Vibrio anguillarum，為革蘭氏陰性菌，感染魚體後會引起弧菌症（Vibriosis），是屬於高感染性病原。根據學術上的研究以及商品化疫苗使用的結果均認為V. anguillarum不論是以注射或浸泡施予方法皆可比其他細菌性疫苗能促製更好的專一性保護效果；此外也發現添加有弧菌菌體的疫苗，可得到較佳的免疫成果，顯示弧菌菌體對於魚類而言為一好的免疫促進劑（佐劑）。
為利用弧菌在魚體免疫上之特性，在本實驗中第一部份將建立一套以弧菌為宿主(host)的表現系統，作為外來抗原的表現載體，應用在魚用疫苗上。在表現載體的構築上，係以質體pEGFP-N1為骨架，再加入tac promoter作為啟動子，構築出新的表現質體pMI。接著將reporter gene gfp 選殖到pMI，構築出pMI-gfp。並且以西方轉漬的方法，確定了GFP蛋白在弧菌中的表現，證實質體pMI的確可用作為弧菌的表現用載體。
為評估以弧菌為疫苗載體之可行性，在實驗的第二部分，我們以建立好的弧菌蛋白表現系統，用來表現石斑魚神經壞死病毒的鞘蛋白（NNV coat protein），並以此表現有抗原蛋白之重組弧菌對石斑魚進行腹腔注射免疫接種，結果在混合弗氏佐劑的條件下，免疫石斑魚後可得血清中抗NNV抗體力價在第六週達最高為178倍;另外在不混合弗氏佐劑的條件下，免疫石斑魚後也可得到血清中抗NNV抗體力價在第六週達最高為163倍。此結果和對照組（以E. coli表現NNV coat protein）相比，混合佐劑和不混佐劑的條件下，免疫石斑魚後只可得到血清中抗NNV抗體力價在第八週達最高，分別為111倍和66倍。除此之外，在不添加佐劑的情況之下，接種表現有NNV鞘蛋白之弧菌，血清中抗NNV抗體在第二週即被明顯誘導出來（p<0.05）;相對於接種表現有NNV鞘蛋白之大腸桿菌，血清中抗NNV抗體在第八週才被明顯誘導出來（p<0.05）。實驗結果證實接種表現有NNV抗原蛋白之弧菌均能在石斑魚體中誘導出較高的抗體力價，顯示以弧菌來攜帶抗原蛋白做為疫苗載體的構想的確是可行的.

Vibrio anguillarum, a Gram-negative bacterium, is one of the most important pathogens causing vibriosis in many species of marine fish. Consequently, many V. anguillarum bacterin vaccines have been developed and successfully used in salmonoids. Based on the usage experience of vibrio bacterin vaccine, it has been found that vibrio bacterin not only able to provoke specific immuno-response, but also stimulates non-specific immune response. In order to take advantages of these effects, we have tried to develop a protein expression system using V. anguillarum as a host for the development of fish subunit vaccine.
An expression vector, pMI, was constructed on pEGFP-N1 by adding a tac promoter. The expression ability of pMI was evaluated by the assay of GFP reporter gene expression and confirmed by western blotting. To evaluate the feasibility of this system for the development of a subunit vaccine, a NNV coat protein gene was cloned and expressed in V. anguillarum. The resulting recombinant NNV coat protein in the whole bacterin was used to immunize fish. Sera samples obtained from vaccinated fish indicated that the anti-NNV specific antibody from those fish immunized with the whole bacterin obtained from recombinant NNV expressed in V. anguillarum was more effective than that those immunized with bacterin obtained from recombinant E. coli.
This indicates that the recombinant V. anguillarum expression system has the advantage of offering a single vaccine preparation including both antigen and adjuvant activities, these creating a novel approach for the development of an efficient fish subunit vaccine.

