鰻弧菌是良好的免疫促進劑，經由口服給予後，可誘發較強的先天性及後天性免疫反應。應用轉型後鰻弧菌所表現的NNV次單位疫苗去對抗神經壞死病毒，其保護效果優於大腸桿菌。顯見應用弧菌作為疫苗抗原表現載體可獲改更高之免疫效果。影響表現量高低關鍵在於啟動子，實驗室已經選殖出熱休克蛋白啟動子，為表現管家基因(housekeeping gene)的強力啟動子。但此啟動子在鰻弧菌內表現NNV外殼蛋白的表現量只占總蛋白的1 %。本研究主要目的是增加鰻弧菌抗原表現量。建構groEp-GFP載體並且分別轉型到大腸桿菌和鰻弧菌。利用EMS (ethyl-methyl sulfate)與PCR方式誘導核苷酸點突變。經過突變處理後，可觀察綠螢光蛋白不同的表現程度。利用螢光篩選並且得到強力啟動子的菌落。利用PCR方式使熱休克啟動子的-35區域造成突變，G到T點突變可增強該啟動子的活性。這些啟動子用於弧菌內來表現NNV外殼蛋白與綠螢光蛋白，結果證明弧菌可成功表現NNV外殼蛋白與綠螢光蛋白，比較原先的表現量(1 %與3 %)後，分別占弧菌總蛋白2 % 與6 %。

Vibrio anguillarum subunit vaccine is an immunostimulant that can trigger stronger innate and humoral immunity after oral administration. It was also found that oral nerve necrosis virus (NNV) subunit vaccine encapsulating NNV coat protein expressed in V. anguillarum provided better protective efficacy for grouper larvae against NNV infection than that of E.coli, indicating that Vibrio as the antigen expression host improved higher immunity protective. Previous, our lab has constructed an expression vector using Vibrio promoter (groEp promoter) of heat shock protein to express NNV antigen. Despite the groEp promoter is strongly promoter, but the expression of NNV coat protein only reaches 1% of the total protein in Vibrio. This study tries to increase the expression of foreign antigen in V. anguillarum by mutating the promoter, the groEp-GFPuv vector is constructed and is transformed to E.coli (Top10) and Vibrio anguillarum. Mutation by PCR method and ethyl methanesulfonic acid (EMS) are used to induce nucleotides mutation in promoter using GFP as indicator, and the high expressing colonies are selected. The -35 region of groEp promoter with G to T single mutation mutated by PCR mutation can enhance the promoter activity. These up-promoters were used to express the NNV subunit vaccine in Vibrio. The result demonstrates that mutated promoter enhance the expression of NNV coat protein and green fluorescence protein, and reaches 2 % of total protein in Vibrio and the green fluorescence protein is 6 %, vs. the previous amount of 1 % and 3 % respectively.

林青丘 (2004)，魚用生物包埋型口服疫苗製備與有效性測試，國立
成功大學生物科技研究所碩士論文。
吳政隆 (2005)，發展以弧菌為載體之石斑魚神經壞死病疫苗，國立
成功大學生物科技研究所碩士論文。
劉曉謙 (2007)，篩選弧菌熱休克蛋白啟動子建立弧菌表現系統，國
立成功大學生物科技研究所碩士論文。
施慶宏 (2008)，利用弧菌表現神經壞死病毒之口服次單位疫苗，國
立成功大學生物科技研究所碩士論文。

Ahne W., Witon J. R. and Kimura T. (1989) Prevention of infectious diseases in aquaculture. Journal of veterinary medicine Series B. 36: 561- 567.

Arai T., Hamashima H. and Hasegawa H. (1985) Isolation of a new drug-resistance plasmid from a strain of Vibrio parahaemolyticus. Microbiol. Immunol. 29: 103-112.

Aravindhan V., Christy A. J., Roy S. and Narayanan P. R. (2009) Mycobacterium tuberculosis groE promoter controls the expression of the bicistronic groESL1 operon and shows differential regulation under stress condition. FEMS Microbiol Lett. 292: 42-49.

Chiang L. and Mekalanos J. (2000) Construction of a Vibrio cholerae Vaccine Candidate Using Transposon Delivery and FLP Recombinase-Mediated Excision. INFECTION & IMMUNITY. 68: 6391-6397.

Cutrin M., Toranzo E. and Barja L. (1995) Genetic transformation of Vibrio anguillarum and Pasteurella piscicida by electroporation. FEMS Microbiol Lett. 128: 75-80.

Grossman A. D., Straus D. B., Walter W. A. And Gross C. A. (2008) σ32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coil. GENES & DEVELOPMENT. 1: 179-184.

Hamashima H., Nakano T., Tamura S. and Arai T. (1990) Genetic transformation of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholera non O-1 with plasmid DNA by electroporation . Microbiol. Immunol. 34: 703-708.

