賴秀穗2006-07-262018-07-092006-07-262018-07-092000http://ntur.lib.ntu.edu.tw//handle/246246/28680豬繁殖與呼吸道症候群(Porcine reproductive and respiratory syndrome, PRRS) 與假性狂犬 病（Peudorabies, PR）為豬重要的傳染病。為了 研究可否利用減毒假性狂犬病病毒做為載體來攜 帶一段PRRS 病毒基因，以便構築雙價疫苗，做為 防疫該兩大疾病之用. 本實驗將PRRS 病毒的GP3 醣蛋白基因（即ORF3 基因）植入取代PR 病毒醣蛋 白基因內，以構築PRRS/PR 複合病毒（chimeric virus）。本實驗首先利用反轉錄聚合脢鏈反應增 幅出PRRS 病毒的GP3 醣蛋白基因片段，再將之植 入pGEX-2T 原核表現載體, 並進行大量表現獲得 分子量約30 kDa 的GST-GP3 融合重組蛋白，然後 利用此重組蛋白製備單株抗體做為篩選和確認 PRRS/PR 複合病毒之用。經過單株抗體的融合製 備，順利獲得抗GP3 重組蛋白單株抗體，該單株抗 體具有辨識PRRS 病毒GP3 醣蛋白的能力；但不具 中和PRRS 病毒功能。另一方面，同樣利用反轉錄 聚合脢鏈反應的選殖方式，將PRRS 病毒的GP3 醣 蛋白基因，選殖入含PR 病毒gG 基因片段的pGX 載體內，再將含GP3 基因的pJ3X 重組質體與減毒 的PRV-gE--TK-病毒完整DNA 共同感染RK-13 細胞 進行同源重組（Homologous recombination）後， 成功構築出PRRS/PR 重組複合病毒（J3X)，經由 南方轉漬試驗、西方免疫轉漬試驗、免疫螢光染色 試驗及免疫過氧化脢染色試驗的驗證得知J3X 複 合病毒基因體內確實嵌有PRRS 病毒的GP3 醣蛋白 基因，並可表現出GP3 重組醣蛋白。J3X 複合病毒 在細胞株所形成的病毒斑，比PR 病毒強毒株的病 毒斑小,未來將進行該複合病毒的抗原性試驗。Porcine reproductive and respiratory syndrome (PRRS) and Pseudorabies (PR) are two impor tant infectious diseases of swine. In order to construct a chimer ic virus, PR virus was used as a vector to car ry a foreign virus gene fragment ORF 3 of PRRS. The total fragment of ORF3 gene was amplified by Rever se-Transcr iption Polymerase Chain Reaction (RT-PCR). The resulting cDNA was inser ted into prokaryotic expression vector (pGEX-2T) system. GST-GP3 (gluthione-S-Transferase-glycoprotein5) expressed fusion proteins having molecular weight about 30 kDa was obtained. The fusion proteins were used as antigens to prepare monoclonal antibodies （Mabs）, and a hybr idoma capabling of secreting the Mabs against the GP3 fusion proteins was successfully produced. The Mabs against GP3 fusion proteins was used for screening and confirming PRRS/PR chimer ic virus car rying the ORF 3 gene expressed proteins. The Mabs can identify native GP3 proteins of PRRS virus, but not to neutralize PRRS virus. . Fur thermore, the RT-PCR products GP3 gene fragments were cloned into the pGX vector contained the gG gene fragment of PR virus. Then, the constructed pJ3X plasmid DNA contained gG gene fragment of PR virus and GP3 gene fragment of PRRS virus and DNA of the attenuated PRV-gE--TK- virus were co-transfected into RK-13 cells. Following homologous recombination in the cells, a PRRS/PR chimer ic virus (designated as J3X chimer ic virus) car r ied GP3 gene fragment in the genome of PR virus was successfully constructed. GP3 recombinant proteins of PRRS/PR chimer ic virus were detected by Southern blotting, Western blotting, Immunofluorescent assay and Immuno-peroxidase staining. Plaques produced by J3X chimer ic virus in RK-13 cell lines were much smaller than those produced by wild-type PR virus. The chimer ic virus J3X will be fur ther investigated on its immunogenicity in pigs.application/pdf32630 bytesapplication/pdfzh-TW國立臺灣大學獸醫學系暨研究所[SDGs]SDG3reporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/28680/1/892313B002069.pdf