羅秉真，南台灣養殖石斑魚病毒性神經壞死症之研究。國立台灣大學動物學研究所碩士論文，台北，1997
Akjlaghi M. Passive immunization of fish against vibriosis, comparision of intraperitoneal, oral and immersion routes. Aquaclture. 180: 191-205. (1999)
Austin B., Alsina M., Austin D. A., Blanch A. R., Grimont F., Grimont P. A. D., Jofre J., Koblavi S., Larsen J. L., Pedersen K., Tiainen T., Verdonck L., Swings J. Identification and typing of Vibrio anguillarum: a comparison of different methods. Systematic and Applied Microbiology. 18: 285–302. (1995)
Bentley W. E., Mirjalili N., Andersen D. C., Davis, R. H. Plasmid encoded protein: the principle factor in the metabolic burden associated with recombinated bacterial. Biotechnol Bioeng. 35: 668-681. (1990)
Blazer V. S. Piscine macrophage function and nutritional influences: a review. Journal of Aquatic Animal Health. 3: 77-86. (1991)
Boonyaratpalin S., Supamattaya K., Kasornchandra J., Hoffmann R. W., Picona - like virus associated with mortality and a spongious encephalopathy in grouper Epinephelus malabaricus. Dis Aquat Org. 26: 75-80. (1996)
Bowden T. J., Menoyoluque D., Bricknell I. R., Wergeland H. Efficacy of different administration routes for vaccination against Vibrio anguillarum in Atlantic halibut. Fish and Shellfish Immunology. 12: 283-285. (2002)
Brosisu J., Μlrich A., Raker M. A., Noller H. F. Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of Escherichia coli. Plasmid. 6: 112-118. (1981)
Busch R. A. Enteric redmonth disease, in Anderson D. P., Dorson M. DuBourget P (eds): Les antigens des micro-organismes pathogens des poisons, Collection Fongdation Marcel Merieux. 201-222. (1982)
Chen D., Ainsworth A. J. Glucan administration potentiates immune defense mechanisms of channel catfish, Ictalurus punctatus. Journal of fish disease. 15: 295–304. (1992)
Davina J. H. M., Parmentier H. K., Timmermans L. P. M. Effect of oral administration of Vibrio bacterian on the intestine of cyprinid fish. Dev Comp Immunol Suppl. 2: 157-166. (1982)
Dixon P. In: Gudding R., Lillehaung A., Midtlyng P. J., Brown, F (Eds), Fish Vaccinology, Dev Bio Stand. p.107.(1997)
Engstad R.E., Robertsen B., Frivold E. Yeast glucan induces increase in activity of lysozyme and complement-mediated haemolytic activity in Atlantic salmon blood. Fish and Shellfish Immunology. 2: 287–297. (1992)
Evelyn T. P. A historical review of fish vaccinology. Dev Biol Stand. 90: 3-12. (1997).
Freidank H. M., Billing H., Wiedmann-Al-Ahmad M. Influence of iron restriction on Chlamydia pneumoniae and C. trachomatis. J Med Microbiol. 50: 223-227. (2001)
Furste J. P., Pansegrau W., Frank R., Blocker H., Scholz P., Bagdasarian M., Lanka E. Molecular cloning of the RP4 primase region in a multi - host - range tac P expression vector. Gene. 48: 119-131. (1986)
Gentschev I., Dietrich G., Spreng S., Kolb-Maurer A., Daniels J., Hessm H., Kaufman S. H., Goebel W. Delivery of protein antigens and DNA by virulence - attenuated strains of Salmonella typhimurium and Listeria monocytogenes. Journal of Biotechnology. 83: 19-26. (2000)
Gudding R., Lillehaug A., Midtlying P. J., Brown F. Immune responses after injection vaccination of fish. Fish vaccinology. 90: 55-57. (1997a)
Gudding R., Lillehaug A., Midtlying P. J., Brown F. Immune responses after immersion vaccination of fish. Fish vaccinology. 90: 59-68. (1997b)
Gudding R., Lillehaug A., Midtlying P. J., Brown F. Immune responses after oral vaccination. Fish vaccinology. 90: 69-78. (1997c)
Gudding R., Lillehaug O., Evensen. Recent developments in Fish vaccinology. Veterinary Lmmunology and Imunopathology. 72: 203-212. (1999)
Hardie L. J., Fletcher T. C., Secombes C. J. The effect of dietary vitamin C on the immune response of Atlantic salmon Salmo salar . Aquaculture. 95: 201–214. (1991)
