CP4-EPSPS转基因蛋白的适体筛选与亲和性分析
|
摘要
本文应用SELEX技术(指数富集配基的系统进化)对CP4-EPSPS转基因蛋白进行适体筛选,旨在筛选出能与该靶目标具有高亲和力、特异性结合的DNA适体,为DNA适体在转基因成分检测中的应用研究奠定基础。
本文在对CP4-EPSPS转基因蛋白的SELEX体外筛选过程中,利用反向筛选技术去除可能存在的与靶蛋白相同的结合位点,缩短针对靶蛋白的特异表位的适体富集过程。经过15轮的筛选,对筛选产物进行克隆、测序,并对测序适体进行一级结构和二级结构的分析、结合率以及特异性的测定、比较。
SELEX筛选结果表明,每轮的ssDNA富集文库与靶蛋白的结合率呈上升趋势,结合显色后吸光度值从第1轮的0.2257上升到第15轮的1.5132,提高了6.7倍。一级结构保守序列显示分别有4对适体序列完全一致和10对几乎完全相同(其中6对完全一致,其余4对只有1、2个碱基的差别),这说明一级结构同源的适体得到了富集。二级结构模拟图以茎环状结构为主,说明这些茎环状结构是适体与靶蛋白进行结合的基础。适体与靶蛋白的结合实验表明,D04号适体的结合率最大,G02号适体的结合率最小。D04号适体特异性实验显示,D04号适体与CP4-EPSPS转基因蛋白结合后的吸光度值最大,说明D04号适体具有较好的特异性,为后续的应用研究奠定了基础。
In this study, in order to lay a foundation for the DNA aptamer to detection the genetically modified component, SELEX method was utilized to screen DNA aptamers that specially bind the target with a high affinity.
In vitro selection process against CP4-EPSPS transgenic protein, reverse screening method was used to remove the common binding site which existed probably to contract the process of aptamer enrichment against specific site on the target. After 15 rounds of selection, the screened products were cloned and sequenced. The primary structure and secondary structure of the sequenced aptamers was analyzed. The combination rate and specificity of the aptamers was also measured and compared.
The SELEX screening results showed that the combination rate of ssDNA enriched library of each round with target protein had increased, and the absorbance value had raised 6.7 times, from 0.2257 of the first round to 1.5132 of the fifteenth round. The conserved sequences of primary structure also revealed that 4 pairs of aptamers had identical sequences and 10 pairs aptamers (6 pairs of 10 pairs aptamers had identical sequences and there was only one or two different base in sequences of another 4 of aptamers.)This implicated that the aptamers with primary structure homology had been enriched.The secondary structure consisted mainly of Stem-loop structure.This suggested that these Stem-loops were the basis for aptamers binding to target protein.The experiments of aptamer binding to CP4-EPSPS transgenic protein showed that the combination rate of NO.D04 aptamer was maximum, and the combination rate of NO.G02 aptamer was minimum. The specificity experiments of NO.D04 aptamer revealed that the absorbance value of NO.D04 aptamer binding to CP4-EPSPS transgenic protein was maximum. This implicated that NO.D04 aptamer had a better specificity. And this had laid a foundation for subsequent applied research.
本文在对CP4-EPSPS转基因蛋白的SELEX体外筛选过程中,利用反向筛选技术去除可能存在的与靶蛋白相同的结合位点,缩短针对靶蛋白的特异表位的适体富集过程。经过15轮的筛选,对筛选产物进行克隆、测序,并对测序适体进行一级结构和二级结构的分析、结合率以及特异性的测定、比较。
SELEX筛选结果表明,每轮的ssDNA富集文库与靶蛋白的结合率呈上升趋势,结合显色后吸光度值从第1轮的0.2257上升到第15轮的1.5132,提高了6.7倍。一级结构保守序列显示分别有4对适体序列完全一致和10对几乎完全相同(其中6对完全一致,其余4对只有1、2个碱基的差别),这说明一级结构同源的适体得到了富集。二级结构模拟图以茎环状结构为主,说明这些茎环状结构是适体与靶蛋白进行结合的基础。适体与靶蛋白的结合实验表明,D04号适体的结合率最大,G02号适体的结合率最小。D04号适体特异性实验显示,D04号适体与CP4-EPSPS转基因蛋白结合后的吸光度值最大,说明D04号适体具有较好的特异性,为后续的应用研究奠定了基础。
In this study, in order to lay a foundation for the DNA aptamer to detection the genetically modified component, SELEX method was utilized to screen DNA aptamers that specially bind the target with a high affinity.
In vitro selection process against CP4-EPSPS transgenic protein, reverse screening method was used to remove the common binding site which existed probably to contract the process of aptamer enrichment against specific site on the target. After 15 rounds of selection, the screened products were cloned and sequenced. The primary structure and secondary structure of the sequenced aptamers was analyzed. The combination rate and specificity of the aptamers was also measured and compared.
