牛流行熱病毒(bovine ephemeral fever virus, BEFV)為子彈型病毒科(rhabdoviridae)成員之一，會感染牛隻造成急性發熱，精神抑鬱(呻吟苦悶)、食慾不振、呼吸症狀、流產、產乳量下降等症狀，影響畜牧業經濟損失。BEF在台灣牛場的盛行率達56.8％，因而死亡或淘汰之牛隻為5％。現行疫苗為民國73年所分離之病毒株經福馬林不活化後，加入氫氧化鋁膠作為佐劑製成不活化疫苗，保護力仍不理想。DNA疫苗是近年來熱門且吸引人的新疫苗研究方向，可提升體液免疫反應及細胞免疫反應，搭配佐劑使用，可提升效力。

　　本研究擬以牛流行熱外套膜醣蛋白G基因為標的基因，結合細胞激素IL-2 (interleukin-2) DNA及GM-CSF (granulocyte/macrophage-colony stimulating factor) DNA之加強作用，研究此DNA疫苗的效力。在轉染細胞48小時後，抽取細胞RNA以RT-PCR進行RNA表現測定，結果顯示三種重組載體DNA皆有進行基因轉錄作用，轉錄之RNA定序結果與各載體攜帶之基因相符，亦顯示本研究所構築的三種重組載體皆可有效在哺乳類動物細胞中進行基因表現。另外，於BHK-21細胞預先轉染載體pcDNA3.1/BEFV-G，於12、24、36、48小時後，以劑量為100TCID50之 BEFV感染細胞，繼續培養48小時後，收集細胞培養液進行病毒效價測定(TCID50)。結果顯示，轉染載體後36小時內，病毒生長未受抑制，僅48小時後略下降，表示G載體於細胞內對於病毒後續感染未造成明顯干擾。

　　動物實驗方面，將BALB/c小鼠肌肉注射各重組載體DNA疫苗及佐劑，每間隔二週追加一劑，共施打三劑，並於每週進行採血，測定抗體反應及中和試驗，顯示同時免疫接種三種載體(pcDNA3.1/BEFV-G, pcDNA3.1/IL-2, pcDNA3.1/GM-CSF) 的組別，血清對於病毒之抗體效價(ELISA) 較為顯著，追加免疫後，血清效價上升更為顯著；其病毒中和性抗體產生亦有明顯反應。

　　Bovine Ephemeral fever Virus (BEFV), a member of the rhabdoviridae, can cause a sudden onset of fever, depression, lameness, respiration symptoms, increase in abortions and reduce milk production in cattle. It can cause significant economic impact in livestock industry. The disease incidence in Taiwan is up to 56.8％ and case fatality is about 5%. The vaccine currently used was formaldehyde-inactivated virus of 1984 isolate in Taiwan, but its protection is not effective enough. DNA vaccine is a novel vaccine developing field. It can elicit persistent humoral- and cell-mediated immune responses. Recent studies have found that certain combinations of DNA vaccine, such as supplementation of cytokine DNA, could amplify immune response significantly.

　　Our research aimed to study the efficacy of the DNA vaccine pcDNA3.1/G using glycoprotein G gene of BEFV as a target gene combined with IL-2 and GM-CSF gene DNAs (pcDNA3.1/IL-2 and pcDNA3.1/GM-CSF, respectively) as adjuvants. First of all, we detected the individual target gene expression ability of these three recombinant vectors in BHK-21 cell culture model. After 48 hours’ transfection, we extracted total cellular RNA and detected equivalent gene expressions. The result showed that all of three recombinant vectors could express efficiently. Their sequencing results also matched perfectly with those recorded in NCBI nucleotide database. If BHK-21 cells were pre-transfected with pcDNA3.1/BEFV-G, then were infected with BEFV (100TCID50) after 12, 24, 36 and 48 hours transfections. Cells were then cultured for more 48 hours. Culture medium was harvested and pooled to detect viral titer (TCID50). The result showed that viral replication was not inhibited for 36 hours pre-transfection group, but inhibited slightly for 48 hours pr-transfection group. It showed that pcDNA3.1/BEFV-G could not cause interference in subsequent viral infection.

　　The female BALB/c mice were passively immunized by intramuscular inoculation with carrying vector, pcDNA3.1/BEFV-G or pcDNA3.1/BEFV-G combined with pcDNA3.1/IL-2 and/or pcDNA3.1/GM-CSF. Booster injection were performed twice at 2-weeks intervals. Blood samples for serology assay were obtained by orbital bleeding on every week. The results showed that mice immunized with combined DNA vectors (pcDNA3.1/BEFV-G, pcDNA3.1/IL-2 and pcDNA3.1/GM-CSF) had better antibody responses than those immunized with pcDNA3.1/BEFV-G alone. After final booster inoculation, serum antibody titers were further upgraded. And virus-neutralizing antibody titers were also dominantly found.

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