鸭源鸡杆菌gtxA突变株构建及其生物特性分析
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摘要
为了了解毒素GtxA对鸭源鸡杆菌(Gallibacterium anatis,G.anatis)生物学特性及致病性的影响,利用自然转化和正向筛选法构建了1株G.anatis gtxA缺失突变株,初步探究突变株的生物学特性。与亲本菌株比较,突变株溶血活性消失,菌落形态及生长速率并未出现显著改变,其对小鼠的致病力下降。外毒素GtxA参与G.anatis致病过程并扮演一定角色;G.anatis gtxA基因突变株的构建及其生物学特性探究为更好的了解其致病机理和研发出更为高效安全疫苗建立了理论基础。
To determine the biological role of toxin GtxA of Gallibacterium anatis,a Gallibacterium anatis gtxA deleted mutant was constructed by natural transformation,The gtxA deleted mutant was confirmed by PCR,SDS-PAGE,Western blot and heredity seability assays.The biological characterics of the mutant strain was investigated.The results demonstrated that the mutant lose hemolytic activity and possess the same growth rate as its parental strain,but it attenuated in mice.These results indicated that GtxA was an important virulence factor of G.anatis.This study have further illumination on the pathogenicity of G.anatis and it will facilitate the development of a reasonable live attenuated vaccines of G.anatis.
To determine the biological role of toxin GtxA of Gallibacterium anatis,a Gallibacterium anatis gtxA deleted mutant was constructed by natural transformation,The gtxA deleted mutant was confirmed by PCR,SDS-PAGE,Western blot and heredity seability assays.The biological characterics of the mutant strain was investigated.The results demonstrated that the mutant lose hemolytic activity and possess the same growth rate as its parental strain,but it attenuated in mice.These results indicated that GtxA was an important virulence factor of G.anatis.This study have further illumination on the pathogenicity of G.anatis and it will facilitate the development of a reasonable live attenuated vaccines of G.anatis.
引文
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[14] 彭志锋,崔丹丹,杨霞,等.鸭源鸡杆菌ompW基因缺失菌株的构建及其药物敏感性分析 [J].畜牧兽医学报,2017,48(7):1306-1313.
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[21] 郭东春,卢艳,刘家森,等.多杀性巴杆菌aroA基因缺失突变株的构建及鉴定 [J].微生物学报,2012,52(4):526-531.
[22] 王继洋,王艳,彭志锋,等.鸭源鸡杆菌相关致病因子研究进展[J].中国兽医学报,2017,37(8):1627-1632.
[23] VOJTOVA J,KAMANOVA J,SEBO P.Bordetella adenylate cyclase toxin:a swift saboteur of host defense [J].Curr Opin Microbiol,2006,9(1):69.
[24] AHMAD M A,CHOMPOUNOOT I,NITAT S,et al.Structuralcharacterization of humanized nanobodies with neutralizing activity against the bordetella pertussis CyaA-Hemolysin:implications for a potential epitope of toxin-protective antigen[J].Toxins,2016,8(4):99.
[25] 谭双,余旭平,袁佳杰,等.革兰阴性菌蛋白分泌系统研究进展 [J].中国兽药杂志,2009,43(9):45-48.
[26] AKTORIES K,LANG A,SCHWAN C,et al.Actin as target for modification by bacterial protein toxins [J].FEBS J,2011,278(23):4526-4543.
[27] DAS M,ITHYCHANDA S,QIN J,et al.Mechanisms of talin-dependent integrin signaling and crosstalk[J].Biochim Biophys Acta,2014,1838(2):579-588.
[2] 王川庆,陈陆,杨霞,等.蛋鸡群卡氏杆菌感染情况的初步研究 [J].河南农业科学,2008,37(3):97-100.
[3] KRISTENSEN B M,FREES D,BOJESEN A M.Expression and secretion of the RTX-toxin GtxA among members of the genus Gallibacterium [J].Vet Microbiol,2011,153(1/2):116-123.
[4] 李欢.鸭源鸡杆菌流行株毒素蛋白GtxA表达及其部分生物学特性的研究[D].郑州:河南农业大学,2014.
[5] SINGH S V,SINGH B R,SINHA D K,et al.Gallibacterium anatis:an emerging pathogen of poultry birds and domiciled birds [J].J Vet Sci Technol,2016,7(3):1000324.