The SELEX screening results showed that the combination rate of ssDNA enriched library of each round with target protein had increased, and the absorbance value had raised 6.7 times, from 0.2257 of the first round to 1.5132 of the fifteenth round. The conserved sequences of primary structure also revealed that 4 pairs of aptamers had identical sequences and 10 pairs aptamers (6 pairs of 10 pairs aptamers had identical sequences and there was only one or two different base in sequences of another 4 of aptamers.)This implicated that the aptamers with primary structure homology had been enriched.The secondary structure consisted mainly of Stem-loop structure.This suggested that these Stem-loops were the basis for aptamers binding to target protein.The experiments of aptamer binding to CP4-EPSPS transgenic protein showed that the combination rate of NO.D04 aptamer was maximum, and the combination rate of NO.G02 aptamer was minimum. The specificity experiments of NO.D04 aptamer revealed that the absorbance value of NO.D04 aptamer binding to CP4-EPSPS transgenic protein was maximum. This implicated that NO.D04 aptamer had a better specificity. And this had laid a foundation for subsequent applied research.
引文
[1]梁克红,李俊,李军国,等.豆粕中转基因成分检测方法的研究[J].中国农业科技导报,2008,10(S2):81-86.
[2]陶震,杨胜利,龚毅.利用MPCR方法快速检测植物转基因背景[J].生物技术通报,2000.6:37-41.
[3]蒋原,祝长青,林宏,等.利用PCR技术检测转基因大豆Roundup Ready的研究[J].检验检疫科学,2001,11(4):11-13.
[4] Marc V,Hans P,Franco G,et al.Real-time quantitative PCR detection of genetically modified maximizer maize and roundup ready soybean in some representative foods[J].J.Agric.Food Chem.1999,47:5261-5266.
[5]金红,程奕,赵昕,等.大豆色拉油中转基因成分检测技术研究[J].华北农学报,2004,19(1):24-27.
[6] Rottm E,Lawrence T S,Wall E M,et al.Detection and quantification of roundup ready soy in foods by conventional and real-time polymerase chain reaction [J].J.Agric.Food Chem.2004,52(16):5223-5232.
[7] Rogan G J,Dud N Y A,Lee T C,et al.Immunodiagnostic methods for detection of 5-enolpyruvyl shikimate-3-phosphate synthase in Roundup Ready soybeans[J].Food Control,1999,10:407-414.
[8] Jun Sheng Lin,Kenneth P McNatty.Aptamer-Based Regionally Protected PCR for Protein Detection[J].Clinical Chemistry,2009,55(9):1686-1693.
[9]邬芳玉.SELEX筛选AFP-L3核酸适配子方法的建立[D].南昌:南昌大学医学院,2006.
[10]马占忠,秦莲花,王玉炯,等.结核分枝杆菌ESAT-6抗原适体的筛选与亲和性分析[J].中华临床医师杂志,2007,1(5):45-48.
[11]甄蓓,张敏丽,宋亚军,等.抗体-适配子混合夹心法检测炭疽芽孢杆菌芽孢的研究[J].生物技术通讯,2002,l3(2):l23-l25.
[12]江树勋,黄新,邵碧英,等.转基因玉米EPSPS蛋白适体的筛选与结构分析[J].热带作物学报,2010,31(4):600-604.
[13]姚姝,张保龙,沈新莲,等.分别转CP4 EPSPS和aroA基因拟南芥对草甘膦的抗性[J].苏农业学报,2006,22(3):302-304.
[14]苏少泉.转基因抗除草剂作物及其食品安全性[J].世界农业,2004,9(305):47-50.
[15]中国农科院知识产权研究中心,EPSPS基因技术发展与知识产权保护分析报告,2009-11-30.
[16] Forlani G,Mangiagalli A.Degradation of the phosphonate herbicide glyphosate in soil:evidence for a possible involvement of uncuhurable microorganisms[J].Soil Biology&Biochemistry,1999,31(7):991-997.
[17]潘良文,陈家华,沈禹飞,等.进口转基因抗草甘膦油菜籽和大豆中CP4-EPSPS基因的检测比较研究[J].生物技术通讯,2001,12(3):175-177,207.
[18] GB/T19495.5-2004,转基因产品检测核酸定量PCR检测方法[S].
[19]兰青阔,王永,赵新,等.LAMP在检测转基因抗草甘膦大豆cp4-epsps基因上的应用[J].安徽农业科学,2008.36(24):10377-10378,10390.
[20] MUNRAY M G,THOMPSON W F,Rapid isolation of high molecular weight plant DNA[J].Nucleic Acid Res,1980,8:4321-4325.
[21] SHIVAPPA R B,SAVAN R,KONO T,et a1.Detection of spring viraemia of carp virus(SVCV)by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio L[J].J Fish Dis,2008,3l(4):249-258.
[22] PANDEY B D,POUDEL A,YODA T.et al.Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients[J].J Med Microbiol,2008,57:439-443.
[23] GB/T19495.8-2004.转基因产品检测蛋白质检测方法[S].
[24]王占成.睾丸酮丛毛单胞菌关键酶基因转水稻后代的遗传分析[D].福州:福建农林大学生命科学学院,2009.
[25] Ellingtion AD,Szostak JW.In vitro selection of RNA molecules that bind specific ligands[J].Nature,1990,346(6287):818-822.
[26]张月侠,宋茂勇,李涛,等.适配体的筛选及在生命分析化学中的应用[J].生物技术,2009,19(5):90-94.
[27]Tuerk C,Gold L.Systematic evolution of ligands by exponential enrichment:RNA ligands to bacteriophage T4 DNA polymerase[J].Science,1990,249(4968):505-510.
[28] Robertson DL,Joyce GF.Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA[J].Nature,1990,344(6265):467-468.
[29]张慧卿,方念,张焜和.适配子筛选技术[J].中国生物工程杂志,2008,28(1):113-118.
