全细菌SELEX法筛选粪肠球菌特异性适配体
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摘要
肠球菌(Enterococcus)是内源性和外源性医院感染的第二大病原菌,检出率仅次于大肠杆菌,从分子水平上发展靶标的高亲和力分子探针对肠球菌的识别和检测具有非常重要的意义。本研究以粪肠球菌为靶标,运用全细菌指数富集的配体系统进化技术(whole-bacteria systematic evolution of ligands by exponential enrichment, whole-bacteria SELEX),从全长为79个核苷酸包含35个随机碱基序列的单链DNA文库中筛选与靶标高亲和力、高特异性结合的适配体,利用荧光分析法监控筛选过程中不同轮次所得次级文库与粪肠球菌的结合力,经12轮筛选和克隆测序,获得了39条适配体序列。进一步对筛选得到的适配体进行序列比对、二级结构分析、流式细胞分析、解离常数(K_d)测定及特异性验证,最终获得一条与粪肠球菌能特异性结合的适配体Apt 21,其K_d值为549.2±147.4 nmol/L。该适配体可作为粪肠球菌检测的识别元件,为建立基于适配体的新型粪肠球菌检测方法奠定了基础。
Enterococcus is the second most common pathogen of endogenous and exogenous nosocomial infections, whose isolation rate is only lower than that of Escherichia coli. It is of great significance to develop molecular probes with high affinity for Enterococcus, which can be used in the identification and detection of Enterococcus. In this study, aptamers against Enterococcus faecalis were screened by whole-bacteria systematic evolution of ligands by exponential enrichment(whole-bacteria SELEX) from a random single-stranded DNA(ssDNA) library with the length of 79 nucleotides containing 35 random bases. During the SELEX process, the affinity of ssDNA sub-library toward E. faecalis was evaluated by fluorescence analysis. After 12 rounds of selection, cloning and sequencing, overall 39 aptamers were screened. Then sequence alignment, secondary structure prediction, flow cytometry analysis, dissociation constant(K_d) determination and specificity evaluation were carried out. Finally, an aptamer named Apt 21 was picked as the optimal aptamer of E. faecalis, with a K_d value of 549.2 ± 147.4 nmol/L. It can be used as the recognition element for E. faecalis to construct novel detection methods for E. faecalis.
Enterococcus is the second most common pathogen of endogenous and exogenous nosocomial infections, whose isolation rate is only lower than that of Escherichia coli. It is of great significance to develop molecular probes with high affinity for Enterococcus, which can be used in the identification and detection of Enterococcus. In this study, aptamers against Enterococcus faecalis were screened by whole-bacteria systematic evolution of ligands by exponential enrichment(whole-bacteria SELEX) from a random single-stranded DNA(ssDNA) library with the length of 79 nucleotides containing 35 random bases. During the SELEX process, the affinity of ssDNA sub-library toward E. faecalis was evaluated by fluorescence analysis. After 12 rounds of selection, cloning and sequencing, overall 39 aptamers were screened. Then sequence alignment, secondary structure prediction, flow cytometry analysis, dissociation constant(K_d) determination and specificity evaluation were carried out. Finally, an aptamer named Apt 21 was picked as the optimal aptamer of E. faecalis, with a K_d value of 549.2 ± 147.4 nmol/L. It can be used as the recognition element for E. faecalis to construct novel detection methods for E. faecalis.
