基于体内诱导表达系统在迟钝爱德华氏菌中构建多价载体疫苗
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摘要
重组细菌载体疫苗作为一种有效的策略可以携带抗原蛋白并激发宿主的产生多重免疫保护力。目前,利用细菌载体活疫苗作为递呈载体是新兴现代疫苗的重要方向,接种这种疫苗后,机体既可产生针对载体细菌来源的病原的免疫保护力,又可产生针对携带抗原来源病原的免疫保护力,能够达到同时预防多种病原感染的作用。然而,活菌载体的安全性、外源基因表达稳定性等问题也是细菌载体疫苗发展中不可忽视的问题。迟钝爱德华氏菌(Edwardsiella tarda)是一种重要的革兰氏阴性鱼类病原菌。该病原菌能引起鱼类的出血性败血症,在本实验室前期工作中,已经从发病大菱鲆体内分离得到一株迟钝爱德华氏菌EIB202强毒株,并对其进行了全基因组测序,并构建了一系列适用于载体活菌疫苗的缺失减毒菌株。在本研究中,我们筛选了53个不同细菌来源的铁调控启动子,并检测了它们在体外培养时的转录活性。将筛选得到的具有较好铁调控活性的启动子Pdps和Pdps在巨噬细胞内进行胞内转录活性分析,最终选取了能够在胞内高效表达的启动子Pdps,并在模式动物斑马鱼上进行了观察检测。实验证明Pdps在体内有一个转录增强的趋势,可以用于体内诱导表达系统的构建。为了证明该系统能够作为成熟的抗原表达系统,我们将来源于嗜水气单胞菌(Aeromonas hydrophila) LSA34中的保护性抗原GAPDH连入含有Pdps的重组质粒中,同时,选取迟钝爱德华氏菌的减毒株WED作为宿主菌,构建了细菌载体疫苗WED/pUTDgap.大菱鲆免疫攻毒实验证明,WED/pUTDgap针对对野生型迟钝爱德华氏菌和嗜水气单胞菌均有超过60%的免疫保护力。
本研究在迟钝爱德华氏菌中成功建立了基于铁诱导启动子的体内诱导抗原表达系统,并在迟钝爱德华氏菌减毒株WED中实现了嗜水气单胞菌保护性抗原蛋白的体内表达,成功构建了一株具有二价免疫保护力的迟钝爱德华氏菌减毒活疫苗候选株。
Recombinant bacterial vector vaccine is an attractive vaccination strategy to induce the immune response to a carried protective antigen, and the main concern of bacterial vector vaccine is to establish a stable antigen expression system in vector bacteria. Edwardsiella tarda is an important facultative intracellular pathogen of both animals and humans, and its attenuated derivates are good bacterial vectors for use in recombinant vaccine design. In this study, we aimed to build an in vivo inducible expression system in E. tarda and establish potential recombinant E. tarda vector vaccine. With wild-type strain E. tarda EIB202 as a vector,53 different bacteria-originated promoters were examined for iron-responsive transcription in vitro, and the promoters Pdps and PyncE showed high transcription activity. These two promoters were further assayed for their transcription profile in vivo, and Pdps demonstrated an enhanced in vivo-inducible transcription in macrophage, zebrafish larvae and zebrafish adults. The gapA34 gene, encoding the protective antigen GAPDH from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the Pdps-based protein expression system, and transformed into attenuated E. tarda strains. The resultant recombinant vector vaccine WED/pUTDgap was evaluated in turbot(Scophtalmus maximus). Over 60%of the vaccinated fish survived when challenging with A. hydrophila LSA34 and E. tarda EIB202, suggesting that the Pdps-based antigen delivery system had great potential in bacterial vector vaccine application.