[30]孟庆玲,陈创夫.SELEX技术及其应用前景[J].塔里木大学学报,2008,20 (3):44-48.
[31]沈倍奋.分子文库[M].第一版.北京:科学出版社,2001:116-132.
[32] Brockstedt U,Uzarowska A,Montpettt A,et al.In vitro evolution of RNA aptamers recognizing careinogenic aromatic amines[J].J Biochem Biophys Res Commun, 2004,313:1004-1008.
[33]甄蓓,杨瑞馥,俞守义.SELEX:一种体外筛选核酸适配子的新技术[J].国外医学分子生物学分册,2001,23(2):126-128.
[34] Ciesiolka J,Illangasekare M,Majerfeld, et al.Affinity selextion-amplification from randomized ribooligonucleotide pools[J].Methods in Enzymology,1996, 267:315-335.
[35]陈敏.单核增生李斯特氏菌适体的筛选、结构分析与特异性的研究[D].福建:福建师范大学,2010.
[36] Latham JA,Johnson R,Toole J.The application of a modified nucleotide in aptamer selection-novel thrombin aptamers containing 5-(1-pentynyl)-2-deoxyuridine[J].Nucleic Acids Res,1994,22:2817-2822.
[37] Bock LC,Griffin LC,Latham JA,et al.Selection of singer-stranded DNA molecules that bind and inhibit human thrombin[J].Nature,1992,355:564-566.
[38] Kubik MF,Stephens AW,Schneider D,et al.High-affinity RNA ligands to humanα-thrombin[J].Nucleic Acids Res,1994,22:2619-2626.
[39] Conrad RC,Giver L,Tian Y,et al.In vitro selection of nucleic acid aptamers that bind proteins[J].Methods in Enzymology,1996,267:336-367.
[40] Vant-Hull B,Payano-Baez A,Davis RH,et al.The mathematics of SELEX against complex targets[J].J Mol Biol,1998,278:579-597.
[41] Fitzwater T,Polisky B.A SELEX primer[J].Methods in Enzymology,1996,267: 275-301.
[42] Tang J,Xie J,Shao N,et al.The DNA aptamers that specifically recognize ricin toxin are selected by two in vitro selection methods[J].Electrophoresis,2006,27(7): 1303-1311.
[43] Mendonsa S D,Bowser M T.In vitro selection of high-affinity DNA ligands for human IgE using capillary electrophoresis[J].Anal Chem,2004,76(18):5387-5392.
[44] Mosing R K,Mendonsa S D,Bowser M T.Capillary electrophoresis-SELEXselection of aptamers with affinity for HIV-1 reverse transcriptase[J].Anal Chem, 2005,77(19):6107-6712.
[45] Mendonsa S D,Bowser M T.In vitro selection of aptamers with affinity for neuropeptide Y using capillary electrophoresis[J].J Am Chem Soc,2005,127(26): 9382-9383.
[46] Stoltenburg R,Reinemann C,Strehlitz B.FluMag-SELEX as an advantageous method for DNA aptamer selection[J].Anal Bioanal Chem,2005,383(1):83-91.
[47] Cox J C,Ellington A D.Automated selection of anti-protein aptamers[J].Bioorg Med Chem,2001,9(10):2525-2531.
[48] Hybarger G,Bynum J,Williams R F,et al.A microfluidic SELEX prototype[J]. Anal Bioanal Chem, 2006, 384 (1) : 191-198.
[49] Wen J D,GrayD M.Selection of genomic sequences that bind tightly to Ff gene 5 protein:primer-free genomic SELEX[J].Nucleic Acids Res,2004,32(22):e182.
[50] Vater A,Jarosch F,Buchner K,et al.Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach:tailored-SELEX[J].Nucleic Acids Res,2003,31(21):e130.
[51] Jarosch F,Buchner K,Klussmann S.In vitro selection using a dual RNA library that allows primerless selection[J].Nucleic Acids Res,2006,34(12):e86.
[52] White R,Rusconi C,Scardino E,et al.Generation of species cross-reactive aptamers using“toggle”SELEX[J].Mol Ther,2001,4(6):567-573.
[53]Wang C,Zhang M,Yang G,et al.Single-stranded DNA aptamers that bind differentiated but not parental cells:subtractive systematic evolution of ligands by exponential enrichment[J].J Biotechnol,2003,102(1):15-22.
[54] Martell R E,Nevins J R,Sullenger B A.Optimizing aptamer activity for gene therapy applications using expression cassette SELEX[J].Mol Ther,2002,6(1): 30-34.
[55] Morris KN,Jensen KN,Julin CM,et al.High affinity ligands from in vitro selection:complex targets[J].Proc Natl Acad Sci USA,1998,95:2902-2907.
[56] Buerger C,Nagel-Wolfrum K,Kunz C,et al.Sequence-specific Peptide Aptamers,Interacting with the Intracellular Domain of the Epidermal Growth Factor Receptor,Interfere with Stat3 Activation and Inhibit the Growth of Tumor Cells[J].J Biol Chem,2003,278(39):37610-37621.
[57] Hofmann H-P,Limmer S,Hornung V,et al.Ni2+-binding RNA motifs with an asym-metric purine-rich internal loop and a G-A base pair[J].RNA,1997,3(11):1289-1300.
[58] ZHANG Xing-Mei,SHAO Ning-Sheng,CHI Mu-Gen,et al.Screening of RNA olecules inhibiting human acetylcholinesterase by virtue of systematic evolution of ligands by exponential enrichmental[J].Acta Pharmacol Sin,2003,24(7): 711-714.
