with light chain and the other one is lgG���k with�R light chain. None of MAbs could neutralize PRRSV. In addition, these MAbs could react with Taiwan and America isolates of PRRSV by immunofluorescent assay, but failed to react with Britain isolate. These results indicate that Taiwan isolates of PRRSV were antigenically closely related to America isolates, but distantly related to Europe isolates. |
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