本研究在迟钝爱德华氏菌中成功建立了基于铁诱导启动子的体内诱导抗原表达系统,并在迟钝爱德华氏菌减毒株WED中实现了嗜水气单胞菌保护性抗原蛋白的体内表达,成功构建了一株具有二价免疫保护力的迟钝爱德华氏菌减毒活疫苗候选株。
Recombinant bacterial vector vaccine is an attractive vaccination strategy to induce the immune response to a carried protective antigen, and the main concern of bacterial vector vaccine is to establish a stable antigen expression system in vector bacteria. Edwardsiella tarda is an important facultative intracellular pathogen of both animals and humans, and its attenuated derivates are good bacterial vectors for use in recombinant vaccine design. In this study, we aimed to build an in vivo inducible expression system in E. tarda and establish potential recombinant E. tarda vector vaccine. With wild-type strain E. tarda EIB202 as a vector,53 different bacteria-originated promoters were examined for iron-responsive transcription in vitro, and the promoters Pdps and PyncE showed high transcription activity. These two promoters were further assayed for their transcription profile in vivo, and Pdps demonstrated an enhanced in vivo-inducible transcription in macrophage, zebrafish larvae and zebrafish adults. The gapA34 gene, encoding the protective antigen GAPDH from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the Pdps-based protein expression system, and transformed into attenuated E. tarda strains. The resultant recombinant vector vaccine WED/pUTDgap was evaluated in turbot(Scophtalmus maximus). Over 60%of the vaccinated fish survived when challenging with A. hydrophila LSA34 and E. tarda EIB202, suggesting that the Pdps-based antigen delivery system had great potential in bacterial vector vaccine application.
引文
[1]张奇亚.我国水生动物疾病研究进展.水生生物学报2002,26：89-103.
[2]黄志斌,刘志军,廖国礼,赵军,石存斌.水产养殖动物疾病控制与安全用药.广东饲料2009,18：43-46.
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[4]Sakezaki R, Murata Y. The new group of the Enterobacteriaceae, the Asakusa group. Jap. J Bacteriol 1962,17:616-617.
[5]张晓君,战文斌,陈珍,房海.牙鲆迟钝爱德华氏菌感染症及其病原的研究.水生生物学报2005,29：31-37.
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[9]Brenner J, Fanning R, Steigerwalt AG. Polynucleotide sequence relatedness in Edwardsiella tarda. J Syst Bacteriol 1974,24:186-190.
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[32]John M, Crean TI, Calderwood SB, Ryan ET. In vitro and in vivo analyses of constitutive and in vivo-induced promoters in attenuated vaccine and vector strains of Vibrio cholerae. Infect Immun 2000,68:1171-1175.
[33]Morin CE, Kaper JB. Use of stabilized luciferase-expressing plasmids to examine in vivo-induced promoters in the Vibrio cholerae vaccine strain CVD 103-HgR. FEMS Immunol Med Microbiol 2009,57:69-79.
[34]Husseiny MI, Hensel M. Evaluation of an intracellular-activated promoter for the generation of live Salmonella recombinant vaccines. Vaccine 2005,23:2580-2590.
[35]Galen JE, Pasetti MF, Tennant S, Ruiz-Olvera P, Sztein MB, Levine MM. Salmonella enterica serovar Typhi live vector vaccines finally come of age. Immunol Cell Biol 2009,87:400-412.
[36]Bury NR, Grosell M. Waterborne iron acquisition by a freshwater teleost fish, zebrafish Danio rerio. J Exp Biol 2003,206:3529-3535.
[37]Payne SM. Iron acquisition in microbial pathogenesis. Trends Microbiol 1993,1:66-9.
[38]McHugh JP, Rodriguez-Quinones F, Abdul-Tehrani H, Svistunenko DA, Poole RK, Cooper CE. Global iron-dependent gene regulation in Escherichia coli A new mechanism for iron homeostasis. J Biol Chem 2003,278:29478-29486.
[39]Stojiljkovic I, Baumler AJ, Hantke K. Fur regulon in gram-negative bacteria Identification and characterization of new iron-regulated Escherichia coli genes by a fur titration assay. J Mol Biol 1994,236:531-545.
