大腸桿菌的基因轉殖是現今用以生產魚用次單位疫苗的主要方法，其優點在於生產技術成熟，產量高且容易製備，然而卻有疫苗本身可能不具有誘發魚隻先天性免疫反應的能力，而導致免疫效果低落的可能性存在。
鰻弧菌 (Vibrio anguillarum) 已知具有誘發魚隻先天免疫反應的能力，因此若能利用鰻弧菌表現外源蛋白，則有可能開發出兼具誘發先天及後天性免疫反應的次單位疫苗，藉此而改善上述缺點。本研究的目的，即在建立利用鰻弧菌表現系統生產魚用次單位疫苗的方法，同時來評估其效能。
首先本研究將含有鰻弧菌熱休克蛋白60基因的啟動子及神經壞死病毒外鞘蛋白的基因植入pGL3-Basic質體，利用電破法將表現質體殖入鰻弧菌的勝任細胞後，利用含高濃度ampicillin的培養皿進行篩選。隨後進行培養條件的最適化，藉以改善外鞘蛋白的表現量。接著將含有外鞘蛋白的鰻弧菌，連同傳統的大腸桿菌次單位疫苗進行豐年蝦的生物包埋後，餵食白身階段的石斑魚苗。在服用4天內及7天後，分別分析TNF-α基因、IL-1β基因與Mx基因的表現量以及抗外鞘蛋白抗體的含量，作為評估疫苗引發先天及後天免疫反應能力的依據，同時在服用後7天進行神經壞死病毒的攻毒實驗，攻毒劑量設定為1×105.5 TCID50 /fish。
研究結果證明，鰻弧菌確實可以利用上述方法表現外源蛋白，產量可達
菌體總蛋白之1~2 %；相對於大腸桿菌的表現量雖明顯偏低，然而其無論在誘發Mx基因表現量的能力、產生抗體的量，以及所能提供石斑幼魚的保護效果等方面均明顯優於後者，顯示弧菌表現系統確實有機會能取代大腸桿菌系統，成為新世代的生產魚用次單位疫苗的平台。

Most piscatorial subunit vaccines are produced by E. coli expression system that is easy to use. However, this conventional oral vaccine can not add additional adjuvant
to induce effective innate immunity, which may result in a lower protective immune response.
Previously, we have demonstrated that Vibrio anguillarum could induce fish innate immunity and could be used as an effective adjuvant. By using V. anguillarum as expression host to synthesize specific antigen and use the whole Vibrio as component of an oral vaccine, it is expected to induce both innate and adaptive immunity simultaneously, thus could confer better protective efficacy.
First we obtained NNV coat protein gene and expressed in V. anguillarum and E. coli respectively. They were then encapsulated in Artemia for the following study. After administrating to grouper larvae, the immune stimulatory effects of V. anguillarum and E. coli were evaluated by analyzing the gene expression levels of TNF-α, IL-1β and Mx genes at 4 day post immunization (DPI), and the
antigen-specific antibody production at 7 DPI respectively, and the protective efficacy of these two vaccines were then evaluated by challenge of 1×105.5
TCID50/fish of NNV at 7 DPI.
The results showed V. anguillarum can induce the Mx mRNA expression in brain at 2nd day after administration. Although the amount of NNV coat protein in V. anguillarum only reached 1~2 % of the total protein, and was much lower than that at least 10 % as in E. coli, however, the new subunit vaccines can elicit higher antibody titer at 7 DPI and provide much better protective efficacy than that of E. coli vaccine, revealing the potential of using Vibrio expression system for the production of piscatorial subunit vaccines.

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。
黃貴民 (1999)，實用石斑魚養殖，水產出版社出版。
黃琪琰 (1995)，水產動物疾病學，水產出版社出版。
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。
蘇偉成 劉富光 (2005)，台灣水產養殖的永續經營，科學發展期刊 385 期，行政院國家科學委員會。
Acosta F., Lockhart K., Gahlawat S. K., Real F. and Ellis A. E. (2004) Mx expression in Atlantic salmon (Salmo salar L.) parr in response to Listonella anguillarum bacterin, lipopolysaccharide and chromosomal DNA. Fish Shellfish Immunol. 17: 255-263.
