病毒的同股轉錄調控單元在日本鰻細胞株及活體中表現外來基因之適用性評估

Abstract

利用攜帶特殊抗原基因之質體DNA作為疫苗是近年來新開發之動物防疫技術。發展DNA疫苗之基本問題在於如何構築能適當表現抗原基因的載體。因此，同股轉錄調節單元（cis-transcriptional regulatory element; TRE）之研究變得十分重要。本篇論文利用氯黴素乙醯轉移酵素（Chloramphenicol acetyltransferase; CAT）為報導基因，評估來自巨細胞病毒（Cytomegalovirus; CMV）、雞肉瘤病毒（Rous sarcoma virus; RSV）及猴病毒（Semian virus 40; SV40）等之TRE在鰻魚細胞株及活體中控制基因表現的能力，以供未來開發鰻魚或其他高經濟魚類DNA疫苗之參考。本研究擬分析之TRE包括CMV的速發早期基因（Immediate early gene; IE）的啟動子（promoter）、RSV的控制轉錄的長端重複序列（long terminal repeat; LTR）及SV40之啟動子和加強子。將上述病毒TRE與CAT基因組合之重組質體以陽離子脂肪球轉染法遞送入鰻魚細胞株EK-1及EO-2中，結果發現，在EK-1細胞株中，三種病毒TRE控制CAT基因表現的能力由強至弱依序為RSV、CMV及SV40，在EO-2細胞株中則以CMV較強，RSV次之，SV40仍為表現最弱者。將攜帶RSV之TRE及CAT的重組質體，以肌肉注射法送入幼鰻體內，結果發現，CAT表現量會隨著注射劑量的增加而上升；CAT基因表現量在注射4天後達到高峰，一直到第8天仍無明顯之下降。以陽離子複合脂肪球懸浮液浸泡鰻線，將重組質體DNA轉染入鰻線的實驗中，發現在轉染後第7天的鰻線樣品中可偵測到CAT基因暫時性的表現。結果顯示RSV的TRE確實可使外來基因在日本鰻細胞株及體內表現，具有更進一步應用於發展DNA疫苗之潛力。

DNA vaccines are vector DNAs which carry a specific antigen gene and have been used in newly developed techniques to vaccinate animals. Recently, these DNA vaccines have shown increasing success in inducing both specific antibody response and cytotoxic T lymphocyte response to protect animals from viral or protozoan pathogens. One of the substantial problems to develop effective DNA vaccines is the construction of an expression vector which may express the antigen gene products at a level sufficient to induce the required immune responses. Thus, the study of the cis-transcriptional regulatory elements (TRE) becomes an essential part in the development of the DNA vaccine. In this article, we use a Escherichia. coli. chloramphenicol acetyltransferase (CAT) as the reporter gene to investigate the potentiality of three viral TREs (CMV IE promoter, RSV LTR, and SV40 promoter and enhancer) in two cell lines derived from Japanese eel (Anguilla japonica). The ability of the three viral TREs to drive CAT gene expression in the EK-1 cell line was RSV>CMV>SV40. CMV IE promoter expressed the CAT gene at a higher level than RSV LTR in the EO-2 cell line. SV40 early promoter and enhancer was the least potent in both of these two cell lines. When plasmid containing RSV TRE and the CAT gene was injected into elver muscle, CAT gene expression level increased with the dosage of plasmid injected. Highest levels of CAT expression were obtained 4 to 6 days after injection and lasted for at least 8 days. Transient expression of the CAT gene in elvers transfected with plasmid containing RSV TRE and the CAT gene led to the dection of CAT in the elvers 7 days posttransfection. We offer these results as helpful in the further development of DNA vaccines to be used in the control of eel diseases.