[40]王启要.海洋鱼类病原溶藻弧菌中铁载体介导的铁摄取系统研究.华东理工大学博士学位论文.2007.4.25.
[41]Wang Q, Yang M, Xiao J, Wu H, Wang X, Lv Y, Zhang Y. Genome sequence of the versatile fish pathogen Edwardsiella tarda provides insights into its adaptation to broad host ranges and intracellular niches. PLoS One 2009,4:e7646.
[42]Sambrook J, Russell DW.黄培堂等译.分子克隆实验指南,第三版.科学出版社,2002.
[43]Saeys Y, Inza I, Larranaga P. A review of feature selection techniques in bioinformatics. Bioinformatics 2007,23:2507-2517.
[44]Park S, Wakabayashi H, and Watanabe Y. Serotype and virulence of Edwardsiella tarda isolated from eel and their environment. Fish Pathol 1983,18:85-89.
[45]Baba-Dikwa A, Thompson D, Spencer NJ, Andrews SC, Watson KA. Overproduction, purification and preliminary X-ray diffraction analysis of YncE, an iron-regulated Sec-dependent periplasmic protein from Escherichia coli. Acta Crystallogr Sect F Struct Biol Cryst Commun 2008,64:966-999.
[46]Wang F, Cheng S, Sun K, Sun L. Molecular analysis of the fur (ferric uptake regulator) gene of a pathogenic Edwardsiella tarda strain. J Microbiol 2008,46:350-355.
[47]Halsey TA, Vazquez-Torres A, Gravdahl DJ, Fang FC, Libby SJ. The ferritin-like Dps protein is required for Salmonella enterica serovar Typhimurium oxidative stress resistance and virulence. Infect Immun 2004,72:1155-1158.
[48]Ishikawa T, Mizunoe Y, Kawabata S, Takade A, Harada M, Wai SN, Hantke K. The iron-binding protein Dps confers hydrogen peroxide stress resistance to Campylobacter jejuni. J Bacteriol 2003, 185:1010-1017.
[49]Olsen KN, Larsen MH, Gahan CG, Kallipolitis B, Wolf XA, Rea R. The Dps-like protein Fri of Listeria monocytogenes promotes stress tolerance and intracellular multiplication in macrophage-like cells. Microbiology 2005,151:925-933.
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[2]黄志斌,刘志军,廖国礼,赵军,石存斌.水产养殖动物疾病控制与安全用药.广东饲料2009,18：43-46.
[3]Pozet F, Morand M, Moussa A, Torhy C, Kinkelin P. Isolation and characterization of a pathogenic icosahedral deoxyribovirus from the catfish Ictalurus melas. Dis Aquat Org 1992,14:35-42.
[4]Sakezaki R, Murata Y. The new group of the Enterobacteriaceae, the Asakusa group. Jap. J Bacteriol 1962,17:616-617.
[5]张晓君,战文斌,陈珍,房海.牙鲆迟钝爱德华氏菌感染症及其病原的研究.水生生物学报2005,29：31-37.
[6]Austin B, Mura DA. Bacterial Fish Pathogens:Disease in Farmed and Wild Fish.3rd ed.1999. Chichester, UK:Parxis Publishing.
[7]Mohant BR, Sahoo PK. Edwardsiellosis in fish:a brief review. J Biosci 2007,32:1331-1344.
[8]Rashid M, Honda K, Naka T, Muroga K. An ecological study on Edwardsiella tarda in flounder farms. Fish Pathol 1994,29:221-227.
[9]Brenner J, Fanning R, Steigerwalt AG. Polynucleotide sequence relatedness in Edwardsiella tarda. J Syst Bacteriol 1974,24:186-190.
[10]Jordan W, Hadley K. Human infection with Edwardsiella tarda. Ann Intern Med 1969,70:283-288.
[11]Newman G. Bacterial vaccine for fish. Ann Rev Fish Dis 1993,32:145-185.
[12]单晓枫,高航,李影.鱼用疫苗研究进展.中国兽药杂志2005,39：19-22.
