羊种布鲁氏菌疫苗株PCR-RFLP鉴定方法的建立
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摘要
为建立一种羊种布鲁氏菌Rev.1疫苗株PCR-RFLP鉴定方法,利用羊种布鲁氏菌Rev.1具有链霉素抗性的特性,选取与链霉素抗性相关编码核糖体蛋白S12的rps L基因为模板设计引物,经PCR扩增后,将产物进行Nci I酶切,发现羊种布鲁氏菌疫苗株Rev.1出现约500 bp条带,而不具有链霉素抗性的羊种布鲁氏菌株16M则出现384 bp条带。结果表明,PCR-RFLP方法能够用于羊种布鲁氏菌Rev.1疫苗株的鉴定,这为今后区分羊种布鲁氏菌疫苗株Rev.1免疫与野株感染提供了基础。
In order to develop a PCR-RFLP method for identifying the brucella melitensis vaccine strain Rev.1,given that the Rev.1 strain was resistant to streptomycin and the rps L gene was related to the streptomycin resistance,a pair of primers were designed according to the sequence of rps L gene in Gen Bank.After PCR amplification,the PCR products were digested with Nci I.Results showed that,a 500 bp band was got in the enzyme-digested products of the vaccine strain Rev.1,while a 384 bp band was got in the enzyme-digested products of wild strain 16 M.As a conclusion,the PCR-RFLP method could be applied in identification of the brucella melitensis vaccine strain Rev.1,which would provide a solid foundation for differentiating brucella melitensis vaccine strain Rev.1 and other wild strains in the future.
In order to develop a PCR-RFLP method for identifying the brucella melitensis vaccine strain Rev.1,given that the Rev.1 strain was resistant to streptomycin and the rps L gene was related to the streptomycin resistance,a pair of primers were designed according to the sequence of rps L gene in Gen Bank.After PCR amplification,the PCR products were digested with Nci I.Results showed that,a 500 bp band was got in the enzyme-digested products of the vaccine strain Rev.1,while a 384 bp band was got in the enzyme-digested products of wild strain 16 M.As a conclusion,the PCR-RFLP method could be applied in identification of the brucella melitensis vaccine strain Rev.1,which would provide a solid foundation for differentiating brucella melitensis vaccine strain Rev.1 and other wild strains in the future.
引文
[1]BLASCO J M.Animal brucellosis:Brucella ovis[M].NIELSEN K,DUNCAN J R,editors.Boca Raton:CRC Press,1990:351-378.
[2]崔步云.中国布鲁氏菌病疫情监测与控制[J].疾病监测,2007,22(10):649-651.
[3]BLASCO J M.A review of the use of B.melitensis Rev.1vaccine in adult sheep and goats[J].Preventive veterinary medicine,1997,31:275-283.
[4]GARIN-BASTUJI B,BLASCO J M,GRAYON M,et al.Brucella melitensis infection in sheep:present and future[J].Veterinary research,1998,29:255-274.
[5]ELBERG S S,FAUNCE K J R.Immunization against Brucella infection.VI.Immunity conferred on goats by a nondependent mutant from a streptomycin-dependent mutant strain of Brucella melitensis[J].Journal of bacteriology,1957,73:211-217.
[6]ALTON G G,JONES L M,ANGUS R D,et al.Techniques for the brucellosis laboratory[J].CAB direct,1988,13(6):420.
[7]BJORKMAN J,SAMUELSSON P,ANDERSSON D I,et al.Novel ribosomal mutations affecting translational accuracy,antibiotic resistance and virulence of Salmonella typhimurium[J].Molecular microbiology,1999,31:53-58.
[8]FINKENM,KIRSCHNERP,MEIERA,et al.Molecular basis of streptomycin resistance in Mycobacterium tuberculosis:alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot[J].Molecular microbiology,1993,9:1239-1246.
[9]KENNEY T J,CHURCH WARDG.Cloning and sequence analysis of the rps L and rps G genes of Mycobacterium smegmatis and characterization of mutations causing resistance to streptomycin[J].Journal of bacteriology,1994,176:6153-6156.
[10]CLOECKAERT A,GRAYON M,GRéPINET O.Identification of Brucella melitensis,vaccine strain Rev.1by PCR-RFLP based on a mutation in the rps L gene[J].Vaccine,2002,20(19/20):2546-2550.
[11]钟旗,范伟兴.用AMOS-PCR对布鲁氏菌种型鉴定的研究[J].中国人兽共患病学报,2007,23(7):683-686.
[12]SHOME R,GUPTA V K,RAO K N,et al.Detection of Brucella melitensis Rev-1 vaccinal antibodies in sheep in India[J].Advances in animal and veterinary sciences,2014,280(2):19-22.
[13]易新萍,马晓菁,闫晶华,等.牛乳样品中布鲁氏菌的分离和鉴定[J].中国预防兽医学报,2013,1(35):28-30.