[59] Mannironi C,Nardo AD,Fruscoloni P,et al.In vitro selection of dopamine RNA ligands[J].Biochemistry,1997,36(32):9726-9734.
[60] Yang Q,Goldstein IJ,Mei HY,et al.DNA ligands that bind tightly and selectively to cellobiose[J].Proc Natl Acad Sci USA,1998,95(10):5462-5467.
[61] Geiger A,Burgstaller P,Vonder Eltz H,et al.RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity[J].Nucleic Acids Research,1996,24(6):1029-1036.
[62] Weill L,Louis D,Sargueil B.Selection and evolution of NTP-specific aptamers[J]. Nucleic Acids Research,2004,32(17):5045-5058.
[63] Butz K,Denk C,Fitscher B,et al.Peptide aptamers targeting the hepatitis B virus core protein:a new class of molecules with antiviral activity[J].Oncogene,2001, 20(45):6579-6586.
[64] Bruno JG,Kiel JL.In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection[J].Biosens Bioelectron,1999,14(15):457-464.
[65] Kusser W.Chemically modified nucleic acid aptamers for in vitro selections: evolving evolution[J].J Biotechnol,2000,74(1):27-38.
[66] Tim Sampson.Aptamers and SELEX:the technology[J].World Patent Information, 2003,25:123-129.
[67] Bridonneau P,Chang YF,Buvoli AVB,et a1.Site-directed selection of oligonuclio-tide antagonists by competitive elution[J].Antisense Nucleic Acid Drug Dev,1999,9(3):1-11.
[68]Padmanabhan K,Padmanabhan KP,Ferrara JD,et al.The structure of alphathrombin inhibited by a 15-mer single-stranded DNA aptamer[J].J Biol Chem,1993,268(24):17651-17654.
[69] Sumedha D.Jayasena aptamers:an emerging class of molecules that rival antibodies in diagnostics[J].Clinical Chemistry,1999,45(9):1628-1650.
[70]漆红兰,李延,李晓霞,等.适体传感器研究新进展[J].化学传感器,2007,27(3): 1-8.
[71] Jayasena SD.Aptamers:an emerging class of molecules that rival antibodies in diagnostics[J].Clinical Chemistry,1999,45(9):1628-1650.
[72]Fredriksson S,Gullberg M,Jarvius J,et a1.Protein detection using proximity-dependent DNA ligation assays[J].Nat Bi Biotechnol,2002.20:473-477.
[73]D A Di Giusto,W A Wlassoff,J J Gooding,et a1.Proximity,extension of circular DNA aptamers with real-time protein detection[J].Nucleic Acids Res,2005,33:e64.
[74] X.L Wang,F Li,Y H Su,et a1.Ultrasensitive detection of protein using an aptamer,based exonclease protection[J].An al Chem,2004.76:5605-5610.
[75]H Zhang,Z Wang,X F Li,et a1.Ultrasensitive detection of proteins by amplification of affinity aptamers[J].An gevew Ghem Int Ed,2006,45:1576-1580.
[76] A-N Kawde,M C Rodriquez,T M H Lee,et al.Label-Iree bio-electronic detection of aptamer-protein,interactions[J].Electrochem Commun,2005,7:537-540.
[77]H M So,K won,Y H Kim,et a1.Single-walled carbon nanotube biosensors using aptamersas molecular recognition elements[J].J Am Chem,Soc,2005,127:11906-11907.
[78] Camille L A Hamula,Jeffrey W,Guthrie,Hongquan Zhang,et a1.Selection and analytical applications of aptamers[J].TrAC Trends in An alytical Chemistry,2006,25(7):681-691.
[79]李卫滨,兰小鹏.适体技术在临床治疗及药物研究中的应用[J].国外医学内科学分册,2006,33(10):449-452.
[80] Santulli-Marotto S,Nair SK, Rusconi C,et al.Multivalent RNA aptamers that inhibit CTLA-4 and enhance tumor immunity[J].Cancer Res,2003,63:7483-7489.
[81] Liu X,Cao G,Ding H,et al.Screening of functional antidotes of RNA aptamers against bovine thrombin.FEBS Lett,2004,562(1-3):125-128.
[82] Biesecker G,Dihel L,Enney K, et al.Derivation of RNA aptamer inhibits of human complement C5[J].Immunopharmacology,1999,42:219-230.
[1]甑蓓,宋亚军,郭兆彪,等.SELEX法筛选炭疽芽孢杆菌芽孢适配子的研究[J].军事医学科学院院刊,2001,25(2):111-114.
[2]刘丰伟.体外筛选铜绿假单胞菌适体的研究及初步应用[D].福州:福建医科大学,2007.
[3]周宇凡,张桂梅,冯作化,等.H22肿瘤细胞aptamer的筛选与结构分析[J].重庆医科大学学报,2006,31(6):841-844.
[4]李卫滨,兰小鹏,杨湘越,等.筛选环孢霉素A适体的SELEX技术的建立[J].实用医学杂志,2007,23(19):2966-2968.
[5]祝军,张景海.生物素-亲和素ELISA法对ANEPⅢ寡核苷酸适配子亲和力的定量测定[J].细胞与分子免疫学杂志,2007,23(6):580-584.
[6]马占忠,王玉炯,秦莲花,等.筛选结核分枝杆菌CFP-10抗原适体的研究[J].中国病原生物学杂志,2008,3(2):86-89.
