维氏气单胞菌的smpB、tmRNA及hfq敲除菌株减毒活疫苗筛选
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摘要
为筛选维氏气单胞菌减毒活疫苗,为水产重要细菌性病害的防控提供新策略,本文通过荧光实时定量PCR检测三种敲除型菌株smpB、tmRNA及hfq中毒力相关基因表达;并采取腹腔注射法对尼罗罗非鱼进行野生型及三种敲除型感染,观察染病罗非鱼症状,计算累计死亡率及LD50。结果表明,维氏气单胞菌野生型与三种敲除型菌株相比,细菌毒力相关蛋白表达均呈现显著差异,部分毒力相关基因显著下调;三种敲除型菌株LD50分别增加1.24、5.32和19倍。由此可见,三种敲除型维氏气单胞菌可作为候选减毒疫苗,下调显著的hfq菌株是制备减毒疫苗的首选。
In order to screen out live attenuated vaccines from Aeromonas veronii so as to provide a new strategy for controlling against bacterial diseases of aquatic products, toxic gene expressions of A. veronii strains deleted smpB, tmRNA or hfq were detected by Quantitative Real-time PCR. The symptom, accumulative death rate and LD50 of the wild Oreochromis niloticus and some infected with deleted strains by Intraperitoneal injection were recorded or calculated. The resulted showed there were significant differences among bacterial toxicity relative protein expressions of three deleted strains and wild A.veronii, some of them obviously went down, LD50 of three strains added up to 1.24, 5.32 and 19 times, respectively.Therefore the deleted A. veronii strains could be developed for candidates of attenuated vaccines, hfq strain was a first choice.
In order to screen out live attenuated vaccines from Aeromonas veronii so as to provide a new strategy for controlling against bacterial diseases of aquatic products, toxic gene expressions of A. veronii strains deleted smpB, tmRNA or hfq were detected by Quantitative Real-time PCR. The symptom, accumulative death rate and LD50 of the wild Oreochromis niloticus and some infected with deleted strains by Intraperitoneal injection were recorded or calculated. The resulted showed there were significant differences among bacterial toxicity relative protein expressions of three deleted strains and wild A.veronii, some of them obviously went down, LD50 of three strains added up to 1.24, 5.32 and 19 times, respectively.Therefore the deleted A. veronii strains could be developed for candidates of attenuated vaccines, hfq strain was a first choice.
引文
[1]简灯.维氏气单胞菌分离鉴定及其灭活疫苗效应的研究[D].重庆:西南大学,2015:1
[2]吴同垒,单晓枫,孟庆峰,等.维氏气单胞菌研究进展[J].中国兽药杂志,2011,45(7):41-44
[3]曾范利,赵丹,康元环,等.不同动物源性维氏气单胞菌气溶素基因的克隆及比较分析[J].中国兽药杂志,2014,48(8):6-10
[4] Roberts M, Enoch D, Harris K, et al. Aeromonas veronii biovar sobria bacteraemia with septic arthritis confirmed by16SrDNA PCR in an immunocompetent adult[J]. J Med Microbiol, 2006,55(2):241-243
[5] Mencacci A, Cenci E, Mazzolla R, et al. Aeromonas veronii biovar veronii septicaemia and acute suppurative cholangitis in a patient with hepatitis B[J]. J Med Microbiol, 2003,52(8):727-730
[6] Huter P, Müller C, Arenz S, et al. Structural basis for ribosome rescue in bacteria[J]. Trends Biochem Sci., 2017,42(8):669-680
[7] Hayes CS, Keiler KC. Beyond ribosome rescue:tm RNA and co-translational processes[J]. FEBS Lett, 2010,584(2):413-419
[8] Vogel J, Luisi BF. Hfq and its constellation of RNA[J]. Nat.Rev.Microbiol., 2011,9(8):578–589
