王金和2006-07-262018-07-092006-07-262018-07-091999http://ntur.lib.ntu.edu.tw//handle/246246/28673依照己定序完成之台灣雞傳染性支氣 管炎病毒分離株A1171 的S1 醣蛋白基因序 列設計引子IBVc1-IBVc2，利用此引子進行 反轉錄聚合脢連鎖反應，增幅出539bp 的 PCR 產物，其序列位置包括不具病毒中和能 力的抗原決定位S1-F、S1 和S2 之間的裂 解訊號及一小部份S2 的N 端。將PCR 產物 與pRSET A,B,C 載體進行接合作用後，轉 化至大腸桿菌勝任細胞內，再利用IPTG 在 原核系統中誘導台灣雞傳染性支氣管炎病 毒分離株S1 醣蛋白基因的表現。表現得到 的S1-F 融合蛋白質分子量約27.5 kD，可 與兔子抗IBV 的多源抗血清反應。將S1-F 融合蛋白當作酵素連結免疫吸附法的抗 原，以I-ELISA 的方法測定血清抗體，証 實S1 基因表現蛋白可有效測定不同雞傳染 性支氣管炎病毒分離株抗血清的抗體。Enzyme-linked immnunosorbent assay (ELISA) using an expression S protein fragment as an antigen was developed for detection of antibodies against IBV. The primers (IBVc1-IBVc2) were designed according to the published entire S1 nucleotide sequence of Taiwan IBV isolate Tw/1171/92. The F frgament of S1 gene was amplified using primers IBVc1-IBVc2, cloned into the pRSET vectors and transformed into competent E. coli BL21 (DE3). A 27.5 kD S1-F fusion protein was successfully expressed, affinity-purified and used as a coated antigen for serum antibody detection by ELISA. Although there was little cross-reactivity in the serum neutralization (SN) test, cross-reactivity was observed in this ELISA system among different IBV strains, H120, Taiwan Group I and Taiwan Group II. The ELISA technique using expression S1-F protein is able to detect antibodies from different IBV serotypes and appears to be suitable for large-scale serological surveys.application/pdf37544 bytesapplication/pdfzh-TW國立臺灣大學獸醫學系暨研究所雞傳染性支氣管炎S1 基因酵素連結免疫吸附法ELISAexpression S1 proteininfectious bronchitisreporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/28673/1/882313B002075.pdf