[6] BAGER R J,NESTA B,PORS S E,et al.The fimbrial protein FlfA from Gallibacterium anatis is a virulence factor and vaccine candidate [J].Infect Immun,2013,81(6):1964.
[7] LALLY E T,HILL R B,KIEBA I R,et al.The interaction between RTX toxins and target cells [J].Trends Microbiol,1999,7(9):356-361.
[8] WATTS J L,SHRYOCK T R,APLEY M,et al.Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals;approved standard-third edition[S].Clinical and Laboratory Standards Institute(CLSI):CLSI document M31-A3,2008.
[9] LINHARTOV I,BUMBA L,BASLER M,et al.RTX proteins:a highly diverse family secreted by a common mechanism [J].Fems Microbiol Rev,2010,34(6):1076-1112.
[10] KAMANOVA J,KOFRONOVA O,MASIN J,et al.Adenylate cyclase toxin subverts phagocyte function by RhoA inhibition and unproductive ruffling [J].J Immunol,2008,181(8):5587-5597.
[11] FU S,OU J,ZHANG M,et al.The live attenuated Actinobacillus pleuropneumoniae triple-deletion mutant ΔapxIC ΔapxIIC ΔapxIV-ORF1 strain,SLW05,Immunizes pigs against lethal challenge with Haemophilus parasuis[J].Clin Vaccine Immunol,2013,20(2):134-139.
[12] KRISTENSEN B M,FREES D,BOJESEN A M.GtxA from Gallibacterium anatis,a cytolytic RTX-toxin with a novel domain organisation [J].Vet Res,2010,41(3):25.
[13] 郑鹿平.我国鸡群鸡杆菌的首次分离鉴定及生物学特性初步研究 [D].郑州:河南农业大学,2010.
[14] 彭志锋,崔丹丹,杨霞,等.鸭源鸡杆菌ompW基因缺失菌株的构建及其药物敏感性分析 [J].畜牧兽医学报,2017,48(7):1306-1313.
[15] POJE G,REDFIELD R J.Transformation of Haemophilus influenzae [J].Methods Mol Med,2003,71(3):57-70.
[16] J 萨姆布鲁克,D W 拉塞尔.分子克隆实验指南[J]. 3版.黄培堂,等译,北京:科学出版社,2008.
[17] 谭秀文,马所峰,刘新勇,等.不同物种输卵管上皮细胞培养、纯度检测及支持小鼠胚胎发育的能力 [J].畜牧兽医学报,2006,37(9):878-882.
[18] 王德云,胡元亮,孔祥峰,等.中药成分对培养的鸡脾脏淋巴细胞增殖的影响 [J].中国家禽,2006,36(19):1210-1214.
[19] 黄勇.细菌基因突变的策略及应用 [J].微生物学通报,2007,34(1):169-172.
[20] TATUM F M,BRIGGS R E.Construction of in-frame aroA deletion mutants of Mannheimia haemolytica,Pasteurella multocida,and Haemophilus somnus by using a new temperature-sensitive plasmid [J].Appl Environ Microbiol,2005,71(11):7196-7202.
[21] 郭东春,卢艳,刘家森,等.多杀性巴杆菌aroA基因缺失突变株的构建及鉴定 [J].微生物学报,2012,52(4):526-531.
[22] 王继洋,王艳,彭志锋,等.鸭源鸡杆菌相关致病因子研究进展[J].中国兽医学报,2017,37(8):1627-1632.
[23] VOJTOVA J,KAMANOVA J,SEBO P.Bordetella adenylate cyclase toxin:a swift saboteur of host defense [J].Curr Opin Microbiol,2006,9(1):69.
[24] AHMAD M A,CHOMPOUNOOT I,NITAT S,et al.Structuralcharacterization of humanized nanobodies with neutralizing activity against the bordetella pertussis CyaA-Hemolysin:implications for a potential epitope of toxin-protective antigen[J].Toxins,2016,8(4):99.
[25] 谭双,余旭平,袁佳杰,等.革兰阴性菌蛋白分泌系统研究进展 [J].中国兽药杂志,2009,43(9):45-48.
[26] AKTORIES K,LANG A,SCHWAN C,et al.Actin as target for modification by bacterial protein toxins [J].FEBS J,2011,278(23):4526-4543.
[27] DAS M,ITHYCHANDA S,QIN J,et al.Mechanisms of talin-dependent integrin signaling and crosstalk[J].Biochim Biophys Acta,2014,1838(2):579-588.