鳗弧菌菌蜕疫苗的初步制备
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摘要
菌蜕作为一种新型疫苗,较好的保留了细菌的原有形态及膜蛋白结构,能够诱导机体产生体液免疫反应及细胞免疫反应。为制备鳗弧菌菌蜕疫苗,本实验通过基因重组技术,构建了含噬菌体PhiX174裂解酶基因E(LysisE)及温控表达系统cIts857的重组载体pRK-857-E,通过结合转移技术,将重组表达载体转移至鳗弧菌,成功构建鳗弧菌菌蜕。结果表明,42℃升温诱导后,菌体OD600于1h时开始下降,3h后趋于平稳,表示细菌裂解基本完成。平板涂布结果显示,活菌数由诱导前的8.21×10~7 CFU/mL,下降至诱导后的2.31×10~3 CFU/mL,裂解效率接近99.999%。电镜对比观察诱导前后的细菌,发现诱导后的鳗弧菌形态未发生变化,可观察到直径为100~300nm的跨膜裂解孔洞,且有细胞质流出。本研究成功构建鳗弧菌菌蜕疫苗,并对其裂解效果进行检测,为弧菌菌蜕疫苗的开发奠定了基础。
As a new vaccine,the bacterial ghosts preserve the original morphology of bacteria and the structure of membrane protein,which can induce the humoral immune response and cellular immune response.In order to prepare the Vibrioanguillarum bacterial ghost vaccine,a vector containing PhiX174 LysisE and temperature-controlling expression system cIts857 was constructed by gene recombination technology.The recombinant expression vector was transferred to V.anguillarumthrough the conjugal transfer technology,to successfully construct the V.anguillarum bacterial ghosts.The results showed that OD600 began to decrease after 1 hour and became stable after 3 hours when induced temperature rose at 42°C,indicating that the bacterial lysis was basically complete.The spread plate results showed that the number of live bacteria count decreased from 8.21×10~7 CFU/mL before induction to 2.31×10~3 CFU/mL after induction.The lysis efficiency was close to 99.999%.The morphology of bacteria showed no change by transmission electron microscopy observation after induction.However,transmembrane lysis pores with a diameter of 100~300 nm could be observed with cytoplasmic outflow.In the study,we successfully constructed the V.anguillarumbacterial ghosts and detected the lysis effect,which will provide the foundation for the development of Vibrio ghosts vaccine.
As a new vaccine,the bacterial ghosts preserve the original morphology of bacteria and the structure of membrane protein,which can induce the humoral immune response and cellular immune response.In order to prepare the Vibrioanguillarum bacterial ghost vaccine,a vector containing PhiX174 LysisE and temperature-controlling expression system cIts857 was constructed by gene recombination technology.The recombinant expression vector was transferred to V.anguillarumthrough the conjugal transfer technology,to successfully construct the V.anguillarum bacterial ghosts.The results showed that OD600 began to decrease after 1 hour and became stable after 3 hours when induced temperature rose at 42°C,indicating that the bacterial lysis was basically complete.The spread plate results showed that the number of live bacteria count decreased from 8.21×10~7 CFU/mL before induction to 2.31×10~3 CFU/mL after induction.The lysis efficiency was close to 99.999%.The morphology of bacteria showed no change by transmission electron microscopy observation after induction.However,transmembrane lysis pores with a diameter of 100~300 nm could be observed with cytoplasmic outflow.In the study,we successfully constructed the V.anguillarumbacterial ghosts and detected the lysis effect,which will provide the foundation for the development of Vibrio ghosts vaccine.
