- 學位論文
牛呼吸道融合病毒重組蛋白之抗原性評估
Evaluation the Antigenicity of Bovine Respiratory Syncytial Virus Recombinant Protein
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摘要
牛呼吸道融合病毒(Bovine respiratory syncytial virus, BRSV)會引起呼吸困難、發燒及間質性肺炎等臨床症狀，也會與其他病毒一起併發，常導致重症牛隻流產、產乳量下降或死亡。文獻中指出牛呼吸道融合病毒有11個結構蛋白質，其中的G蛋白與F蛋白，在病毒感染及致病機制扮演很重要的角色。G蛋白是負責病毒與呼吸道上皮細胞相互連結；F蛋白會與細胞膜上的蛋白融合，幫助病毒進入細胞中，且兩者皆能引起很好的免疫中和作用。本研究已成功構築G基因及F基因至pFastBac HT B載體，且利用昆蟲桿狀病毒表現系統成功表現兩種蛋白，並以SDS-PAGE和西方點墨法確認其分子量大小及抗原性。搭配水包油包水佐劑，免疫於小鼠，以細胞激素試驗偵測mRNA表現量，利用T檢定分析結果，免疫重組G蛋白組及BRSV減毒組，在第4週之IFN-γ表現量與Sf-9控制組具有顯著性差異(P<0.05)。IL-12表現量與Sf-9控制組也具有顯著性差異(P<0.01)。以ELISA偵測小鼠血清中抗體含量，結果顯示重組G蛋白與其他組別皆具有顯著性差異(p<0.001)，重組F蛋白組別與Sf-9控制組也具有顯著性差異(P<0.01)。以Western dot偵測小鼠血清中抗體與BRSV病毒之結合效果，結果顯示重組G蛋白及BRSV減毒組與PBS對照組具有顯著性差異 (P<0.01)。初步結果顯示，重組G蛋白具有抗原性及發展為次單位疫苗之潛力。 關鍵字：牛呼吸道融合病毒、G蛋白、F蛋白、重組蛋白
並列摘要
Bovine respiratory syncytial virus (BRSV) can cause dyspnea, fever and interstitial pneumonia and other clinical symptom. It also occurs with other viruses, and often leading to severe cattle abortion, milk production decline or death. Some research indicated that the BRSV has 11 structural proteins, in which the fusion (F) and the attachment glycoprotein (G) are crucial for virus infectivity and pathogenesis of BRSV. The G protein is responsible for the attachment of the virus to respiratory epithelial cells; the F protein fuse viral and cellular membranes, followed by the insertion of the viral RNA. Both of them are good neutralizing antigen. This study was successfully constructed G and F gene to pFastBac HT B vector, and these two proteins were successfully expressed by the baculovirus expression system, and confirm the molecular size and antigenicity by SDS-PAGE and western blot. Combine with water-in-oil-in-water adjuvant immunize in mice, using T-test analysis results, the IFN-γ expression of immune recombinant G protein group and the BRSV attenuated group at the 4th week was significantly higher than the Sf-9 control group (P<0.05). There was also a significant difference in the expression of IL-12 (P<0.01). ELISA results showed that the recombinant G protein was significantly different than other groups (p<0.001), and the recombinant F protein group was also significantly different than the Sf-9 control group (P< 0.01). Western dot was used to detect the binding effect of antibody in serum to BRSV virus. The results showed that the recombinant G protein and BRSV attenuated group were significantly different from the PBS control group (P<0.01). Preliminary results indicate that the antigenicity of recombinant G proteins could be the subunit vaccine candidates. Keyword: Bovine respiratory syncytial virus (BRSV)、G protein、F protein、Recombinant proteins
參考文獻
參考文獻
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