Ahne W., Winton J. R. and Kimura T. (1989) Prevention of infectious diseases in aquaculture. Journal of veterinary medicine Series B. 36: 561-567.
Anderson E. D., Mourich D. V., Fahrenkrug S. C., LaPatra S., Shepherd J. and Leong J. C. (1996) Genetic immunization of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus. Mol Mar Biol Biotechnol. 5: 114-122.
Cabello F. C. (2006) Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ Microbiol. 8: 1137–1144.
Campbell R., Adams A., Tatner M. F., Chair M. and Sorgeloos P. (1993) Uptake of Vibrio anguillarum vaccine by Artemia salina as a potential oral delivery system to fish fry. Fish Shellfish Immunol. 3: 451-459.
Chen Y. M., Su Y. L., Shie P. S., Huang S. L., Yang H. L. and Chen T. Y. (2008) Grouper Mx confers resistance to nodavirus and interacts with coat protein. Dev Comp Immunol. 32: 825–836.
Cohen S. N., Chang A. C. Y., Boyer H. W. and Helling R. B. (1972)Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci. 69: 2110-2114.
De baulny M. O., Quentel C., Fournier V., Lamour F. and LeGouvello R. (1996) Effect of long-term oral administration of beta-glucan as an immunostimulant
or an adjuvant on some non-specific parameters of the immune response of turbot Scophthalmus maximus. Dis Aquat Org. 26: 139-147.
Duff D. C. B. (1942) The oral immunization of trout against bacterium samonicida. J. Immunol. 44: 87-94.
Ellis A. E. (1997) Immunization with bacterial antigens: furunculosis. Dev Biol Stand. 90: 107-116.
Frost P. and Ness A. (1997) Vaccination of Atlantic salmon with recombinant VP2 of infectious pancreatic necrosis virus (IPNV), added to a multivalent vaccine, suppresses viral replication following IPNV challenge. Fish Shellfish Immunol. 7: 609.
Gudding R., Lillehaug A., Midtlyng P. J. and Brown F. (1997) Fish Vaccinology. Dev Bio Stand Karger Basel. 107–116.
Horne M. T. (1997) Technical aspects of the administration of vaccines. Dev Biol Stand. 90: 79-89.
Iwamoto T., Mise K., Takeda A., Okinaka Y., Mori K., Arimoto M., Okuno T. and Nakai T. (2005) Characterization of Striped jack nervous necrosis virus subgenomic RNA3 and biological activities of its encoded protein B2. J General Virol. 86: 2807–2816.
Johnson K. A. and Amend D. F. (1983). Efficacy of Vibrio anguillarum and Yersinia ruckeri bacterins applied by oral and anal intubation of salmonids. J Fish Dis. 6: 473-476.
Joosten P. H. M., Tiemersma E., Threels A., Caumartin-Dhieux C. and Rombout J. H. W. M. (1997) Oral vaccination of fish against Vibrio anguillarum using alginate microparticles. Fish Shellfish Immunol. 7: 471-485.
Jorgensen J. B., Johansen L., Steiro K. and Johansen A. (2003) CpG DNA inducesprotective antiviral immune responses in Atlantic Salmon (Salmo salar L.) J Virol. 77: 11471–11479.
Kanemori M., Yanagi H. and Yura T. (1999) Marked instability of the σ32 heat shock transcription factor at high temperature. J Biol Chem. 274: 22002–22007.
Klein G., Walczak R., Krasnowska E., Blaszczak A. and Lipińska B. (1995) Characterization of heat-shock response of the marine bacterium Vibrio harveyi. Mol Microbiol. 16: 801-11.
Le Morvan C., Troutaud D. and Deschaux P. (1998) Differential effects of temperature on specific and nonspecific immune defences in fish. J Exp Biol. 201: 165–168.