引文
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[16]Witte A,Wanner G,Blsi U,et al.Endogenous transmembrane tunnel formation mediated by phi X174lysis protein E[J].Journal of Bacteriology,1990,172(7):4109-4114
[17]朱开玲,陈吉祥,李筠,等.鳗弧菌灭活疫苗对海水养殖大菱鲆的免疫预防研究[J].高技术通讯,2004,14(2):76-80
[18]康玉军,杨姣姣,苟彦龙,等.鳗弧菌灭活疫苗对杂交鲟的免疫效果初探[J].甘肃畜牧兽医,2016,46(17):126-128
[19]Kwon S R,Yue J K,Dong J L,et al.Generation of Vibrio anguillarum,Ghost by Coexpression of PhiX 174Lysis E,gene and Staphylococcal Nuclease A,Gene[J].Molecular Biotechnology,2009,42(2):154-9
[20]李绍戊,王荻,刘红柏,等.嗜水气单胞菌菌蜕系统的构建及其免疫效果研究[J].西北农林科技大学学报(自然科学版),2016,44(4):17-23
[21]曹际,马林,张文畅,等.大黄鱼源溶藻弧菌的鉴定及其菌蜕制备[J].微生物学通报,2018,45(1):129-137
[22]Koller V J,Dirsch V M,Beres H,et al.Modulation of bacterial ghosts-induced nitric oxide production in macrophages by bacterial ghost-delivered resveratrol[J].Febs Journal,2013,280(5):1214-1225
[23]吴忆春.猪大肠杆菌O_(139)菌影的制备及其裂解效率[J].中国兽医科学,2013(5):476-479
[24]Chaehun R,Sungjun P,Kihong K,et al.Production of recombinant ghost bacterial vaccine against streptococcal disease of olive flounder[J].Process Biochemistry,2010,45(3):317-322
[2]李立华.我国水产养殖病害控制技术现状与发展趋势[J].科学技术创新,2016(8):274-274
[3]李宁求,付小哲,石存斌,等.大宗淡水鱼类病害防控技术现状及前景展望[J].动物医学进展,2011,32(4):113-117
[4]陆盼盼,郭松林,关瑞章,等.致病性弧菌外膜蛋白及其免疫原理研究进展[J].生物技术通报.2014(04):30-34
[5]张新中,张世秀,李海平,等.海水养殖动物致病性鳗弧菌(Vibroanguillarum)的研究综述[J].渔业信息与战略,2007,22(1):16-19
[6]Duff D C B.The Oral Immunization of Trout Against Bacterium Salmonicida[J].Journal of Immunology,1942,44(1):87-94
[7]Mayrhofer P,Tabrizi C A,Walcher P,et al.Immobilization of plasmid DNA in bacterial ghosts[J].Journal of Controlled Release Official Journal of the Controlled Release Society,2005,102(3):725
[8]吴淑勤,谢骏,石存斌.水产疫苗---鱼类防疫之星[J].江西水产科技,2005(2):1-5
[9]Str9mbestor M,Mustamki N,Heinikainen S,et al.Introduction of Yersinia ruckeri biotype 2into Finnish fish farms[J].Aquaculture,2010,308(1/2):1-5
[10]杨先乐,陈远新.鱼用疫苗的现状及其发展趋势[J].水产学报,1996,20(2):159-167
[11]Witte A,Wanner G,Sulzner M,et al.Dynamics of PhiX174protein E-mediated lysis of Escherichia coli[J].Archives of Microbiology,1992,157(4):381-388
[12]吴淑勤,陶家发,巩华,等.渔用疫苗发展现状及趋势[J].中国渔业质量与标准,2014,4(1):1-13
[13]孙勇琦,江世华,马希艳.活苗和灭活苗优缺点及使用时注意事项[J].中国畜禽种业,2012,8(12):20
[14]Corbeil S,K G,LaPatra SE.Fish DNA vaccine against infectious hematopoietic necrosis virus:efficacy of various routes of immunization[J].Fish Shellfish Immunol,2000(10):711-723
[15]Bernhardt T G,Roof W D,Young R.Genetic Evidence that the BacteriophageφX174Lysis Protein Inhibits Cell Wall Synthesis[J].Proceedings of the National Academy of Sciences of the United States of America,2000,97(8):4297-4302
[16]Witte A,Wanner G,Blsi U,et al.Endogenous transmembrane tunnel formation mediated by phi X174lysis protein E[J].Journal of Bacteriology,1990,172(7):4109-4114
[17]朱开玲,陈吉祥,李筠,等.鳗弧菌灭活疫苗对海水养殖大菱鲆的免疫预防研究[J].高技术通讯,2004,14(2):76-80
[18]康玉军,杨姣姣,苟彦龙,等.鳗弧菌灭活疫苗对杂交鲟的免疫效果初探[J].甘肃畜牧兽医,2016,46(17):126-128
[19]Kwon S R,Yue J K,Dong J L,et al.Generation of Vibrio anguillarum,Ghost by Coexpression of PhiX 174Lysis E,gene and Staphylococcal Nuclease A,Gene[J].Molecular Biotechnology,2009,42(2):154-9
[20]李绍戊,王荻,刘红柏,等.嗜水气单胞菌菌蜕系统的构建及其免疫效果研究[J].西北农林科技大学学报(自然科学版),2016,44(4):17-23
[21]曹际,马林,张文畅,等.大黄鱼源溶藻弧菌的鉴定及其菌蜕制备[J].微生物学通报,2018,45(1):129-137
[22]Koller V J,Dirsch V M,Beres H,et al.Modulation of bacterial ghosts-induced nitric oxide production in macrophages by bacterial ghost-delivered resveratrol[J].Febs Journal,2013,280(5):1214-1225
[23]吴忆春.猪大肠杆菌O_(139)菌影的制备及其裂解效率[J].中国兽医科学,2013(5):476-479
[24]Chaehun R,Sungjun P,Kihong K,et al.Production of recombinant ghost bacterial vaccine against streptococcal disease of olive flounder[J].Process Biochemistry,2010,45(3):317-322