[14]丁家波,冯忠武.动物布鲁氏菌病疫苗应用现状及研究进展[J].生命科学,2013,25(1):91-99.
[15]付湘云,马晓菁,等.羊用布鲁氏菌疫苗研究进展[J].动物医学进展,2015,36(5):91-94.
[16]BLASCO J M.A review of the use of B.melitensis Rev.1vaccine in adult sheep and goats[J].Preventive veterinary medicine,1997,31(3/4):275-283.
[17]JENSEN A E,EWALT D R,CHEVILLE N F,et a1.Application of pulse-field gel electrophoresis for differentiation of vaccine strain RB51 from field isolates of Brucella abortus from cattle,bison,and elk[J].American journal of veterinary research,1995,56(3):308-312.
[18]RIDLER A L,LEYLAND M J,FENWICK S G,et a1.Demonstration of polymorphism among Brucella ovis field isolates by pulsed-field gel electrophoresis[J].Veterinary microbiology,2005,108(1/2):69-74
[19]骆利敏,崔步云,芮勇宇,等.布鲁菌脉冲场凝胶电泳图谱分型研究[J].中华微生物学和免疫学杂志,2005,25(12):1043.
[2]崔步云.中国布鲁氏菌病疫情监测与控制[J].疾病监测,2007,22(10):649-651.
[3]BLASCO J M.A review of the use of B.melitensis Rev.1vaccine in adult sheep and goats[J].Preventive veterinary medicine,1997,31:275-283.
[4]GARIN-BASTUJI B,BLASCO J M,GRAYON M,et al.Brucella melitensis infection in sheep:present and future[J].Veterinary research,1998,29:255-274.
[5]ELBERG S S,FAUNCE K J R.Immunization against Brucella infection.VI.Immunity conferred on goats by a nondependent mutant from a streptomycin-dependent mutant strain of Brucella melitensis[J].Journal of bacteriology,1957,73:211-217.
[6]ALTON G G,JONES L M,ANGUS R D,et al.Techniques for the brucellosis laboratory[J].CAB direct,1988,13(6):420.
[7]BJORKMAN J,SAMUELSSON P,ANDERSSON D I,et al.Novel ribosomal mutations affecting translational accuracy,antibiotic resistance and virulence of Salmonella typhimurium[J].Molecular microbiology,1999,31:53-58.
[8]FINKENM,KIRSCHNERP,MEIERA,et al.Molecular basis of streptomycin resistance in Mycobacterium tuberculosis:alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot[J].Molecular microbiology,1993,9:1239-1246.
[9]KENNEY T J,CHURCH WARDG.Cloning and sequence analysis of the rps L and rps G genes of Mycobacterium smegmatis and characterization of mutations causing resistance to streptomycin[J].Journal of bacteriology,1994,176:6153-6156.
[10]CLOECKAERT A,GRAYON M,GRéPINET O.Identification of Brucella melitensis,vaccine strain Rev.1by PCR-RFLP based on a mutation in the rps L gene[J].Vaccine,2002,20(19/20):2546-2550.
[11]钟旗,范伟兴.用AMOS-PCR对布鲁氏菌种型鉴定的研究[J].中国人兽共患病学报,2007,23(7):683-686.
[12]SHOME R,GUPTA V K,RAO K N,et al.Detection of Brucella melitensis Rev-1 vaccinal antibodies in sheep in India[J].Advances in animal and veterinary sciences,2014,280(2):19-22.
[13]易新萍,马晓菁,闫晶华,等.牛乳样品中布鲁氏菌的分离和鉴定[J].中国预防兽医学报,2013,1(35):28-30.
[14]丁家波,冯忠武.动物布鲁氏菌病疫苗应用现状及研究进展[J].生命科学,2013,25(1):91-99.
[15]付湘云,马晓菁,等.羊用布鲁氏菌疫苗研究进展[J].动物医学进展,2015,36(5):91-94.
[16]BLASCO J M.A review of the use of B.melitensis Rev.1vaccine in adult sheep and goats[J].Preventive veterinary medicine,1997,31(3/4):275-283.
[17]JENSEN A E,EWALT D R,CHEVILLE N F,et a1.Application of pulse-field gel electrophoresis for differentiation of vaccine strain RB51 from field isolates of Brucella abortus from cattle,bison,and elk[J].American journal of veterinary research,1995,56(3):308-312.
[18]RIDLER A L,LEYLAND M J,FENWICK S G,et a1.Demonstration of polymorphism among Brucella ovis field isolates by pulsed-field gel electrophoresis[J].Veterinary microbiology,2005,108(1/2):69-74
[19]骆利敏,崔步云,芮勇宇,等.布鲁菌脉冲场凝胶电泳图谱分型研究[J].中华微生物学和免疫学杂志,2005,25(12):1043.