[13]Iwama G, Nakanishi T. The Fish Immune System:Organism, Pathogen, and Environment. Academic Press,1996.
[14]Gudding R, Lillehug A, Evensen O. Recent developments in fish vaccinology. Vet Immunol Immunopathol 1999,72:203-212.
[15]Cohen D, Orr N, Ham M, Ashkenazi S, Robin G, Green MS. Safety and immunogenicity of two different lots of the oral, killed enterotoxigenic Escherichia coli-cholera toxin B subunit vaccine in Israeli young adults. Infect Immun 2000,68:4492-4497.
[16]Joosten HM, Tiemersma E, Threels A, Caumartin-Dhieux C, Rombout JHWM. Oral vaccination of fish against Vibrio anguillarum using alginate microparticles. Fish Shellfish Immun.1997,7: 471-485.
[17]Kanellos T, Sylvester D, Howard R, Russell H. DNA is as effective as protein at inducing antibody in fish. Vaccine 1999,17:965-972.
[18]Deas A, Rodriguez S, Perez-Prieto I. Immunogenic and protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish. Fish Shellfish Immunol 2010,28:562-570.
[19]Tonheim C, Bogwald J, Dalmo A. What happens to the DNA vaccine in fish? A review of current knowledge. Fish Shellfish Immunol 2008,25:1-18.
[20]Gottschalk M, Laurent S, Lewandowski R. Vaccine development:strategies for coping with the antigenic diversity of bacteria. Rev Sci Technol 2007,26:91-103.
[21]Hagan DT, Valiante NM. Recent advances in the discovery and delivery of vaccine adjuvants Nat Rev Drug Discov 2003,2:727-35.
[22]Dunstan J, Simmons P, Strugnell A. Use of in vivo-regulated promoters to deliver antigens from attenuated Salmonella enterica var. Typhimurium. Infect Immun 1999,67:5133-5141.
[23]Loessner H, Endmann A, Leschner S, Bauer H, Zelmer A, Zur LS. Improving live attenuated bacterial carriers for vaccination and therapy. Int J Med Microbiol 2008,298:21-26.
[24]Leckenby MW, Spear AM, Neeson BN, Williamson ED, Cranenburgh RM, Atkins HS. Enhanced vaccine antigen delivery by Salmonella using antibiotic-free operator-repressor titration-based plasmid stabilisation compared to chromosomal integration. Microb Pathog 2009,46:201-206.
[25]Pappalardo F, Halling-Brown MD, Rapin N, Zhang P, Alemani D, Emerson A, Paci P, Duroux P, Pennisi M. ImmunoGrid. An integrative environment for large-scale simulation of the immune system for vaccine discovery, design and optimization. Brief Bioinform 2009,10:330-340.
[26]Nagarajan AG, Balasundaram SV, Janice J, Karnam G, Eswarappa SM, Chakravortty D. SopB of Salmonella enterica serovar Typhimurium is a potential DNA vaccine candidate in conjugation with live attenuated bacteria. Vaccine 2009,27:2804-2811.
[27]Wang S, Li Y, Shi H, Sun W, Roland KL, Curtiss RR. Comparison of a regulated delayed antigen synthesis system with in vivo-inducible promoters for antigen delivery by live attenuated Salmonella vaccines. Infect Immun 2011,79:937-949.
[28]Stratford R, McKelve ND, Hughes NJ, Aldred E, Wiseman C, Curtis J, Leschner S, Bauer H. Optimization of Salmonella enterica serovar typhi Delta aroC Delta ssaV derivatives as vehicles for delivering heterologous antigens by chromosomal integration and in vivo inducible promoters. Infect Immun 2005,73:362-368.
[29]LoVullo ED, Molins-Schneekloth CR, Schweizer HP, Jr Pavelka MS. Single-copy chromosomal integration systems for Francisella tularensis. Microbiology 2009,155:1152-1163.