[9]徐巧林,宋艳钊,郭岩彬.细菌非编码RNA及其分子伴侣Hfq[J].微生物学报,2018,58(9):1511-1520
[10] Robertson GT, Roop RM. The Brucella abortus host factor I(HF-I)protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice[J]. Mol Microbiol, 1999,34(4):690-700
[11] Sittka A, Pfeiffer V, Tedin K, et al. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium[J].Mol Microbiol, 2007,63(1):193-217
[12] Ding Y, Davis BM, Waldor MK. Hfq is essential for Vibrio cholerae virulence and downregulates sigma expression[J].Mol Microbiol, 2004,53(1):345-354
[13] Ansong C, Yoon H, Porwollik S, et al. Global systems-level analysis of Hfq and SmpB deletion mutants in Salmonella:implications for virulence and global protein translation[J]. PLoS ONE, 2009,4(3):e4809
[14] Karasova D, Sebkova A, Vrbas V, et al. Comparative analysis of Salmonella enterica serovar Enteritidis mutants with a vaccine potential[J]. Vaccine, 2009,27(38):5265-5270
[15]顾兵,张政,李玉萍,等.半数致死量及其计算方法概述[J].中国职业医学,2009,36(6):507-509
[16]康元环,张冬星,杨滨僮,等.维氏气单胞菌最新研究进展[J].中国人兽共患病学报,2018,34(5):452-410
[17]李征.减毒活疫苗的应用及其研究进展[J].中国生物制品学杂志,2018,31(2):205-209
[2]吴同垒,单晓枫,孟庆峰,等.维氏气单胞菌研究进展[J].中国兽药杂志,2011,45(7):41-44
[3]曾范利,赵丹,康元环,等.不同动物源性维氏气单胞菌气溶素基因的克隆及比较分析[J].中国兽药杂志,2014,48(8):6-10
[4] Roberts M, Enoch D, Harris K, et al. Aeromonas veronii biovar sobria bacteraemia with septic arthritis confirmed by16SrDNA PCR in an immunocompetent adult[J]. J Med Microbiol, 2006,55(2):241-243
[5] Mencacci A, Cenci E, Mazzolla R, et al. Aeromonas veronii biovar veronii septicaemia and acute suppurative cholangitis in a patient with hepatitis B[J]. J Med Microbiol, 2003,52(8):727-730
[6] Huter P, Müller C, Arenz S, et al. Structural basis for ribosome rescue in bacteria[J]. Trends Biochem Sci., 2017,42(8):669-680
[7] Hayes CS, Keiler KC. Beyond ribosome rescue:tm RNA and co-translational processes[J]. FEBS Lett, 2010,584(2):413-419
[8] Vogel J, Luisi BF. Hfq and its constellation of RNA[J]. Nat.Rev.Microbiol., 2011,9(8):578–589
[9]徐巧林,宋艳钊,郭岩彬.细菌非编码RNA及其分子伴侣Hfq[J].微生物学报,2018,58(9):1511-1520
[10] Robertson GT, Roop RM. The Brucella abortus host factor I(HF-I)protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice[J]. Mol Microbiol, 1999,34(4):690-700
[11] Sittka A, Pfeiffer V, Tedin K, et al. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium[J].Mol Microbiol, 2007,63(1):193-217
[12] Ding Y, Davis BM, Waldor MK. Hfq is essential for Vibrio cholerae virulence and downregulates sigma expression[J].Mol Microbiol, 2004,53(1):345-354
[13] Ansong C, Yoon H, Porwollik S, et al. Global systems-level analysis of Hfq and SmpB deletion mutants in Salmonella:implications for virulence and global protein translation[J]. PLoS ONE, 2009,4(3):e4809
[14] Karasova D, Sebkova A, Vrbas V, et al. Comparative analysis of Salmonella enterica serovar Enteritidis mutants with a vaccine potential[J]. Vaccine, 2009,27(38):5265-5270
[15]顾兵,张政,李玉萍,等.半数致死量及其计算方法概述[J].中国职业医学,2009,36(6):507-509
[16]康元环,张冬星,杨滨僮,等.维氏气单胞菌最新研究进展[J].中国人兽共患病学报,2018,34(5):452-410
[17]李征.减毒活疫苗的应用及其研究进展[J].中国生物制品学杂志,2018,31(2):205-209