[7] Dielfenbach,C.W.Dveksler,G.S.PCR技术实验指南,科学出版社.1998,8.
[8]潘良文,陈家华,沈禹飞,等.进口转基因抗草甘膦油菜籽和大豆中CP4-EPSPS基因的检测比较研究[J].生物技术通讯,2001,12(3):175-177,207.
[9]姚姝,张保龙,沈新莲,等.分别转CP4 EPSPS和aroA基因拟南芥对草甘膦的抗性[J].苏农业学报,2006,22(3):302-304.
[10]兰青阔,王永,赵新,等.LAMP在检测转基因抗草甘膦大豆cp4-epsps基因上的应用[J].安徽农业科学,2008,36(24):10377-10378,10390.
[11]江树勋,黄新,邵碧英,等.转基因玉米EPSPS蛋白适体的筛选与结构分析[J].热带作物学报,2010,31(4):600-604.
[12] Ciesiolka J,Illangasekare M,Majerfeld,et al.Affinity selextion-amplification from randomized ribooligonucleotide pools[J].Methods in Enzymology,1996,267: 315-335.
[13]陈敏.单核增生李斯特氏菌适体的筛选、结构分析与特异性的研究[D].福建:福建师范大学,2010.
[14] Conrad RC,Giver L,Tian Y,et al.In vitro selection of nucleic acid aptamers that bind proteins[J].Methods in Enzymology,1996,267:336-367.
[15]Jun Sheng Lin,Kenneth P McNatty.Aptamer-Based Regionally Protected PCRfor Protein Detection[J].Clinical Chemistry,2009,55(9):1686-1693.
[16]Bruno JG,Kiel JL.In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection[J].Biosens Bioelectron,1999,14(15):457-464.
[17] Fulusaki E,Hasunuma T,Kajiyama S,et al.SELEX for tubulin affords specific T-rich DNA aptamers[J].Bioorg Med Chem Lett,2001,11:2927-30.
[18] Jun Sheng Lin,Kenneth P.McNatty.Aptamer-Based Regionally Protected PCR for Protein Detection[J].Clinical Chemistry,2009,55(9):1686-1693.
[1]马占忠,王玉炯,秦莲花,等.筛选结核分枝杆菌CFP-10抗原适体的研究[J].中国病原生物学杂志,2008,3(2):86-89.
[2]柳志强,孙志浩.串珠镰孢霉D-泛解酸内酯水解酶基因的克隆及在大肠杆菌中的表达[J].生物工程学报,2005,21(3):390-395.
[3]赵征,文玲英,金岩,等.ADAM28基因原核表达载体的构建和在大肠杆菌中的融合表达[J].牙体牙髓牙周病学杂志,2005,15(5):246-250.
[4]梁毅.结构生物学[M].第一版.北京:科学出版社,2005,27-31.
[1]刘丰伟.体外筛选铜绿假单胞菌适体的研究及初步应用[D].福州:福建医科大学,2007.
[2]邬芳玉.SELEX筛选AFP-L3核酸适配子方法的建立[D].南昌:南昌大学医学院,2006.
[3] Kubik MF,Stephens AW,Schneider D,et al.High-affinity RNA ligands to humanα-thrombin[J].Nucleic Acids Res,1994,22:2619-2626.
[4] Lin Y,Padmapriya A,Morden KM,et al.Peptide conjugation to an in vitro-selection DNA ligand improves enzyme inhibition[J].Proc Natl Acad Sci USA,1995, 92:11044-11048.
[5] Jun Sheng Lin,Kenneth P.McNatty.Aptamer-Based Regionally Protected PCR for Protein Detection[J].Clinical Chemistry,2009,55(9):1686-1693.
[2]陶震,杨胜利,龚毅.利用MPCR方法快速检测植物转基因背景[J].生物技术通报,2000.6:37-41.
[3]蒋原,祝长青,林宏,等.利用PCR技术检测转基因大豆Roundup Ready的研究[J].检验检疫科学,2001,11(4):11-13.
[4] Marc V,Hans P,Franco G,et al.Real-time quantitative PCR detection of genetically modified maximizer maize and roundup ready soybean in some representative foods[J].J.Agric.Food Chem.1999,47:5261-5266.
[5]金红,程奕,赵昕,等.大豆色拉油中转基因成分检测技术研究[J].华北农学报,2004,19(1):24-27.
[6] Rottm E,Lawrence T S,Wall E M,et al.Detection and quantification of roundup ready soy in foods by conventional and real-time polymerase chain reaction [J].J.Agric.Food Chem.2004,52(16):5223-5232.
[7] Rogan G J,Dud N Y A,Lee T C,et al.Immunodiagnostic methods for detection of 5-enolpyruvyl shikimate-3-phosphate synthase in Roundup Ready soybeans[J].Food Control,1999,10:407-414.
[8] Jun Sheng Lin,Kenneth P McNatty.Aptamer-Based Regionally Protected PCR for Protein Detection[J].Clinical Chemistry,2009,55(9):1686-1693.
[9]邬芳玉.SELEX筛选AFP-L3核酸适配子方法的建立[D].南昌:南昌大学医学院,2006.
[10]马占忠,秦莲花,王玉炯,等.结核分枝杆菌ESAT-6抗原适体的筛选与亲和性分析[J].中华临床医师杂志,2007,1(5):45-48.
