- 學位論文
石斑魚病毒之分子學研究：乙型野田病毒線性B細胞抗原決定區與虹彩病毒抗細胞凋亡CARD蛋白
Molecular Study of Grouper Viruses: Linear B-cell Epitope of Betanodavirus and Anti-apoptotic CARD Protein of Iridovirus
摘要
乙型野田病毒為引起多種重要海水養殖魚種病毒性神經壞死症的感染原，其感染結果導致全球性的海水魚苗高死亡率。外套蛋白是乙型野田病毒唯一的結構蛋白，具有自行組裝成病毒顆粒的能力。本研究利用一株具有高效價中和能力的單株抗體，RG-M18，來辨識病毒外套蛋白上的線性B細胞抗原決定區。分析一系列大腸桿菌/PET表現系統生產的重組蛋白後，證實RG-M18單株抗體辨識的線性抗原決定區位於此病毒外套蛋白C端介於胺基酸殘基195至338之間，另用一套重疊的人工胜肽被合成來評估對RG-M18單株抗體的結合能力，以確認最小的抗原決定區範圍，分析辨識出195VNVSVLCR202為最小的抗原決定區範圍。利用點漬矩陣和酵素免疫分析法來對比分析這個抗原決定區的丙胺酸掃描突變，結果顯示纈胺酸197，纈胺酸199和半胱胺酸201為抗體結合關鍵性的胺基酸。若將RGNNV，BFNNV和TPNNV基因型病毒上的白胺酸200替換成SJNNV基因型上的甲硫胺酸200並不會影響結合親和力，推測RGM18單株抗體可以辨識乙型野田病毒屬的所有病毒基因型。由競爭實驗的結果顯示此抗原決定區的合成多抗原胜肽具有完全抑制龍膽石斑神經壞死病毒在石斑腦細胞株中的繁殖能力。這些研究結果提供了這株中和性單株抗體新的保護機制，並對乙型野田病毒的疫苗學和胜肽藥物開發具有更寬廣的含意。在虹彩病毒CARD蛋白的抗細胞凋亡研究方面：石斑魚虹彩病毒(GIV)屬於虹彩病毒科、Ranavirus屬,而此病毒科之基因體皆包含了一個具有抗細胞凋亡功能的半胱胺酸蛋白酶聚集功能區基因(caspase recruitment domain, CARD)。石斑魚虹彩病毒之CARD基因可轉錄出91個胺基酸、分子量為10.505 KDa的蛋白,且此蛋白與人類ICEBERG還有其他的病毒所帶有的CARD蛋白高度相似。在本論文中利用北方墨點法證明GIV-CARD於感染細胞後4小時開始轉錄;另一方面，其轉錄完全被cycloheximide所抑制，而非aphidicolin，結果顯示GIV-CARD是一個早期基因。在細胞中的GIV-CARD-EGFP與GIV-CARD-FLAG重組蛋白，可觀察到重組蛋白皆會從細胞質進入細胞核，但在GIV-CARD蛋白上並沒有明顯的核定位序列。在感染GIV的石斑腎臟細胞株中利用RNA干擾技術抑制GIV-CARD的表現量亦可發現同時在早期感染階段也降低其他五個病毒基因的表現量，並且也降低了石斑魚虹彩病毒感染的能力。免疫螢光染色法顯示細胞中所表現的GIV-CARD重組蛋白有效的抑制細胞凋亡的發生。 HeLa細胞經由UV照射或使用anti-Fas抗體處理後分別誘發內在途徑和外在途徑的細胞凋亡。然而，過度表現的重組GIV-CARD蛋白可在HeLa細胞中分別抑制經由粒線體及死亡受體訊號傳遞下的細胞凋亡。最後在本論文提出在HeLa細胞中所表現的GIV-CARD重組蛋白顯著降低經由anti-Fas抗體所誘發的caspase-8和-9的活性。總結而言，這些結果顯示GIV-CARD通過內在途徑和外在途徑抑制細胞凋亡。
並列摘要
In molecular study of linear B-cell epitope in betanodavirus: Betanodavirus is a causative agent of viral nervous necrosis syndrome in many important aquaculture marine fish larvae, resulting in high global mortality. The coat protein of betanodavirus is the sole structural protein, and it can assemble the virion particle by itself. In this study, we used a high-titer neutralizing mAB, RG-M18, to identify the linear B-cell epitope on the viral coat protein. By mapping a series of recombinant proteins generated using the E. coli PET expression system, we demonstrated that the linear epitope recognized by RG-M18 is located at the C-terminus of the coat protein, between amino acid residues 195 and 338. To define the minimal epitope region, a set of overlapping peptides were synthesized and evaluated for RG-M18 binding. Such analysis identified the 195VNVSVLCR202 motif as the minimal epitope. Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine197, Valine199, and Cysteine201 are critical for antibody binding. Substitution of Leucine200 in the RGNNV, BFNNV, and TPNNV genotypes with Methionine200 (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of betanodaviruses. In competition experiments, synthetic multiple antigen peptides of this epitope dramatically suppressed giant grouper nervous necrosis virus (GGNNV) propagation in grouper brain cells. The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for betanodavirus vaccinology and antiviral peptide drug development. In molecular study of anti-apoptotic CARD protein in iridovirus: Grouper iridovirus (GIV) belongs to the genus Ranavirus of the family Iridoviridae; the genomes of such viruses contain an anti-apoptotic caspase recruitment domain (CARD) gene. The GIV-CARD gene encodes a protein of 91 amino acids with a molecular mass of 10,505 Daltons, and shows high similarity to other viral CARD genes and human ICEBERG. In this study, we used Northern blot to demonstrate that GIV-CARD transcription begins at 4 h post-infection; furthermore, we report that its transcription is completely inhibited by cycloheximide but not by aphidicolin, indicating that GIV-CARD is an early gene. GIV-CARD-EGFP and GIV-CARD-FLAG recombinant proteins were observed to translocate from the cytoplasm into the nucleus, but no obvious nuclear localization sequence was observed within GIV-CARD. RNA interference-mediated knockdown of GIV-CARD in GK cells infected with GIV inhibited expression of GIV-CARD and five other viral genes during the early stages of infection, and also reduced GIV infection ability. Immunostaining was performed to show that apoptosis was effectively inhibited in cells expressing GIV-CARD. HeLa cells irradiated with UV or treated with anti-Fas antibody will undergo apoptosis through the intrinsic and extrinsic pathways, respectively. However, over-expression of recombinant GIV-CARD protein in HeLa cells inhibited apoptosis induced by mitochondrial and death receptor signaling. Finally, we report that expression of GIV-CARD in HeLa cells significantly reduced the activities of caspase-8 and -9 following apoptosis triggered by anti-Fas antibody. Taken together, these results demonstrate that GIV-CARD inhibits apoptosis through both intrinsic and extrinsic pathways.
並列關鍵字
betanodavirus ； epitope ； peptide ； monoclonal antibody ； giant grouper ； nervous necrosis virus ； peptide entry inhibitor ； apoptosis ； CARD ； grouper ； iridovirus ； caspase參考文獻
延伸閱讀
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