鰻弧菌 (Vibrio anguillarum)，是水產養殖漁業中常見的病原菌。研究報告指出鰻弧菌可能是一種免疫促進劑。本實驗室已證明以弧菌為宿主 (host) 之疫苗，免疫效果優於以大腸桿菌之宿主疫苗，但是以弧菌為宿主之口服疫苗面臨抗原表現量不佳之瓶頸。
影響蛋白表現量的重要因素之一是啟動子的選擇，實驗第一部分，首先以篩選弧菌之啟動子為目標。過去的文獻已經證明細菌之熱休克蛋白基因 groEL (hsp60) 為一種管家基因 (housekeeping gene)，為大量表現之蛋白，而且此類蛋白的啟動子已經有應用在疫苗上之研究，因此實驗設計退化性引子 (degeneracy primer)，篩選出鰻弧菌的 groEL 熱休克蛋白基因，接著利用 genomic walking 的方式得到 groE 熱休克蛋白基因調節序列。實驗第二部分，以大腸桿菌為模式了解熱休克蛋白基因調節序列的功能。將調節序列放入沒有啟動子之載體，證實在大腸桿菌中可以驅動持續表現報導基因-luciferase，繼代4小時後表現量達到最大量，佔大腸桿菌蛋白表現量約15%。實驗第三部分，將構築好之質體 (plasmid vector) 送入鰻弧菌中，證實在鰻弧菌中以 37˚C 熱休克可以誘導表現報導基因- luciferase，誘導6小時後表現量最大，佔弧菌蛋白表現量約7%，因此利用 groE 啟動子建立鰻弧菌的抗原表現系統是可行的途徑。

Vibrio anguillarum, is a common opportunist bacterial pathogen in marine farm fish. V. anguillarum was found to induce specific immune response and also provide cross protection on some other bacterial diseases, indicating that V. anguillarum could be an potential immuno-stimulator. Our lab has developed a cloning system using V. anguillarum as an expression host for the construction of a fish subunit vaccine. We used GFP (green fluorescent protein) as antigen and transformed onto V. anguillarum, and demonstrated that this subunit vaccine induced stronger specific immune response compared to that in E.coli. However, this system facing a low antigen expression problem.
Bacterial groEL (hsp60) is a housekeeping gene. The groE promoter from mycobacterium has demonstrated to be useful for the development of subunit vaccine. In this experiment, we attempted to clone the groE promoter by genomic walking from a groEL gene of V. anguillarum. Then, we construct the groE promoter upstream to a luciferase gene on a promoter-free vector in E. coli. Luciferase expression achieved highest level 4 hours after incubation, the amount of expression of luciferase was estimated about 15% of the tatol protein of E. coli. Finally, the promoter was introduced onto V. anguillarum as a host to evaluate the promoter activity. The result indicated V. anguillarum expressed luciferase at highest level 6 hours after heat shock under 37˚C. The amount of expression is about 7% of tatol protein of V. anguillarum as evaluated by Image Quant software. These results demonstrated the potential of using groE promoter for antigen expression in the V. anguillarum and for the development of an oral subunit vaccine.

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