[11]甄蓓,张敏丽,宋亚军,等.抗体-适配子混合夹心法检测炭疽芽孢杆菌芽孢的研究[J].生物技术通讯,2002,l3(2):l23-l25.
[12]江树勋,黄新,邵碧英,等.转基因玉米EPSPS蛋白适体的筛选与结构分析[J].热带作物学报,2010,31(4):600-604.
[13]姚姝,张保龙,沈新莲,等.分别转CP4 EPSPS和aroA基因拟南芥对草甘膦的抗性[J].苏农业学报,2006,22(3):302-304.
[14]苏少泉.转基因抗除草剂作物及其食品安全性[J].世界农业,2004,9(305):47-50.
[15]中国农科院知识产权研究中心,EPSPS基因技术发展与知识产权保护分析报告,2009-11-30.
[16] Forlani G,Mangiagalli A.Degradation of the phosphonate herbicide glyphosate in soil:evidence for a possible involvement of uncuhurable microorganisms[J].Soil Biology&Biochemistry,1999,31(7):991-997.
[17]潘良文,陈家华,沈禹飞,等.进口转基因抗草甘膦油菜籽和大豆中CP4-EPSPS基因的检测比较研究[J].生物技术通讯,2001,12(3):175-177,207.
[18] GB/T19495.5-2004,转基因产品检测核酸定量PCR检测方法[S].
[19]兰青阔,王永,赵新,等.LAMP在检测转基因抗草甘膦大豆cp4-epsps基因上的应用[J].安徽农业科学,2008.36(24):10377-10378,10390.
[20] MUNRAY M G,THOMPSON W F,Rapid isolation of high molecular weight plant DNA[J].Nucleic Acid Res,1980,8:4321-4325.
[21] SHIVAPPA R B,SAVAN R,KONO T,et a1.Detection of spring viraemia of carp virus(SVCV)by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio L[J].J Fish Dis,2008,3l(4):249-258.
[22] PANDEY B D,POUDEL A,YODA T.et al.Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients[J].J Med Microbiol,2008,57:439-443.
[23] GB/T19495.8-2004.转基因产品检测蛋白质检测方法[S].
[24]王占成.睾丸酮丛毛单胞菌关键酶基因转水稻后代的遗传分析[D].福州:福建农林大学生命科学学院,2009.
[25] Ellingtion AD,Szostak JW.In vitro selection of RNA molecules that bind specific ligands[J].Nature,1990,346(6287):818-822.
[26]张月侠,宋茂勇,李涛,等.适配体的筛选及在生命分析化学中的应用[J].生物技术,2009,19(5):90-94.
[27]Tuerk C,Gold L.Systematic evolution of ligands by exponential enrichment:RNA ligands to bacteriophage T4 DNA polymerase[J].Science,1990,249(4968):505-510.
[28] Robertson DL,Joyce GF.Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA[J].Nature,1990,344(6265):467-468.
[29]张慧卿,方念,张焜和.适配子筛选技术[J].中国生物工程杂志,2008,28(1):113-118.
[30]孟庆玲,陈创夫.SELEX技术及其应用前景[J].塔里木大学学报,2008,20 (3):44-48.
[31]沈倍奋.分子文库[M].第一版.北京:科学出版社,2001:116-132.
[32] Brockstedt U,Uzarowska A,Montpettt A,et al.In vitro evolution of RNA aptamers recognizing careinogenic aromatic amines[J].J Biochem Biophys Res Commun, 2004,313:1004-1008.
[33]甄蓓,杨瑞馥,俞守义.SELEX:一种体外筛选核酸适配子的新技术[J].国外医学分子生物学分册,2001,23(2):126-128.
[34] Ciesiolka J,Illangasekare M,Majerfeld, et al.Affinity selextion-amplification from randomized ribooligonucleotide pools[J].Methods in Enzymology,1996, 267:315-335.
[35]陈敏.单核增生李斯特氏菌适体的筛选、结构分析与特异性的研究[D].福建:福建师范大学,2010.
[36] Latham JA,Johnson R,Toole J.The application of a modified nucleotide in aptamer selection-novel thrombin aptamers containing 5-(1-pentynyl)-2-deoxyuridine[J].Nucleic Acids Res,1994,22:2817-2822.
[37] Bock LC,Griffin LC,Latham JA,et al.Selection of singer-stranded DNA molecules that bind and inhibit human thrombin[J].Nature,1992,355:564-566.
[38] Kubik MF,Stephens AW,Schneider D,et al.High-affinity RNA ligands to humanα-thrombin[J].Nucleic Acids Res,1994,22:2619-2626.
[39] Conrad RC,Giver L,Tian Y,et al.In vitro selection of nucleic acid aptamers that bind proteins[J].Methods in Enzymology,1996,267:336-367.
[40] Vant-Hull B,Payano-Baez A,Davis RH,et al.The mathematics of SELEX against complex targets[J].J Mol Biol,1998,278:579-597.
[41] Fitzwater T,Polisky B.A SELEX primer[J].Methods in Enzymology,1996,267: 275-301.
[42] Tang J,Xie J,Shao N,et al.The DNA aptamers that specifically recognize ricin toxin are selected by two in vitro selection methods[J].Electrophoresis,2006,27(7): 1303-1311.
[43] Mendonsa S D,Bowser M T.In vitro selection of high-affinity DNA ligands for human IgE using capillary electrophoresis[J].Anal Chem,2004,76(18):5387-5392.