Hsu C.T., Ting C.Y., Ting C.J., Chen T.Y., Lin C.P., Whang-Peng J. & Hwang J. (2000) Vaccination against gonadotropin-releasing hormone (GnRH) using toxin receptor-binding domain-conjugated GnRH repeats. Cancer Res. 60: 3701- 3705.

Ivics Z. and Katzer A. (2007) Targeted Sleeping Beauty transposition in human cells. Mol Ther. 15: 1137-1144.

Jana S. and Karan G Deb JK. (2005) Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol. 67: 289-298

Johnson Ⅲ J. M., Ding W., Henkhaus J. and Fix D. (2001) Identification of a mutation causing increased expression of the tas gene in Escherichia coil FX-11. Mutation Research. 479: 121-130.

Klein G., Walczak R., Krasnowska E., Blaszczak A. and Lipinska B. (1995) Characterization of heat-shock response of the marine bacterium Vibrio harveyi. Mol Microbiol. 16: 801-811.

Kourennaia O. V. And deHaseth P. L. (2007) Substitution of a Highly Conserved Histidine in the Escherichia coil Heat Shock Transcription Factor, σ32, Affects Promoter Utilization In Vitro and Leads to Overexpression of the Biofilm-Associated Flu Protein In Vivo. Bacteriology. 189: 8430-8436.

Kuchanny A. D. and Lipinska B. (2003) Cloning and characterization of groE heat-shock operon of the marine bacterium Vibrio harveyi. Microbiology. 149: 1483-1492.

Lai Y.P., Huang J., Wang L.F., Li J., Wu Z.R. (2004) A new approach to random mutagensis in vitro. Biotechnology and Bioengineering. 86: 622-627.

Lampe D. J., Churchill M. and Robertson H. (1996) A purified mariner transposase is sufficient to mediate transposition in vitro. EMBO. 15: 5470-5479.
Lampe D. J. (2009) Bacterial genetic methods to explore the biology of mariner transposons. Genetics. 149: 179-187.

Marcus H., Ketley J. M., Kaper J. B. and Holmes R. K. (1990) Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholera by electroporation and osmotic shock. FEMS Microbiol. Lett. 68: 149-154.

Nishizawa T., Takano R. and Muroga K. (1999) Mapping a neutralizing epitope on the coat protein of striped jack nervous necrosis virus. General Virology. 80: 3023-3027.

Obrist M., Milek S., Klauck E., Hengge R. and Narberhaus F. (2007) Region 2.1 of the Escherichia coli heat-shock sigma factor RpoH (σ32) is necessary but not sufficient for degradation by the FtsH protease. Microbiology. 153: 2560-2571.

Palazzoli F., Merly F., Bigot Y. (2009) Transposon tools: worldwide
landscape of intellectual property and technological developments. Genetica. 479, 121-130.

Pathangey L., Kohler J., Isoda R. and Brown T. (2009) Effect of expression level on immune responses to recombinant oral Salmonella enterica serovar Typhimurium vaccines. Vaccine. 27: 2707-2711.

Rodriguez F., Rist W., Rudiger S. and Bukau B. (2008) Molecular Basis for Regulation of the Heat Shock Transcription Factorσ32 by the DnaK and DnaJ Chaperones. Molecular Cell. 32: 347-358.

Sakai M., Atsuta S. and Kobayashi M. (1995) Efficacies of combined vaccine for Vibrio anguillarum and Streptococcus sp. Fisheries Sci. 61: 359-360.

Sakai M. (1999) Current research status of fish immunostimulants. Aquaculture. 172: 63-92

Salinas I., Lockhart K., Bowden T. J., Collet B., Secombes C.J. and Ellis A.E. (2004) An assessment of immunostimulants as Mx inducers in Atlantic salmon ( Salmo salar L.) parr and the effect of temperature on the kinetics of Mx responses. Fish Shellfish Immunol. 17: 159-170.

Singer J. T., Phennicie R. T., Sullivan M. J., Porter L. A., Shaffer V. J. and Kim C. H. (2010) Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System. Microbiology. 76: 3467-3474.

Wang H. and Griffiths M.W. (2009) Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation. Microbiology. 48: 349-354.

Zhi N., Wan Z., Liu X. and Kajigaya S. (2010) Codon Optimization of Human Parvovirus B19 Capsid Gene Greatly Increases Their Expression in Nonpermissive Cells. Virology. 84: 13059-13062.

Zhou L., Liu Q., Wang Q., Ma Y., Xu Y., Yang Z., Zhao Y. and Zhang Y. (2008) Secretory delivery of heterologous proteins in attenuated Vibrio anguillarum for potential use in vaccine design. Appl Microbiol Biotechnol. 79: 1027-1034.