引文
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[7] Hufnagel M,Sixel K,Hammer F,et al.Detection of opsonic antibodies against Enterococcus faecalis cell wall carbohydrates in immune globulin preparations [J].Infection,2014,42(4):749-755
[8] 黎满香,林荣高,薛立群,等.粪肠球菌PCR检测方法的建立 [J].中国兽医科学(Li MX,Lin RG,Xue LQ,et al.Establishment of a PCR assay for detection of Enterococcus faecalis [J].Chin Vet Sci),2010,40(12):1255-1258
[9] Verbeke F,Debunne N,Janssen S,et al.Detection and quantification of Enterococcus faecalis RNPP-type quorum sensing peptides in bacterial culture media by UHPLC-MS [J].J Pharm Biomed Anal,2018,160 :55-63
[10] Irvine D,Tuerk C,Gold L.SELEXION:Systematic evolution of ligands by exponential enrichment with integrated optimization by non-linear analysis [J].J Mol Biol,1991,222(3):739-761
[11] Green R,Ellington AD,Szostak JW.In vitro genetic analysis of the Tetrahymena self-splicing intron [J].Nature,1990,347(6291):406-408
[12] Yüce M,Ullah N,Budak H.Trends in aptamer selection methods and applications [J].Analyst,2015,140(16):5379-5399
[13] Jenison RD,Gill SC,Pardi A,et al.High-resolution molecular discrimination by RNA [J].Science,1994,263(5152):1425-1429
[14] Jayasena SD.Aptamers:an emerging class of molecules that rival antibodies in diagnostics [J].Clin Chem,1999,45(9):1628-1650
[15] Lavu PS,Mondal B,Ramlal S,et al.Selection and characterization of aptamers using a modified whole cell bacterium SELEX for the detection of Salmonella enterica Serovar Typhimurium [J].ACS Comb Sci,2016,18(6):292-301
[16] Huang Y,Wang X,Duan N,et al.Selection and characterization,application of a DNA aptamer targeted to Streptococcus pyogenes in cooked chicken [J].Anal Biochem,2018,551 :37-42
[17] Song S,Wang X,Xu K,et al.Selection of highly specific aptamers to Vibrio parahaemolyticus using cell-SELEX powered by functionalized graphene oxide and rolling circle amplification [J].Anal Chim Acta,2019,1052 :153-162
[18] Dwivedi HP,Smiley RD,Jaykus LA.Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX [J].Appl Microbiol Biotechnol,2010,87(6):2323-2334
[19] Wheeler LA,Trifonova R,Vrbanac V,et al.Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras [J].J Clin Invest,2011,121(6):2401-2412
[20] Pieken WA,Olsen DB,Benseler F,et al.Kinetic characterization of ribonuclease-resistant 2′-modified hammerhead ribozymes [J].Science,1991,253(5017):314-317
[21] Pan Q,Wang Q,Sun X,et al.Aptamer against mannose-capped lipoarabinomannan inhibits virulent Mycobacterium tuberculosis infection in mice and rhesus monkeys [J].Mol Ther,2014,22(5):940-951
[22] Hamula CL,Zhang H,Guan LL,et al.Selection of aptamers against live bacterial cells [J].Anal Chem,2008,80(20):7812-7819
[23] Li B,Wei H,Dong S.Sensitive detection of protein by an aptamer-based label-free fluorescing molecular switch [J].Chem Commun(Camb),2007,(1):73-75
[24] Jin Y,Bai J,Li H.Label-free protein recognition using aptamer-based fluorescence assay [J].Analyst,2010,135(7):1731-1735
[25] 陈尔凝,赵新颖,屈锋.细菌的核酸适配体筛选的研究进展 [J].色谱(Chen EN,Zhao XY,Qu F.Research advances of aptamers selection for bacterium targets [J].Chin J Chromatogr),2016,34(4):389-396
[26] Fang X,Tan W.Aptamers generated from cell-SELEX for molecular medicine:a chemical biology approach [J].Acc Chem Res,2010,43(1):48-57
[27] Kamal R,Hadi A,Milena F,et al.Development of cell-specific aptamers:recent advances and insight into the selection procedures [J].Molecules,2017,22(12):2070-2082
[28] Catuogno S,Esposito CL.Aptamer cell-based selection:overview and advances [J].Biomedicines,2017,5(3).pii:E 49
[2] Ahmed MO,Baptiste KE.Vancomycin-resistant Enterococci:a review of antimicrobial resistance mechanisms and perspectives of human and animal health [J].Microb Drug Resist,2018,24(5):590-606
[3] 杨青,陈晓,孔海深,等.Mohnarin 2010年度报告:尿标本细菌耐药监测 [J].中华医院感染学杂志(Yang Q,Chen X,Kong HS,et al.Mohnarin report of 2010:surveillance of resistance of pathogens from urine specimens [J].Chin J Nosocomiol),2012,22(3):476-480
