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標題: 利用單源抗體分析家禽里奧病毒株S1133結構蛋白質 σA 之抗原決定位
Mapping of epitopes on structural protein σA of avian reovirus strain S1133 using monoclonal antibodies
作者: 白萬泉
Pai, Wan-Chain
關鍵字: avian reovirus;家禽里奧病毒;monoclonal antibody;epitope;單源抗體;抗原決定位
出版社: 獸醫微生物學研究所
將負責指揮轉譯家禽里奧病毒(avian reovirus)S1133 株結構蛋白質σA 的基因片段選殖,插入由 T7 promoter所調控的大腸桿菌表現載體 pET32a 之中。於轉殖成功的大腸桿菌中加入 IPTG,啟動 T7 promoter 而製造出分子量約 62 kDa 的融合型蛋白質。所誘導表現出的蛋白質經由 S-Tag western blot kit 以及抗 σA 的陽性血清,以 Western blot 進行確認。融合型蛋白先以金屬離子親和性層析法(metal affinity chromatography)進行純化後,再以 recombinant enterokinase 將融合生態(fusion peptide)切除。將表現的σA蛋白免疫BALB/c小白鼠並製備出融合瘤細胞。由融合瘤細胞所分泌抗σA的抗體經由ELISA、Western blot篩選,並經由免疫沈澱法及immunodot分析確認。融合細胞後獲得10株的單源抗體,其中5株的抗體亞型為IgG1; 4株為IgG2a;1 株為IgG2b。經由免疫墨點法分析,4株單源抗體辨識線性的抗原決定位,其餘6株則為辨識結構性的抗原決定位。將這些單源抗體與horseradish peroxidase連結後進行競爭性分析發現有2個抗原決定位的存在(A及B),抗原決定位A為結構性的抗原決定位;抗原決定位 B為線性的抗原決定位。2株經由與horseradish peroxidase連結且辨識不同抗原決定位之單源抗體分別與不同株之家禽里奧病毒進行交叉反應,顯示這些單源抗體可以和不同家禽里奧病毒來源之σA結合。

The coding region of avian reovirus S1133 genomic segment S2, encoding the structural protein σA, was placed under the control of the T7 promoter in the Escherichia coli expression vector pET32a. Induction of the T7 promoter by adding IPTG led to the synthesis of a 62 kDa fusion protein in transformed E. coli. The expressed protein was identified by Western blotting with S-Tag western blot kit and specific antiserum toσA. After purifying the fusion protein by metal affinity chromatography, the fusion peptide was cleaved by recombinant enterokinase and used for the immunization of BALB/c mice for hybridoma generation. The antibodies directed toσA were screened by ELISA, Western blot and identified by immunoprecipitation analysis and immunodot assay. Ten monoclonal antibodies (Mabs) from fusions were obtained; five of them were the subtype IgG1, four were the subtype IgG2a, and one is the subtype IgG2b. By the immunodot assay, four Mabs recognize the linear epitope and the others recognize the conformational epitope. Competitive binding assay using these Mabs coupled with horseradish peroxidase(HRP-Mabs) demonstrated the existence of two distinct epitopes(A and B). Epitope A is defined as conformational epitope, and B is linear epitope. Two HRP-Mabs recognizing distinct epitopes were also used to react with heterologous ARV strains. The results reveal that these Mabs have ability to bind σA derived from different ARV strains, suggesting that both epitopes A and B shared by all all ARV strains tested.
Appears in Collections:微生物暨公共衛生學研究所

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