神經壞死病毒調控石斑魚腦細胞基因表現之研究

Abstract

病毒感染宿主細胞後，藉由種種機制去調控宿主細胞基因之表現，使宿主細胞的某些基因被活化或抑制，以便營造出一個可供病毒複製及組裝的場所。近幾年來，病毒性神經壞死症（viral nervous necrosis disease, VNN disease）對台灣石斑魚養殖造成了嚴重的損失，其致病原為神經壞死病毒（nervous necrosis virus, NNV）。為了研究此病毒感染宿主細胞後，宿主細胞基因表現及其產物受調控的變化，首先建立神經壞死病毒的宿主細胞－石斑魚腦細胞株（GB3 cell line）之噬菌體表現cDNA基因庫（phage display cDNA library），並採用免疫染色法來篩選受到調控之表現基因。利用此方法總共篩選出46個在NNV病毒感染後，具有差異表現的GB3細胞基因，其中有18個基因屬於促進表現（up-expression），27個基因屬於抑制表現（down-regular），1個為持續表現（constant expression）基因。這些基因序列與NCBI基因庫資料進行比對分析之後，在誘導表現的基因群中，可發現數種基因與蛋白質的轉譯有關，如translation elongation factor 1-delta、ribosomal protein S18、ribosomal protein S10、ribosomal protein LP0，也有與免疫相關的基因被誘導，如natural killer cell enhancement factor，它可能與病毒感染後細胞啟動的防禦機制有關，以及與能量生成有關的基因，如ADP/ATP translocase、Glyceraldehyde 3-phosphate dehydrogenase (GAPDH); 另外在抑制表現的基因群中，有數種基因不斷的重複出現，可推測其本身在細胞中的含量較多，例如和神經訊息傳遞有關的endozepine、S100-like calcium binding protein，在腦細胞中含量確實較高，另外也發現與核酸代謝有關的nucleoside-diphosphate kinase受到抑制，此基因表現受到抑制會使細胞週期停止進行，另外也有一部份的蛋白質轉譯相關基因是屬於抑制表現的，如60S ribosomal protein L27、ribosomal protein L23，由此可推測在各種不同的蛋白質轉譯相關基因中，病毒會選擇其中數種來進行病毒蛋白的合成。接著以RT-PCR進一步確認免疫染色法所篩選出的差異表現基因，在病毒感染不同時間之後，其基因之轉錄也同樣具有差異表現，因此本實驗提供了一種新的方式，可在蛋白質的層次下進行基因差異表現的研究，在比較兩著的差異後，也可以進一步探討mRNA轉譯蛋白質的機制，同時也能夠對NNV的感染特性有更多的瞭解，以便往後對抗NNV此種病毒的危害研究上，能有更多的貢獻。

Following infection, viruses use different mechanisms to control host cell’s genes expression. Some of the host genes may be activated or suppressed. And its makes the host cell to be an appropriate place for virus to replicate and assemble. Recently, viral nervous necrosis disease causes a great damage to grouper aquaculture in Taiwan. The pathogen of this disease has been identified as nervous necrosis virus (NNV). In order to understand the infection characteristics of this virus, first we established a phage display cDNA library of virus’s host cell, grouper brain cell (GB3), and used immunostaining method to screen the differentially-expressed genes in response to NNV infection. These screening resulted in the identification of 46 differentially-expressed genes. There were 18 genes been activated, 27 been suppressed and 1 was constantly expressing gene. Analyzing and comparing these genes sequences with NCBI genetic database, it was observed that some of the up-regulated relate to protein translation, like translation elongation factor 1-delta 、ribosomal protein S18、ribosomal protein S10 and ribosomal protein LP0. There has also immune-relate gene: natural killer cell enhancement factor, which may activate the host cell’s immuno-system. Besides, ADP/ATP translocase and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes which relate to energy production have also been found to be up-regulated. A many down-regulated genes, endozepine and S100-like calcium binding protein, which relate to neuro-transmittance and abundant in brain cell, emerge frequently. The nucleic acid-metabolic gene, which related to cell-cycle, nucleoside diphosphate kinase and some protein translation-related genes such as 60S ribosomal protein L27 and ribosomal protein L23 were down- regulated. From this result, we assume that the selected protein translation-related genes may be utilized by the virus to produce viral proteins. The results of RT-PCR, besides confirming the immunostaining screening, showed the differentially expressed genes at transcription level. This experiment provides a new approach to study the difference in host gene expression at transcription and translation levels after virus infection. Taken together, these results revealed the up- and down-regulated genes in response to NNV infection, and this information could be helpful to proceed further to understand the molecular mechanisms behind the NNV-host interaction and to develop control measures against NNV.