艰难梭菌毒素B适配子的筛选和鉴定
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摘要
目的筛选和鉴定艰难梭菌毒素B适配子(Apt)。方法体外合成80bp的单链DNA(ssDNA)随机文库,重组表达艰难梭菌毒素B蛋白,利用指数富集配体系统进化(SELEX)技术筛选艰难梭菌毒素B的核酸适配子。使用DNA MAN5.2.2.0软件和mfold软件筛选出的适配子序列进行同源性和二级结构的分析,并利用非竞争酶联免疫吸附试验(ELISA法)测定其亲和常数。结果经过12轮筛选,文库中随机ssDNA与艰难梭菌毒素B的结合率从0.8%增加到39.6%,筛选出51条适配子,二级结构最稳定的是Apt-B20,其亲和常数为3.76×10~(-7)。结论利用SELEX技术成功筛选出高亲和力的Apt,为临床上艰难梭菌感染的实验室快速诊断提供一定的参考依据。
Objective To select and screen the aptamers of Clostridium difficile(Cd)toxin B.MethodsBy using the recombinant Cdtoxin B protein for the screening target,80 bp single stranded DNA(ssDNA)library were synthetised in vitro and used to select and screen the aptamers of Cdtoxin B by systematic evolution of ligands by exponential(SELEX)technology.The homology analysis and secondary structure on the aptamers were analysed by the software of DNA MAN5.2.2.0 and mfold,and the affinity constants of toxin B aptamer were measured by non-competitive ELISA.Results After 12 cycles of selection,the binding affinity of toxin B and ssDNA in the library increased to 39.6%(for 0.8%at first).Fifty-one aptamers were selected randomly and sequenced,and the aptamer with the most stable secondary structure was the Apt-B20,whose affinity constant was 3.76×10-7.Conclusion The aptamers against Cdtoxin B was successfully obtained by SELEX method,which maybe provided certain reference tools for the laboratory diagnosis of Cdinfection.
Objective To select and screen the aptamers of Clostridium difficile(Cd)toxin B.MethodsBy using the recombinant Cdtoxin B protein for the screening target,80 bp single stranded DNA(ssDNA)library were synthetised in vitro and used to select and screen the aptamers of Cdtoxin B by systematic evolution of ligands by exponential(SELEX)technology.The homology analysis and secondary structure on the aptamers were analysed by the software of DNA MAN5.2.2.0 and mfold,and the affinity constants of toxin B aptamer were measured by non-competitive ELISA.Results After 12 cycles of selection,the binding affinity of toxin B and ssDNA in the library increased to 39.6%(for 0.8%at first).Fifty-one aptamers were selected randomly and sequenced,and the aptamer with the most stable secondary structure was the Apt-B20,whose affinity constant was 3.76×10-7.Conclusion The aptamers against Cdtoxin B was successfully obtained by SELEX method,which maybe provided certain reference tools for the laboratory diagnosis of Cdinfection.
引文
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[15]JANK T,GIESEMANN T,AKTORIES K.Rho-glucosylating Clostridium difficile toxins A and B:new insights into structure and function[J].Glycobiology,2007,17(4):15-22.
[16]DAVIES D R,GELINAS A D,ZHANG C,et al.Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets[J].Proc Natl Acad Sci U S A,2012,109(49):19971-19976.
[17]ROHLOFF J C,GELINAS A D,JARVIS T C,et al.Nucleic acid ligands with protein-like side chains:modified aptamers and their use as diagnostic and therapeutic agents[J].Mol Ther Nucleic Acids,2014,3:e201.
[18]WANG Z W,WU H B,MAO Z F,et al.In vitro selection and identification of ssDNA aptamers recognizing the Ras protein[J].Mol Med Rep,2014,10(3):1481-1488.
[19]LOOMANS E E,ROELEN A J,VAN DAMME H S,et al.Assessment of the functional affinity constant of monoclonal antibodies using an improved enzyme-linked immunosorbent assay[J].J Immunol Methods,1995,184(2):207-217.
[20]OCHSNER U A,KATILIUS E,JANJIC N.Detection of clostridium difficile toxins a,B and binary toxin with slow off-rate modified aptamers[J].Diagn Microbiol Infect Dis,2013,76(3):278-285.