[4] 李光辉,朱德妹,汪复,等.2010年中国CHINET血流感染的病原菌分布及耐药性 [J].中国感染与化疗杂志(Li GH,Zhu DM,Wang F,et al.Bacterial distribution and susceptibility in bloodstream infections in China antibiotic resistance surveillance program CHINET 20l0 [J].Chin J Infect Chemother),2012,12(4):251-258
[5] Gao W,Howden BP,Stinear TP.Evolution of virulence in Enterococcus faecium,a hospital-adapted opportunistic pathogen [J].Curr Opin Microbiol,2018,41 :76-82
[6] 欧都·吾吐那生,齐亚银,卜三平,等.动物源粪肠球菌快速鉴定方法的建立 [J].石河子大学学报 (自科版)(Ou Du ·WTNS,Qi YY,Bu SP,et al.Establishment of a rapid indentification method of Enterococcus faecalis from different animal source [J].J Shihezi Univ Nat Sci),2015,33(5):574-577
[7] Hufnagel M,Sixel K,Hammer F,et al.Detection of opsonic antibodies against Enterococcus faecalis cell wall carbohydrates in immune globulin preparations [J].Infection,2014,42(4):749-755
[8] 黎满香,林荣高,薛立群,等.粪肠球菌PCR检测方法的建立 [J].中国兽医科学(Li MX,Lin RG,Xue LQ,et al.Establishment of a PCR assay for detection of Enterococcus faecalis [J].Chin Vet Sci),2010,40(12):1255-1258
[9] Verbeke F,Debunne N,Janssen S,et al.Detection and quantification of Enterococcus faecalis RNPP-type quorum sensing peptides in bacterial culture media by UHPLC-MS [J].J Pharm Biomed Anal,2018,160 :55-63
[10] Irvine D,Tuerk C,Gold L.SELEXION:Systematic evolution of ligands by exponential enrichment with integrated optimization by non-linear analysis [J].J Mol Biol,1991,222(3):739-761
[11] Green R,Ellington AD,Szostak JW.In vitro genetic analysis of the Tetrahymena self-splicing intron [J].Nature,1990,347(6291):406-408
[12] Yüce M,Ullah N,Budak H.Trends in aptamer selection methods and applications [J].Analyst,2015,140(16):5379-5399
[13] Jenison RD,Gill SC,Pardi A,et al.High-resolution molecular discrimination by RNA [J].Science,1994,263(5152):1425-1429
[14] Jayasena SD.Aptamers:an emerging class of molecules that rival antibodies in diagnostics [J].Clin Chem,1999,45(9):1628-1650
[15] Lavu PS,Mondal B,Ramlal S,et al.Selection and characterization of aptamers using a modified whole cell bacterium SELEX for the detection of Salmonella enterica Serovar Typhimurium [J].ACS Comb Sci,2016,18(6):292-301
[16] Huang Y,Wang X,Duan N,et al.Selection and characterization,application of a DNA aptamer targeted to Streptococcus pyogenes in cooked chicken [J].Anal Biochem,2018,551 :37-42
[17] Song S,Wang X,Xu K,et al.Selection of highly specific aptamers to Vibrio parahaemolyticus using cell-SELEX powered by functionalized graphene oxide and rolling circle amplification [J].Anal Chim Acta,2019,1052 :153-162
[18] Dwivedi HP,Smiley RD,Jaykus LA.Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX [J].Appl Microbiol Biotechnol,2010,87(6):2323-2334
[19] Wheeler LA,Trifonova R,Vrbanac V,et al.Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras [J].J Clin Invest,2011,121(6):2401-2412
[20] Pieken WA,Olsen DB,Benseler F,et al.Kinetic characterization of ribonuclease-resistant 2′-modified hammerhead ribozymes [J].Science,1991,253(5017):314-317
[21] Pan Q,Wang Q,Sun X,et al.Aptamer against mannose-capped lipoarabinomannan inhibits virulent Mycobacterium tuberculosis infection in mice and rhesus monkeys [J].Mol Ther,2014,22(5):940-951
[22] Hamula CL,Zhang H,Guan LL,et al.Selection of aptamers against live bacterial cells [J].Anal Chem,2008,80(20):7812-7819
[23] Li B,Wei H,Dong S.Sensitive detection of protein by an aptamer-based label-free fluorescing molecular switch [J].Chem Commun(Camb),2007,(1):73-75
[24] Jin Y,Bai J,Li H.Label-free protein recognition using aptamer-based fluorescence assay [J].Analyst,2010,135(7):1731-1735
[25] 陈尔凝,赵新颖,屈锋.细菌的核酸适配体筛选的研究进展 [J].色谱(Chen EN,Zhao XY,Qu F.Research advances of aptamers selection for bacterium targets [J].Chin J Chromatogr),2016,34(4):389-396
[26] Fang X,Tan W.Aptamers generated from cell-SELEX for molecular medicine:a chemical biology approach [J].Acc Chem Res,2010,43(1):48-57
[27] Kamal R,Hadi A,Milena F,et al.Development of cell-specific aptamers:recent advances and insight into the selection procedures [J].Molecules,2017,22(12):2070-2082
[28] Catuogno S,Esposito CL.Aptamer cell-based selection:overview and advances [J].Biomedicines,2017,5(3).pii:E 49