Lin C.C., Lin J.H., Chen M.S. and Yang H.L. (2007) An oral nervous necrosis virus vaccine that induces protective immunity in larvae of grouper (Epinephelus coioides).Aquaculture.268: 265–273.
Lin C.H., John J. A.C., Lin C.H. and Chang C.Y. (2006) Inhibition of nervous necrosis virus propagation by fish Mx proteins.Biochemical and Biophysical Research Communications. 351: 534–539.
Livak K. J. and Schmittgen T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 25: 402–408.
Midtlyng P. J. (1997) Vaccinated fish welfare: protection versus side-effects. Dev Biol Stand. 90: 371-9.
Mori K., Nakai T., Muroga K., Arimoto M., Mushiake K. and Furusawa I. (1992) Properties of a new virus belonging to nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virol. 187: 368-71.
Munday B. L., Kwang J. and Moody N. (2002) Betanodavirus infections of teleost fish: a review. J Fish Dis. 25:127-142.
Norqvist A., Hagstrom A. and Wolf-Watz H. (1989) Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio
anguillarum. Appl Environ Microbiol. 55: 1400-1405.
Olafesen J. A. and Hansen G. H. (1992) Intact antigen uptake in intestinal epithelialcells of marine fish larvae. J Fish Biol. 40: 141-156.
Peddie S., Zou J. and Secombes C. J. (2002) Immunostimulation in the rainbow trout (Oncorhynchus mykiss) following intraperitoneal administration of
Ergosan. Vet Immuno. Immunopathol. 86: 101-113.
Quentel C. and Vigneulle M. (1997) Antigen uptake and immune responses after oral vaccination. Dev Biol Stand. 90: 69-78.
Rojo I., Ilarduy O. M., Estonba A. and Pardo M. A. (2007) Innate immune gene expression in individual zebrafish after Listonella anguillarum inoculation. Fish Shellfish Immunol. 23: 1285-1293.
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.
Seppola M., Larsen A. N., Steiro K., Robertsen B. and Jensen I. (2008) Characterisation and expression analysis of the interleukin genes, IL-1β, IL-8 and IL-10, in Atlantic cod (Gadus morhua L.). Mol Immunol. 45: 887-897.
Shao Z. J. (2001) Aquaculture pharmaceuticals and biologicals: current perspectives and future possibilities. Adv Drug Deliv Rev. 50: 229-43.
Shimizu T., Nitta M., Itoh Y., Yanagihara Y. and Mifuchi I. (1981) Mitogenicity and adjuvanticity of a marine bacterium, Vibrio anguillarum, in mice. Microbiol
Immunol. 25: 929-937.
Stover C. K., de la Cruz V. F., Fuerst T. R., Burlein J. E., Benson L. A., Bennett L. T., Bansal G. P., Young J. F., Lee M. H. and Hatfull G. F. (1991) New use of
BCG for recombinant vaccines. Nature. 351: 456-460.
Straus D., Walter W. and Gross C. A. (1990) DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of sigma 32. Genes & Dev. 4: 2202-2209.
Tanaka S., Mori K., Arimoto M., Iwamoto T. and Nakai T. (2001) Protective immunity of sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis. J Fish Dis. 24:15-22.
Tebbit G.L. and Goodrich T.D. (1982) Vibriosis and the development of bacterins for its control. Collection Fondation Marcel Merieux. 225-248.
Vervarcke S., Ollevier F., Kinget R. and Michoel A. (2004) Oral vaccination of African catfish with Vibrio anguillarum O2: effect on antigen uptake and immune response by absorption enhancers in lag time coated pellets. Fish Shellfish Immunol. 16: 407-414.
Yoshikoshi K. and Inoue K. (1990) Viral nervous necrosis in hatchery-reared larvae and juvenile of Japanese parrotfish, Oplegnatbus fasciatus (Temminck & Schlegel). J Fish Dis. 13:69-77.
Zou J., Holland J., Pleguezuelos O., Cunningham C. and Secombes C. J. (2000) Factors influencing the expression of interleukin-1β in cultured rainbow trout (Oncorhynchus mykiss ) leucocytes. Dev Comp Immunol. 24: 575-582.