[44] Mosing R K,Mendonsa S D,Bowser M T.Capillary electrophoresis-SELEXselection of aptamers with affinity for HIV-1 reverse transcriptase[J].Anal Chem, 2005,77(19):6107-6712.
[45] Mendonsa S D,Bowser M T.In vitro selection of aptamers with affinity for neuropeptide Y using capillary electrophoresis[J].J Am Chem Soc,2005,127(26): 9382-9383.
[46] Stoltenburg R,Reinemann C,Strehlitz B.FluMag-SELEX as an advantageous method for DNA aptamer selection[J].Anal Bioanal Chem,2005,383(1):83-91.
[47] Cox J C,Ellington A D.Automated selection of anti-protein aptamers[J].Bioorg Med Chem,2001,9(10):2525-2531.
[48] Hybarger G,Bynum J,Williams R F,et al.A microfluidic SELEX prototype[J]. Anal Bioanal Chem, 2006, 384 (1) : 191-198.
[49] Wen J D,GrayD M.Selection of genomic sequences that bind tightly to Ff gene 5 protein:primer-free genomic SELEX[J].Nucleic Acids Res,2004,32(22):e182.
[50] Vater A,Jarosch F,Buchner K,et al.Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach:tailored-SELEX[J].Nucleic Acids Res,2003,31(21):e130.
[51] Jarosch F,Buchner K,Klussmann S.In vitro selection using a dual RNA library that allows primerless selection[J].Nucleic Acids Res,2006,34(12):e86.
[52] White R,Rusconi C,Scardino E,et al.Generation of species cross-reactive aptamers using“toggle”SELEX[J].Mol Ther,2001,4(6):567-573.
[53]Wang C,Zhang M,Yang G,et al.Single-stranded DNA aptamers that bind differentiated but not parental cells:subtractive systematic evolution of ligands by exponential enrichment[J].J Biotechnol,2003,102(1):15-22.
[54] Martell R E,Nevins J R,Sullenger B A.Optimizing aptamer activity for gene therapy applications using expression cassette SELEX[J].Mol Ther,2002,6(1): 30-34.
[55] Morris KN,Jensen KN,Julin CM,et al.High affinity ligands from in vitro selection:complex targets[J].Proc Natl Acad Sci USA,1998,95:2902-2907.
[56] Buerger C,Nagel-Wolfrum K,Kunz C,et al.Sequence-specific Peptide Aptamers,Interacting with the Intracellular Domain of the Epidermal Growth Factor Receptor,Interfere with Stat3 Activation and Inhibit the Growth of Tumor Cells[J].J Biol Chem,2003,278(39):37610-37621.
[57] Hofmann H-P,Limmer S,Hornung V,et al.Ni2+-binding RNA motifs with an asym-metric purine-rich internal loop and a G-A base pair[J].RNA,1997,3(11):1289-1300.
[58] ZHANG Xing-Mei,SHAO Ning-Sheng,CHI Mu-Gen,et al.Screening of RNA olecules inhibiting human acetylcholinesterase by virtue of systematic evolution of ligands by exponential enrichmental[J].Acta Pharmacol Sin,2003,24(7): 711-714.
[59] Mannironi C,Nardo AD,Fruscoloni P,et al.In vitro selection of dopamine RNA ligands[J].Biochemistry,1997,36(32):9726-9734.
[60] Yang Q,Goldstein IJ,Mei HY,et al.DNA ligands that bind tightly and selectively to cellobiose[J].Proc Natl Acad Sci USA,1998,95(10):5462-5467.
[61] Geiger A,Burgstaller P,Vonder Eltz H,et al.RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity[J].Nucleic Acids Research,1996,24(6):1029-1036.
[62] Weill L,Louis D,Sargueil B.Selection and evolution of NTP-specific aptamers[J]. Nucleic Acids Research,2004,32(17):5045-5058.
[63] Butz K,Denk C,Fitscher B,et al.Peptide aptamers targeting the hepatitis B virus core protein:a new class of molecules with antiviral activity[J].Oncogene,2001, 20(45):6579-6586.
[64] Bruno JG,Kiel JL.In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection[J].Biosens Bioelectron,1999,14(15):457-464.
[65] Kusser W.Chemically modified nucleic acid aptamers for in vitro selections: evolving evolution[J].J Biotechnol,2000,74(1):27-38.
[66] Tim Sampson.Aptamers and SELEX:the technology[J].World Patent Information, 2003,25:123-129.
[67] Bridonneau P,Chang YF,Buvoli AVB,et a1.Site-directed selection of oligonuclio-tide antagonists by competitive elution[J].Antisense Nucleic Acid Drug Dev,1999,9(3):1-11.
[68]Padmanabhan K,Padmanabhan KP,Ferrara JD,et al.The structure of alphathrombin inhibited by a 15-mer single-stranded DNA aptamer[J].J Biol Chem,1993,268(24):17651-17654.
[69] Sumedha D.Jayasena aptamers:an emerging class of molecules that rival antibodies in diagnostics[J].Clinical Chemistry,1999,45(9):1628-1650.
[70]漆红兰,李延,李晓霞,等.适体传感器研究新进展[J].化学传感器,2007,27(3): 1-8.
[71] Jayasena SD.Aptamers:an emerging class of molecules that rival antibodies in diagnostics[J].Clinical Chemistry,1999,45(9):1628-1650.
