陳秀男2006-07-262018-07-062006-07-262018-07-062005-07-31http://ntur.lib.ntu.edu.tw//handle/246246/20570本計畫針對感染石斑魚的神經壞死病毒(Nervous Necrosis Virus ；NNV)，以市售的RNA 抽取套組(Trizol)，抽取其RNA，進而利用針對病毒RNA2 的專一性引子，以RT-PCR 的方 式增殖病毒蛋白質鞘的基因片段，再與含真核生物啟動子的表現質體(pTarget T mammalian Express vector)接合，將此質體轉形(Transform)至大腸桿菌中，培養後純化含有病毒鞘蛋白 基因的大腸桿菌質體，以此質體為疫苗轉移感染(Transfer)至石斑魚腦細胞株(GBC1)，以西 方雜交式判斷此細胞株是否產生神經壞死病毒的蛋白質鞘，以體外的方式(In vitro)探討此疫 苗的效果。實驗結果顯示：利用專一性引子NNV-F 及NNV-R 可有效的增殖病毒蛋白質鞘 片段基因，其產物約1345bp，包含完整的增殖病毒蛋白質鞘基因，此段DNA 經與質體結 合後轉形(Transform)至大腸桿菌中，純化的質體以Lipofectin 及電穿透作用(Electroporation) 皆可有效的送入GBC1 細胞株中，利用大鼠抗NNV 抗體以西方雜交的方式，可確認細胞 產生病毒鞘蛋白約42KDa，因此可應用至魚體實驗，進而利用此DNA 疫苗處理石斑魚苗， 以防治NNV 的感染。Nervous Necrosis Virus (NNV) has been demonstrated to be main causes for the cultured marine fish worldwide. In recent decade, Taiwan, cultured groupers have been found to be infected by NNV with a massive mortality especially for the horal fish. The present study attempts to develop the DNA vaccines for protect NNV infection in marine fish. It is successful to extract viral RNA and amplify a 1330 bp product using specific primers (NNV-F&R) against NNV RNA2 by RT-PCR. After gene cloning, the purified plasmid that contained viral capsid protein genome was used as DNA vaccine and transfected into GBC1 cell line by lipofectin or Electroporation. The protection efficacy of DNA vaccine is then evaluated using rat anti-NNV antibody by western blot hybridization method. It is verified that the transfected cell produce 42KDa protein is same as the molecular weight of NNV capsid protein. Therefore, it is hopeful that developed DNA vaccine may be beneficial to grouper aquaculture industry in Taiwan.application/pdf275843 bytesapplication/pdfzh-TW國立臺灣大學漁業科學研究所[SDGs]SDG3[SDGs]SDG14journal articlehttp://ntur.lib.ntu.edu.tw/bitstream/246246/20570/1/932313B002057.pdf