鮰爱德华菌T6SS eivpP基因缺失突变株的构建
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摘要
为研究鮰爱德华菌Edwardsiella ictaluriⅥ型分泌系统(T6SS)EivpP与其他分泌蛋白之间的相互作用及其在细菌致病过程中的作用机制,在克隆到eivpP基因上、下游同源臂的基础上,采用融合PCR技术制备了eivpP基因缺失用的融合片段,并成功克隆到自杀质粒pDM4内,再将重组自杀质粒转化到大肠杆菌S17-1λ(pir)中,与鮰爱德华菌野生菌株ATCC33202共培养。经同源重组和蔗糖负筛获得鮰爱德华菌eivpP基因缺失突变阳性克隆,经PCR鉴定结果显示,突变株未检测出eivpP基因,即eivpP基因彻底敲除成功。本试验结果可为研究eivpP基因在鮰爱德华菌致病过程中的作用机制提供数据资料,为鮰爱德华菌弱毒疫苗的研发提供理论依据。
In order to study the interaction between EivpP and other secreted proteins of Edwardsiella ictaluri type Ⅵ secretion system(T6 SS) and its mechanism in the pathogenesis of bacteria, the fusion fragment containing the upstream and downstream homologous arms for the eivpP gene deletion was prepared by fusion PCR and successfully cloned into the suicide plasmid pDM4. The recombinant suicide plasmid was transformed into Eschericha coli S17-1λ(pir), co-cultured with E.ictaluri wild strain ATCC33202, and E. ictaluri eivpP gene deletion mutation positive clone was obtained by homologous recombination and sucrose reverse screening. The results of PCR identification showed that the eivpP gene was not detected in the mutant strain, that is, the eivpP gene was successfully knocked out. The findings provide data with research of the role of eivpP gene in the pathogenesis of E.ictaluri, and provide a theoretical basis for the development of E.ictaluri vaccine as well.
In order to study the interaction between EivpP and other secreted proteins of Edwardsiella ictaluri type Ⅵ secretion system(T6 SS) and its mechanism in the pathogenesis of bacteria, the fusion fragment containing the upstream and downstream homologous arms for the eivpP gene deletion was prepared by fusion PCR and successfully cloned into the suicide plasmid pDM4. The recombinant suicide plasmid was transformed into Eschericha coli S17-1λ(pir), co-cultured with E.ictaluri wild strain ATCC33202, and E. ictaluri eivpP gene deletion mutation positive clone was obtained by homologous recombination and sucrose reverse screening. The results of PCR identification showed that the eivpP gene was not detected in the mutant strain, that is, the eivpP gene was successfully knocked out. The findings provide data with research of the role of eivpP gene in the pathogenesis of E.ictaluri, and provide a theoretical basis for the development of E.ictaluri vaccine as well.
引文
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[14] 熊烈,钱淑岚,朱成云,等.假单胞菌基因敲除方法研究进展[J].化学与生物工程,2017,34(8):5-9.
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[19] 刘霞,高鹤,杨琳,等.副溶血性弧菌基因敲除方法的建立及应用[J].中国实验动物学报,2011,19(3):188-192.
[20] Sana T G,Lugo K A,Monack D M.T6SS:the bacterial ‘fight club’ in the host gut[J].PLoS Pathogens,2017,13(6):e1006325.
[21] Wang Xin,Wang Qiyao,Xiao Jingfan,et al.Edwardsiella tarda T6SS component evpP is regulated by esrB and iron,and plays essential roles in the invasion of fish[J].Fish & Shellfish Immunology,2009,27(3):469-477.
[22] Peterson B C,Flora C,Wood M,et al.Vaccination of full-sib channel catfish families against enteric septicemia of catfish with an oral live attenuated Edwardsiella ictaluri vaccine[J].Journal of the World Aquaculture Society,2016,47(2):207-211.
[2] Dong H T,Nguyen V V,Phiwsaiya K,et al.Concurrent infections of Flavobacterium columnare and Edwardsiella ictaluri in striped catfish,Pangasianodon hypophthalmus in Thailand[J].Aquaculture,2015,448:142-150.
