牛流行熱病毒（Bovine ephemeral fever virus, BEFV）在分類上屬於子彈型病毒科(Rhabdoviridae)可經由庫蠓等節肢動物感染牛隻；為一具外套膜之單股負性RNA病毒，有六個基因：N、P、M、G、L及GNS，其中前五個基因產物是存在於病毒顆粒中；而GNS醣蛋白則只會在受病毒感染的細胞中偵測到，其功能未知。本研究先將BEFV中GNS基因選殖後，探討其干擾性RNA設計成short hairpin RNA（shRNA）對於病毒複製與G基因表現之干擾效果，並於細胞培養中共轉染G與GNS載體（即pcDNA3.1/BEFV-G及pcDNA3.1/BEFV-GNS），分析二基因間之關係。首先根據西元2001年BEFV台灣分離株醣蛋白GNS基因的已知核酸序列，設計引子進行RT-PCR反應、選殖及構築GNS完整基因於pcDNA3.1/His-TOPO載體中，再設計三段shRNA序列，構築於pENTR/U6載體中（即pU6-shRNA62，pU6-shRNA114及pU6-shRNA1217），利用RNA polymerase III U6 promoter驅動其表現。BHK-21細胞轉染實驗結果確認此三組GNS之pU6-shRNAs皆能專一性抑制GNS基因之表現（抑制程度約為50 %）。將pcDNA3.1/BEFV-G及pcDNA3.1/BEFV-GNS載體不同先後順序共轉染於BHK-21細胞株中，48小時後利用RT-PCR分析G及GNS基因表現，結果顯示與單獨轉染G或GNS基因相比，先轉染G再轉染GNS基因後，不論G或GNS基因表現都被抑制約20 %；但不論是先轉染GNS再轉染G基因，或是同時轉染G和GNS基因之實驗組別，其G和GNS基因表現都至少被抑制50 %以上，顯示G和GNS基因會互相抑制，但機制不明。之後再將三組GNS之pU6-shRNAs轉染於已含G及GNS基因之細胞48小時後，發現GNS基因表現被抑制了約50 %，而G基因表現也被抑制約20 %。將上述干擾性RNA載體分別轉染細胞後，再接種100 TCID50劑量病毒挑戰之，培養48小時後發現三組pU6-shRNAs不能抑制病毒G基因之表現，但可使病毒效價降低約10倍，顯示本研究設計之BEFV GNS基因干擾性RNA可專一性抑制GNS基因表現，也可降低BEFV之病毒效價。

Bovine ephemeral fever virus (BEFV) is caused by an arthropod-borne rhabdovirus. It contains an envelope and a single-strand, negative-sense RNA genome containing six genes: N, P, M, G, L and GNS. The former 5 genes’ products were associated with virion, but the GNS protein was only found in virus-infected cells. GNS function was still unknown. This study was aimed to clone GNS gene and design short hairpin RNAs (shRNAs) for offering RNA interference (RNAi) in BEFV replication and GNS gene expression. The interaction between G and GNS gene were also studied. G and GNS vectors (i.e., pcDNA3.1/BEFV-G and pcDNA3.1/BEFV-GNS) were cotransfected into the cells with different strategies. The design of GNS primers were based on Taiwan BEFV isolate (2001 strain). RT-PCR reactions were adopted and GNS cDNA was obtained and cloned to pcDNA3.1/His-TOPO vector. Three shRNAs for GNS were constructed into three pENTR/U6 vectors (i.e., pU6-shRNA62, pU6-shRNA114, and pU6-shRNA1217). All shRNA vectors were shown to be able to silence specifically the expression of GNS gene. BHK-21 cells were cotransfected with G and GNS simultaneously or sequentially and then incubated for 48 hrs. Their expressions of G and GNS genes were detected by RT-PCR. The transfections with G first and GNS later could inhibit 20% of the gene expressions of both G and GNS. The transfections with GNS first and G later and with GNS and G simultaneously could inhibit the gene expressions of both G and GNS above 50% fever. This result revealed the inhibitory interaction between G and GNS. Transfections with three pU6-shRNAs into BHK-21 cells already transfected with G and GNS before showed approximately 50% inhibition of GNS and 20% inhibition of G after 48 hrs incubation. BHK-21 cells transfected with three pU6-shRNAs, then challenged with BEFV viral dose of 100 TCID50 showed 10-fold reduction of virus titer compared with control after 48 hrs circulation.

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