[21]NG E W,SHIMA D T,CALIAS P,et al.Pegaptanib,a targeted anti-VEGF aptamer for ocular vascular disease[J].Nat Rev Drug Disco,2006,5(2):123-132.
[2]SULLIVAN N M,PELLETT S,WILKINS T D.Purification and characterization of toxins A and B of Clostridium difficile[J].Infect Immun,1982,35(3):1032-1040.
[3]PLANCHE T,WILCOX M.Reference assays for Clostridium difficile infection:one or two Gold standards?[J].J Clin Pathol,2011,64(1):1-5.
[4]CROBACH M J,DEKKERS O M,WILCOX M H,et al.European society of clinical microbiology and infectious diseases(ESCMID):data review and recommendations for diagnosing clostridium difficile-infection(CDI)[J].Clin Microbiol Infect,2009,15(12):1053-1066.
[5]RENE P,FRENETTE C P,SCHILLER I,et al.Comparison of eight commercial enzyme immunoassays for the detection of Clostridium difficile from stool samples and effect of strain type[J].Diagn Microbiol Infect Dis,2012,73(1):94-96.
[6]MONTEIRO A A,PIRES R N,BAETHGEN L F,et al.Discrepancies among three laboratory methods for Clostridium difficile detection and a proposal for their optimal use[J].FEMS microbiology letters,2014,350(2):133-137.
[7]BUNKA D H,STOCKLEY P G.Aptamers come of ageat last[J].Nat Rev Microbiol,2006,4(8):588-596.
[8]GELINAS A D,DAVIES D R,JANJIC N.Embracing proteins:structural themes in aptamer-protein complexes[J].Curr Opin Struct Biol,2016,36:122-132.
[9]MAYER G.The chemical biology of aptamers[J].Angew Chem,2009,48(15):2672-2689.
[10]PARK K S.Nucleic acid aptamer-based methods for diagnosis of infections[J].Biosen Bioelectron,2017,102:179-188.
[11]吴爱武,唐海先,王丽斯,等.广州地区艰难梭菌耐药检测与耐药机制分析[J].广东医学,2015,36(12):1859-1863.
[12]蔡媛媛.高尔基体蛋白73的原核表达及其适配子的筛选与鉴定[D].广州:广州医学院,2012.
[13]BEATTY J D,BEATTY B G,VLAHOS W G.Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay[J].J Immunol Methods,1987,100(1/2):173-179.
[14]BISHOP A L,HALL A.Rho GTPases and their effector proteins[J].Biochem J,2000,348(2):241-255.
[15]JANK T,GIESEMANN T,AKTORIES K.Rho-glucosylating Clostridium difficile toxins A and B:new insights into structure and function[J].Glycobiology,2007,17(4):15-22.
[16]DAVIES D R,GELINAS A D,ZHANG C,et al.Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets[J].Proc Natl Acad Sci U S A,2012,109(49):19971-19976.
[17]ROHLOFF J C,GELINAS A D,JARVIS T C,et al.Nucleic acid ligands with protein-like side chains:modified aptamers and their use as diagnostic and therapeutic agents[J].Mol Ther Nucleic Acids,2014,3:e201.
[18]WANG Z W,WU H B,MAO Z F,et al.In vitro selection and identification of ssDNA aptamers recognizing the Ras protein[J].Mol Med Rep,2014,10(3):1481-1488.
[19]LOOMANS E E,ROELEN A J,VAN DAMME H S,et al.Assessment of the functional affinity constant of monoclonal antibodies using an improved enzyme-linked immunosorbent assay[J].J Immunol Methods,1995,184(2):207-217.
[20]OCHSNER U A,KATILIUS E,JANJIC N.Detection of clostridium difficile toxins a,B and binary toxin with slow off-rate modified aptamers[J].Diagn Microbiol Infect Dis,2013,76(3):278-285.
[21]NG E W,SHIMA D T,CALIAS P,et al.Pegaptanib,a targeted anti-VEGF aptamer for ocular vascular disease[J].Nat Rev Drug Disco,2006,5(2):123-132.