臺灣博碩士論文加值系統

本實驗室所分離出 IPNV-E1S(Eel No.1 spleen) 病毒株,分類上屬
於 Birnaviridae ;其具有兩段雙股的 RNA,基因體分別為 A 段約 3.2
Kb及 B 段約 2.9 Kb。 A 段主要 轉譯三種蛋白質,依序為 VP2 是病毒
的主要外鞘蛋白質(capsid protein).VP4 具有蛋 白質水解酵素(
protease)活性.VP3是內部結構蛋白質;而 B 段則僅轉譯 RNA-dependent
RNA polymerase,VP1.因VP2中含有兩段雙股RNA病毒科的血清型決定區域,
對於 IPNV 的檢測及疫苗製造非常重要;因此, 針對 A 段中三種蛋白質
基因進行選殖,並與其它 病毒株的類緣關係作一分析比較。 雙股
RNA 變性不易,在諸多方法中,發現以添加最終濃度為 2mM CH3HgOH 效果
最 好,能長時間維持變性狀態,以利反轉錄反應的進行。首先針對這三
個基因分別設計出 多組專一性引子,在 42 ℃下進行第一股 cDNA 的合
成,再以 SuperTaq DNA polymerase 合成第二股 cDNA ,並連接至 pCR3
載體上,經轉形作用,得到 3 個選殖株,分別命名為 pVP2、 pVP4 和
pVP3, pVP2 大小為 1220 bp、pVP4 為 849 bp 、pVP3 為 783 bp; 與
N1 和 Jasper 等病毒株比對相似性,分別為 88.5 ％ 及 79.2 ％, 86.7
％ 及 77.3 ％ 和 86.5 ％ 及 76.4 ％ ;另外,將 pVP2 之 349-807
bp 的 hypervariable region 所轉譯成的胺基酸序列與其它病毒株比
對,發現 E1S 病毒株與 N1 和 Sp 病 毒株類緣關係最近,與核酸序列比
對的結果一致。在 pVP3 中也發現一段與 dsRNA 的 複製和包裝有關的
terminal inverted repeat。 綜論, A 段核酸序列幾近完全得知,
僅剩 5''端和 3''端各缺約 330 bp 和 15 bp, 由核酸及胺基酸序列比對
得知 E1S 與 N1 病毒株類緣最接近。

IPNV-E1S,the Ab type of aquatic birnavirus, has a
bisegmented dsRNA genome which consists of 3.2 Kb A segment and
2.9 Kb B segment. The large ORF of A segment mainly encodes
three viral proteins including VP2 (capsidprotein),VP4
(protease) and VP3 (internal structure protein). B segment
encodes RNA-dependent RNA polymerase. The serotype-specific
epitope contained in VP2 is critical to the detection and
vaccine production of IPNV. For determining the function of
these viral proteins, it is necessary to clone their genes,
respectively. E1S-VP2,VP4 and VP3 cDNA synthesis was
performed with specific primer,reverse transcriptase and
SuperTaq DNA polymerase by PCR,then ligated to pCR3 and
transformed into E.coli. Three inserted colnes (pVP2, pVP4 and
pVP3)had been selected sucessfully. pVP2 (1220 bp) had 88.5 ％
and 79.2 ％, pVP4(849 bp) had 86.5 ％and 77.3 ％, and pVP3 (783
bp) had 86.5 ％ and 76.4 ％ homology of N1 and Jasper strain of
IPNV. In addition,the homology of the dedued amino acid
sequences of the hypervariable region in pVP2 compared withthat
of other strains reveals the close relationship of E1S, N1 and
Sp strains. The complete nucleotide sequence of A segment
in E1S strain was almost obtained except for 330 bp of 5''-end
and 15 bp of 3''-end when compared with N1strain. The results of
comparison in nucleotide and amino acid sequences bothshow that
E1S strain is close to N1 and Sp strains in phylogeny.