[30]Wang S, Li Y, Scarpellin G, Kong W, Shi H, Baek CH, Gunn B, Wanda Y, Roland L, Zhang X, Senechal W, Curtiss R. Salmonella vaccine vectors displaying delayed antigen synthesis in vivo to enhance immunogenicity. Infect Immun 2010,78:3969-3980.
[31]Bullifent HL, Griffin KF, Jones M, Yates A, Harrington L, Titball RW. Antibody responses to Yersinia pestis F1-antigen expressed in Salmonella typhimurium aroA from in vivo-inducible promoters. Vaccine 2000,18:2668-2676.
[32]John M, Crean TI, Calderwood SB, Ryan ET. In vitro and in vivo analyses of constitutive and in vivo-induced promoters in attenuated vaccine and vector strains of Vibrio cholerae. Infect Immun 2000,68:1171-1175.
[33]Morin CE, Kaper JB. Use of stabilized luciferase-expressing plasmids to examine in vivo-induced promoters in the Vibrio cholerae vaccine strain CVD 103-HgR. FEMS Immunol Med Microbiol 2009,57:69-79.
[34]Husseiny MI, Hensel M. Evaluation of an intracellular-activated promoter for the generation of live Salmonella recombinant vaccines. Vaccine 2005,23:2580-2590.
[35]Galen JE, Pasetti MF, Tennant S, Ruiz-Olvera P, Sztein MB, Levine MM. Salmonella enterica serovar Typhi live vector vaccines finally come of age. Immunol Cell Biol 2009,87:400-412.
[36]Bury NR, Grosell M. Waterborne iron acquisition by a freshwater teleost fish, zebrafish Danio rerio. J Exp Biol 2003,206:3529-3535.
[37]Payne SM. Iron acquisition in microbial pathogenesis. Trends Microbiol 1993,1:66-9.
[38]McHugh JP, Rodriguez-Quinones F, Abdul-Tehrani H, Svistunenko DA, Poole RK, Cooper CE. Global iron-dependent gene regulation in Escherichia coli A new mechanism for iron homeostasis. J Biol Chem 2003,278:29478-29486.
[39]Stojiljkovic I, Baumler AJ, Hantke K. Fur regulon in gram-negative bacteria Identification and characterization of new iron-regulated Escherichia coli genes by a fur titration assay. J Mol Biol 1994,236:531-545.
[40]王启要.海洋鱼类病原溶藻弧菌中铁载体介导的铁摄取系统研究.华东理工大学博士学位论文.2007.4.25.
[41]Wang Q, Yang M, Xiao J, Wu H, Wang X, Lv Y, Zhang Y. Genome sequence of the versatile fish pathogen Edwardsiella tarda provides insights into its adaptation to broad host ranges and intracellular niches. PLoS One 2009,4:e7646.
[42]Sambrook J, Russell DW.黄培堂等译.分子克隆实验指南,第三版.科学出版社,2002.
[43]Saeys Y, Inza I, Larranaga P. A review of feature selection techniques in bioinformatics. Bioinformatics 2007,23:2507-2517.
[44]Park S, Wakabayashi H, and Watanabe Y. Serotype and virulence of Edwardsiella tarda isolated from eel and their environment. Fish Pathol 1983,18:85-89.
[45]Baba-Dikwa A, Thompson D, Spencer NJ, Andrews SC, Watson KA. Overproduction, purification and preliminary X-ray diffraction analysis of YncE, an iron-regulated Sec-dependent periplasmic protein from Escherichia coli. Acta Crystallogr Sect F Struct Biol Cryst Commun 2008,64:966-999.
[46]Wang F, Cheng S, Sun K, Sun L. Molecular analysis of the fur (ferric uptake regulator) gene of a pathogenic Edwardsiella tarda strain. J Microbiol 2008,46:350-355.
[47]Halsey TA, Vazquez-Torres A, Gravdahl DJ, Fang FC, Libby SJ. The ferritin-like Dps protein is required for Salmonella enterica serovar Typhimurium oxidative stress resistance and virulence. Infect Immun 2004,72:1155-1158.
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