- 學位論文
豬環狀病毒二型ORF1，ORF2及ORF3基因表現蛋白的抗原性分析
Antigenic Analysis of ORF1, OR2 and ORF3 of Porcine Circovirus Type 2 Expressed Viral Peptides
摘要
豬環狀病毒二型(porcine circovirus type 2)引起豬的PMWS症狀，豬場的感染率很高，造成養豬業很大的損失，且目前無疫苗可預防。本實驗應用聚合酶鏈反應(`polymerase chain reasction, PCR)由PCV2感染豬隻的淋巴結乳劑，經由特異的引子增幅PCV2ORF1、ORF2及ORF3的DNA片段，並以大腸桿菌表現載體pRSET A和DNA疫苗載體pcDNA3選殖出含有ORF1、ORF2及ORF3片段的pRSET A+PCV2 ORFs和pcDNA3+PCV2 ORfs的重組DNA。前者使用大腸桿菌進行蛋白質的表現，並將表現出的蛋白質純化後，進行SDS-page和西方墨點法(Western Blotting)的分析；而後者則在CHO細胞內確定有表現後，以肌肉注射的方式進行實驗兔子的免疫，並使用MagicShuttle protein(MGT)來增加DNA 轉染細胞的效力，再以免疫螢光試驗測定兔子血清內PCV2抗體和中和抗體的力價。結果結果如下，大腸桿菌表現出的ORF1及ORF2可溶性蛋白質和ORF3不可溶性蛋白質可以被PCV2多株抗體所辨識，但ORF2蛋白質無法被PCV2 ORF2單源抗體所辨識。在蛋白質的大小方面，只有ORF1蛋白質和ORF2蛋白質的大小符合預期，ORF3蛋白質的大小和預期大小差異很大。以pcDNA3構築的PCV2 3個ORFs的DNA疫苗，經免疫兔子後， ORF2和ORF3組的血清中可測到PCV2的抗體，而ORF1則無。具有PCV2特異性抗體的血清，經病毒中和抗體試驗結果，僅ORF2激發的血清以及含PCV2淋巴結乳劑免疫的血清具有中和10倍TCID50 PCV2的能力。
並列摘要
Porcine circovirus type 2 (PCV2)cause PMWS in weaning piglets, and was found highly infectious in pug farms. Polymerase chain reactions (PCR) was used to amplify ORF1, ORF2 and ORf3 of PCV2 from infected lymph node emulsion by using specific primers. E. coli expression vector pRSET A and DNA vaccine vactor pcDNA3 were used as the plasmid to clone the 3 ORFs of PCV2, respectively. Recombinant proteins of ORF1, ORF2 and ORF3 of PCV2 were expressed in E. coli were analyzed by SDS-PAGE and western blotting. The DNA vector vaccine were used to immulized rabbits by intramuscular injection. MagicShuttle protein(MGT), a kind of DNA binding protein, were also used to increase the efficiency of DNA transfection. PCV2 antibody and viral neutralizing antibody of immunized rabbits serum were tested by indirect immunofluorescent assay (IFA). The result of E. coli expression showed that the soluble form of ORF1 and ORF2 recombinant proteins, and the inclusion form of ORF3 recombinant protein could be recognized by PCV2 polyclonal antibody but not by PCV2 ORF2 monoclonal antibody. The size of ORF1 and ORF2 recombinant proteins were correct, but that of ORF3 was bigger than the expected size. Sera from DNA vector vaccine immunized rabbits showed that ORF2 and ORF3 immunized sera had PCV2 antibody titers, but not found in ORF1 immunized sera. Viral neutralizing antibody activities were only detected in sera collected in ORF2 and PCV2 infected lymoh node emulsion immunized sera：the neutralizing activities was only found in using low amount of PCV2 (10 TCID50).