Haseth P. L., Zupancic M. L., Record M. T. RNA polymerase - promoter interactions: The comings and goings of RNA polymerase. J Bacteriol. 180: 3019–3025. (1998)
Iwama G., Nakanishi T. The fish immune system, organ, pathogen and environment. Academic Press San Diego. 1–380. (1996)
Jørgensen J. B., Lunde H., Robertsen B. Peritoneal and head kidney cell response to intraperitoneally injected yeast glucan in Atlantic salmon, Salmo salar. Journal of fish disease. 16: 313–325. (1993)
Juan M., Cutrin, Alicia E., Toranzo, Juan L., Barja. Genetic transformation of Vibrio anguillarum and Pasteurella piscicida by electroporation. FEMS Microbiology Letters. 128: 75-80. (1995)
Kajita Y., Sakai M., Atsuta S., Kobayashi M. The immunomodulatory effects of levamisole on rainbow trout, Oncorhynchus mykiss. Fish Pathol. 25: 93–98. (1990)
Kajita Y., Sakai M., Kobayashi M., Kawauchi H. Enhancement of non - specific cytotoxic activity of leucocytes in rainbow trout Oncorhynchus mykiss injected with growth hormone. Fish and Shellfish Immunology. 2: 155–157. (1992b)
Kitao T., Yoshida T. Effect of an immunopotentiator on Aeromonas salmonicida infection in rainbow trout Salmo gairdneri. Vet Immunol Immunopathol. 12: 287–291. (1986)
Kochetkov S. N., Rusakova E. E., Tunitskaya V. L. Recent studies of T7 RNA polymerase mechanism. FEBS Lett. 440: 264–267. (1998)
Lovell R. T. Elevated levels of dietary ascorbic acid increase immune responses in channel catfish. J Nutr. 115: 123–131. (1985)
Lorenzen N., Olesen N. J., Jorgensen P. E. Neutralization of egtved virus pathogenucity to cell culture and fish monoclonal antibodies to the viral G protein. J Gen Viral. 71: 561-567. (1990)
Lorenzen N., Olesen N. J., Koch C. Protective immunity to VHS virus in rainbow trout. Aquaclture .172: 41-61. (1997a)
Lorenzen N., Olesen, N. J., In: Gudding R., Lillehaug A., Midtlyng P. J., Brown F. (Eds), Fish Vaccinology, Dev Biol Stand. p.201. (1997b)
Maza L. M., Peterson E. M. Vaccines for Chlamydia trachomatis infections. Curr Opin Investig Drugs. 3: 980-986. (2002)
Mertens N., Remaut E., Fiers W. Tight transcriptional control mechanism ensures stable high - level expression from T7 promoter - based expression plasmids. Biotechnology. 13: 175–179. (1995)
Masahiro Sakai. Current research status of fish immunostimulants. Aquaclture. 172: 63-92. (1999)
Mclean E., Ash R. Modified uptake of the protein antigen horseradish peroxidase, following oral delivery to rainbow trout, Oncorhynchus mykiss. Aquaculture. 87: 373-379. (1990)
Mori K. I., Nakai T., Muroga K., Arimoto M., Mushiake K., Furusaea I. Properties of a new virus belonging to nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology. 187: 368-371. (1992)
Mori K. I., Nakai T., Nagahara M., Muroga K., Mekuchi T., Kanno T. A viral disease in hatchery - reared larvae and juveniles of redspotted grouper. Gyobyo kenkyu. 26: 209-210. (1991)
Munday B. L., Nakai T., Nguyen H. D. Antigenic relationship of the picorna - like virus of larval barramundi, lates calcarifer Bloch to the nodavirus of larval striped jacj, pseudocaranx dentex. Aust Vet J. 71: 384-385. (1994)
Muroga K. Viral and bacterial disease in larval and juvenile marine fish and shellfish: a review. Fish Pathol. 30: 71-85. (1995)
Nakanishi T., Ototake M. Antigen uptake and immune responses after immersion vaccination. Dev Biol Stand. 90: 59-68. (1997)
Norqvist A., Hagstrom A., Wolf-Watz H. Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum. Appl Environ Microbiol. 55: 1400–1405. (1989)
Olafesen J. A., Hansen G. H. Intact antigen uptake in intestinal epithelial cells of marine fish larvae. J Fish Biol. 40: 141-156. (1992)
Reiner G., Langner A., Cohen S. N., Bujard H. Cloning and analysis of strong promoters is made possible by the downstream placement of a RNA terminator signal. Proc Natl Acad Sca USA. 78: 4936-4940. (1981).