[3] Iwanowicz L R,Griffin A R,Cartwright D D,et al.Mortality and pathology in brown bullheads Amieurus nebulosus associated with a spontaneous Edwardsiella ictaluri outbreak under tank culture conditions[J].Diseases of Aquatic Organisms,2006,70(3):219-225.
[4] Keskin O,Se?er S,Izgür M,et al.Edwardsiella ictaluri infection in rainbow trout (Oncorhynchus mykiss)[J].Turkish Journal of Veterinary and Animal Sciences,2004,28(4):649-653.
[5] 肖克宇,江为民,舒新华,等.鮰爱德华氏菌变异株C9605及对鳖的致病性研究[J].微生物学通报,1998,25(5):262-265.
[6] 战文斌.水产动物病害学[M].北京:中国农业出版社,2004.
[7] 殷战,徐伯亥.鱼类细菌性疾病的研究[J].水生生物学报,1995,19(1):76-83.
[8] WooP T K,Bruno D W.Fish diseases and disorders:Vol.3 Vieal,Bacterial and Fungal Infections [M].2nd ed.Wallingford:CABI,2011.
[9] Williams M L,Gillaspy A F,Dyer D W,et al.Genome sequence of Edwardsiella ictaluri 93-146,a strain associated with a natural channel catfish outbreak of enteric septicemia of catfish[J].Journal of Bacteriology,2012,194(3):740-741.
[10] 魏畅,李华,叶仕根,等.鮰爱德华菌hcp基因的克隆及重组表达[J].大连海洋大学学报,2013,28(5):424-430.
[11] 张利娟,叶仕根,杨晓宇,等.革兰氏阴性细菌Ⅵ型分泌系统的研究进展[J].大连海洋大学学报,2015,30(6):692-698.
[12] Zheng Jun,Leung K Y.Dissection of a type VI secretion system in Edwardsiella tarda[J].Molecular Microbiology,2007,66(5):1192-1206.
[13] Chen Hao,Yang Dahai,Han Fajun,et al.The bacterial T6SS effector EvpP prevents NLRP3 inflammasome activation by inhibiting the Ca2+-dependent MAPK-jnk pathway[J].Cell Host & Microbe,2017,21(1):47-58.
[14] 熊烈,钱淑岚,朱成云,等.假单胞菌基因敲除方法研究进展[J].化学与生物工程,2017,34(8):5-9.
[15] 万海英,汤华.基因敲除技术现状及应用[J].医学分子生物学杂志,2007,4(1):86-90.
[16] 谢承佳,何冰芳,李霜.基因敲除技术及其在微生物代谢工程方面的应用[J].生物加工过程,2007,5(3):10-14.
[17] 陶果,信吉阁,肖晶,等.基因敲除技术最新研究进展及其应用[J].安徽农业科学,2013,41(29):11605-11608,11644.
[18] 李敏,杨谦.一种高效构建同源重组DNA片段的方法——融合PCR[J].中国生物工程杂志,2007,27(8):53-58.
[19] 刘霞,高鹤,杨琳,等.副溶血性弧菌基因敲除方法的建立及应用[J].中国实验动物学报,2011,19(3):188-192.
[20] Sana T G,Lugo K A,Monack D M.T6SS:the bacterial ‘fight club’ in the host gut[J].PLoS Pathogens,2017,13(6):e1006325.
[21] Wang Xin,Wang Qiyao,Xiao Jingfan,et al.Edwardsiella tarda T6SS component evpP is regulated by esrB and iron,and plays essential roles in the invasion of fish[J].Fish & Shellfish Immunology,2009,27(3):469-477.
[22] Peterson B C,Flora C,Wood M,et al.Vaccination of full-sib channel catfish families against enteric septicemia of catfish with an oral live attenuated Edwardsiella ictaluri vaccine[J].Journal of the World Aquaculture Society,2016,47(2):207-211.