一種新的100 kDa蝦白點症病毒蛋白質之研究

Abstract

本實驗室建立之白點症病毒（white spot syndrome virus，WSSV）Hind III基因庫（library）中，包含一段全長約4.4kb的基因，命名為pmh15，此片段接入pUC19載體中，已由病毒基因體完成確認與定序工作。Pmh15包含有六個開放譯讀區（open reading frames，ORFs），其中最長的ORF1由2535個去氧核糖核?酸所組成，可轉譯成845個氨基酸，蛋白質分子量大小經電腦預測之理論值為92kDa。推測轉譯起始位置，即methionine start codon的鄰近序列為AGTATGG，符合真核生物轉譯起始原則（Kozak rule），轉譯終點位置3’方向46核?酸處則存在一個polyadenylation consensus signal（polyA signal）AATAAA。經反轉錄?聚合酵素鏈反應（reverse transcriptase polymerase chain reaction）進行基因轉錄時序分析，可知ORF1屬於病毒感染之早期表現基因（early gene）。 將包含前258個氨基酸部份之DNA片段以聚合酵素鏈反應（polymerase chain reaction，PCR）放大後，接入pQE載體進行活體蛋白質表現（in vivo expression），於大腸桿菌（E. coli）中可誘發出一40 kDa的蛋白質產物，並經N端定序確認其氨基酸序列。誘發出的蛋白質純化後注射至紐西蘭白兔體內生產抗體以進行進一步的分析。 Pmh15-ORF1全長則接入pcDNA3載體，經由離體轉錄/轉譯實驗（in vitro transcription/translation）生產出100kDa之蛋白質。利用生產自白點症病毒100kDa蛋白質N端之抗體進行西方轉印雜合實驗（Western blot hybridization）可偵測到離體轉錄/轉譯之蛋白質產物，以及此100kDa蛋白質於白點症病毒感染之病蝦組織中確實存在。 而在電腦比對方面，WSSV 100 kDa蛋白質發現有三個推測性的穿越細胞膜區域（transmembrane domains）及8個可能被醣化（N-glycosylation）與31個可能被磷酸化（phosphorylation）之位置。然而，WSSV 100 kDa蛋白質並沒有發現與任何桿狀病毒（baculovirus）或其他大型DNA病毒已發表的膜蛋白（envelope protein）及穿越膜蛋白（transmembrane proteins）有任何相似之處，顯示白點症病毒在分類上極有可能為桿狀病毒中新的一屬，或甚至所有病毒種類中新的一科。

WSSV genomic library of Hind III was constructed in pUC19, and contained a 4.4 kb genomic fragment named pmh15. The pmhl5 nucleotide sequences have been identified and sequenced from the viral genome. The pmhl5 was composed of six open reading frames (ORFs), including the longest ORF—pmh15 ORF1 with 2535 nucleotides. And the pmh15-ORF1 encodes 845 amino acid polypeptide of a theoretical size of 92 kDa from computer predictions. The sequence surrounding the methionine start codon (AGTATGG) conformed with the Kozak rule for efficient eukaryotic translation initiation and a polyadenylation consensus (polyA) signal is present 46 nt downstream of the translational stop codon. RT-PCR was used to investigate the temporal expression of the pmh15-ORF1, the results show that the pmh15-ORF1 gene may express early in the course of WSSV infection. A DNA fragment containing the partial pmh15-ORF1 was amplified by PCR reaction for cloning from the start codon to amino acid 258, then ligated into pQE for in vivo expression. The partial pmh15-ORF1 was expressed in E. coli, which produced a 40 kDa protein product. And the N terminal amino acids of the expressed protein were sequenced as prediction. The purified protein was injected into a New Zealand white rabbit to produce antibodies for subsequent analysis. The full-length pmh15-ORF1 was constructed in vector pcDNA3 for in vitro transcription/translation (TNT), and a major product was 100 kDa. The antibody against the N terminus of the WSSV 100 kDa protein could recognize the TNT product and a 100 kDa protein in WSSV-infected shrimp tissues. The 100 kDa protein contains three possible transmembrane domains, 8 putative N-glycosylation sites and 31 possible phosphorylation sites. This viral protein showed no significant homology to any published envelope or transmembrane proteins of baculovirus and other large DNA viruses, that suggests WSSV could be a representative of a new baculovirus genus (whispovirus) or even a new virus family (Whispoviridae).