Robin B., Timothy R. Expression of the B subunit of E. coli heat-labile enterotoxin in a marine Vibrio and in a mutant that is pleiotropically defective in the secretion of extracellular protein. Journal of Microbiology. 138: 719-724. (1992)
Sakai M., Kamiya H., Ishii S., Atsuta S., Kobayashi M. The immunostimulating effects of chitin in rainbow trout, Oncorhynchus mykiss. Diseases in Asian Aquaculture. Fish Health Section, Asian Fisheries Society, Manila, Philippines. 1: 413–417. (1992)
Sakai M., Otubo T., Atsuta S., Kobayashi M. Enhancement of resistance to bacterial infection in rainbow trout, Oncorhynchus mykiss Walbaum by oral administration of bovine lactoferrin. Journal of fish disease. 16: 239–247. (1993)
Sakai M., Atsuta S., Kobayashi M. Efficacies of combined vaccine for Vibrio anguillarum and Streptococcus sp. Fisheries Sci. 61: 359–360. (1995)
Sakai M., Kajita Y., Kobayashi M., Kawauchi H. Immunostimulating effect of growth hormone: in vivo administration of growth hormone in rainbow trout enhances resistance to Vibrio anguillarum infection. Vet Immunol Immunopathol. 57: 147–152. (1997)
Santos N. M., Taverne-Thiele J. J., Barnes A. C., Muiswinkel W. B., Elli A. E., Rombout J. H. The gill is a major organ for antibody secreting cell production following direct immersion of sea bass ( Dicentrarchus labrax, L. ) in a Photobacterium damselae ssp. piscicida bacterin: an ontogenetic study. Fish and Shellfish Immunology. 11: 65-74. (2001)
Shimizu T., Nitta M., Mifuchi I. Antitumer activity of marine bacteria , Vibrio anguillarum, in mice. Gann. 70: 429-433. (1979).
Shimizu T., Nitta M., Itoh Y., Yanagihara Y., Mifuchi I. Mitogenicity and adjuvanticity of a marine bacterium, Vubrio anguillarum, in mice. Microbiol Immunol. 25: 929-937. (1981)
Siwicki A. K., Miyazaki T., Komatsu I., Matsuzato T. In vitro influence of heat extract from firefly squid Watasenia scintillans on the phagocyte and lymphocyte activities in rainbow trout Oncorhynchus mykiss. Fish Pathol. 31: 1–7. (1996)
Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct the selective high level expression of cloned genes. J Mol Biol. 189:113–130. (1986)
Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185: 60-89. (1990)
Tanaka S., Mori K., Arimoto M., Iwamoto T., Nakai T. Protective immunity of sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis. Journal of Fish Diseases. 24: 15-22. (2001)
Tebbit G. L., Goodrich T. D. Vibriosis and the development of bacterins for its control, in Anderson DP, Dorson M, Dubourget P (eds): Les antigens des micro-organismse pathogens des poisson, Collection Fondation Marcel Merieux, 225-248. (1982)
Toranzo A. E., Barja J. L. A review of the taxonomy and seropizootiology of Vibrio anguillarum, with special reference to aquaculture in the northwest of Spain. Dis Aquat Org. 9: 73-82. (1990)
Toranzo A. E., Santos Y., Barja J. L. Immunization with vacterial antigens: Vibrio infections. Dev Biol Stand. 90: 93-105. (1997)
Turner M. S., Giffard P. M. Expression of Chlamydia psittaci- and human immunodeficiency virus-derived antigens on the cell surface of Lactobacillus fermentum BR11 as fusion to bspA. Infect Immun. 67: 5486-5489. (1999)
William S., Rosenberg A., Dunn J., Dubendorff J. Use of T7 RNA polymerase to direct expression of cloned genes.In Method in Enzymology, Goeddel D.V.edAcademic press, p185. (1991)
Winton J. R., In: Gudding R., Lillehaug A., Midtlyng P. J., Brown F(Eds.), Fish vaccinology, Dev. Bio. Stand. p.211 (1997)
Yano T., Mangindaan E. P., Matsuyama H. Enhancement of the resistance of carp Cyprinus carpio to experimental Edwardsiella tarda infection, by some b- 1, 3 - glucans. Nippon Suisan Gakkaishi. 55: 1815–1819. (1989)
Yoshikoshi K., Inoue K. Virus nervous necrosis in hatchery-reared larve and juveniles of Japanase parrotfish, Oplegnathus fasciatus . Journal of fish disease. 13: 69-77. (1990).
Yoshida T., Sakai M., Kitao T., Khlil S. M., Araki S., Saitoh R., Ineno T., Inglis V. Immunodulatory effects of the fermented products of chicken egg, EF203, on rainbow trout, Oncorhynchus mykiss. Aquaculture. 109: 207–214. (1993).
Yusa K., Koesharyanl I., Roza D., Mori K., Nakai T. Immune response of Mpbacj grouper, Cromileptes altivelis (Valenciennes) injected with the recombinant coat protein of betanodavirus. Journal of fish disease. 25: 53-56. (2002)