[72]Fredriksson S,Gullberg M,Jarvius J,et a1.Protein detection using proximity-dependent DNA ligation assays[J].Nat Bi Biotechnol,2002.20:473-477.
[73]D A Di Giusto,W A Wlassoff,J J Gooding,et a1.Proximity,extension of circular DNA aptamers with real-time protein detection[J].Nucleic Acids Res,2005,33:e64.
[74] X.L Wang,F Li,Y H Su,et a1.Ultrasensitive detection of protein using an aptamer,based exonclease protection[J].An al Chem,2004.76:5605-5610.
[75]H Zhang,Z Wang,X F Li,et a1.Ultrasensitive detection of proteins by amplification of affinity aptamers[J].An gevew Ghem Int Ed,2006,45:1576-1580.
[76] A-N Kawde,M C Rodriquez,T M H Lee,et al.Label-Iree bio-electronic detection of aptamer-protein,interactions[J].Electrochem Commun,2005,7:537-540.
[77]H M So,K won,Y H Kim,et a1.Single-walled carbon nanotube biosensors using aptamersas molecular recognition elements[J].J Am Chem,Soc,2005,127:11906-11907.
[78] Camille L A Hamula,Jeffrey W,Guthrie,Hongquan Zhang,et a1.Selection and analytical applications of aptamers[J].TrAC Trends in An alytical Chemistry,2006,25(7):681-691.
[79]李卫滨,兰小鹏.适体技术在临床治疗及药物研究中的应用[J].国外医学内科学分册,2006,33(10):449-452.
[80] Santulli-Marotto S,Nair SK, Rusconi C,et al.Multivalent RNA aptamers that inhibit CTLA-4 and enhance tumor immunity[J].Cancer Res,2003,63:7483-7489.
[81] Liu X,Cao G,Ding H,et al.Screening of functional antidotes of RNA aptamers against bovine thrombin.FEBS Lett,2004,562(1-3):125-128.
[82] Biesecker G,Dihel L,Enney K, et al.Derivation of RNA aptamer inhibits of human complement C5[J].Immunopharmacology,1999,42:219-230.
[1]甑蓓,宋亚军,郭兆彪,等.SELEX法筛选炭疽芽孢杆菌芽孢适配子的研究[J].军事医学科学院院刊,2001,25(2):111-114.
[2]刘丰伟.体外筛选铜绿假单胞菌适体的研究及初步应用[D].福州:福建医科大学,2007.
[3]周宇凡,张桂梅,冯作化,等.H22肿瘤细胞aptamer的筛选与结构分析[J].重庆医科大学学报,2006,31(6):841-844.
[4]李卫滨,兰小鹏,杨湘越,等.筛选环孢霉素A适体的SELEX技术的建立[J].实用医学杂志,2007,23(19):2966-2968.
[5]祝军,张景海.生物素-亲和素ELISA法对ANEPⅢ寡核苷酸适配子亲和力的定量测定[J].细胞与分子免疫学杂志,2007,23(6):580-584.
[6]马占忠,王玉炯,秦莲花,等.筛选结核分枝杆菌CFP-10抗原适体的研究[J].中国病原生物学杂志,2008,3(2):86-89.
[7] Dielfenbach,C.W.Dveksler,G.S.PCR技术实验指南,科学出版社.1998,8.
[8]潘良文,陈家华,沈禹飞,等.进口转基因抗草甘膦油菜籽和大豆中CP4-EPSPS基因的检测比较研究[J].生物技术通讯,2001,12(3):175-177,207.
[9]姚姝,张保龙,沈新莲,等.分别转CP4 EPSPS和aroA基因拟南芥对草甘膦的抗性[J].苏农业学报,2006,22(3):302-304.
[10]兰青阔,王永,赵新,等.LAMP在检测转基因抗草甘膦大豆cp4-epsps基因上的应用[J].安徽农业科学,2008,36(24):10377-10378,10390.
[11]江树勋,黄新,邵碧英,等.转基因玉米EPSPS蛋白适体的筛选与结构分析[J].热带作物学报,2010,31(4):600-604.
[12] Ciesiolka J,Illangasekare M,Majerfeld,et al.Affinity selextion-amplification from randomized ribooligonucleotide pools[J].Methods in Enzymology,1996,267: 315-335.
[13]陈敏.单核增生李斯特氏菌适体的筛选、结构分析与特异性的研究[D].福建:福建师范大学,2010.
[14] Conrad RC,Giver L,Tian Y,et al.In vitro selection of nucleic acid aptamers that bind proteins[J].Methods in Enzymology,1996,267:336-367.
[15]Jun Sheng Lin,Kenneth P McNatty.Aptamer-Based Regionally Protected PCRfor Protein Detection[J].Clinical Chemistry,2009,55(9):1686-1693.
[16]Bruno JG,Kiel JL.In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection[J].Biosens Bioelectron,1999,14(15):457-464.
[17] Fulusaki E,Hasunuma T,Kajiyama S,et al.SELEX for tubulin affords specific T-rich DNA aptamers[J].Bioorg Med Chem Lett,2001,11:2927-30.
[18] Jun Sheng Lin,Kenneth P.McNatty.Aptamer-Based Regionally Protected PCR for Protein Detection[J].Clinical Chemistry,2009,55(9):1686-1693.
[1]马占忠,王玉炯,秦莲花,等.筛选结核分枝杆菌CFP-10抗原适体的研究[J].中国病原生物学杂志,2008,3(2):86-89.
[2]柳志强,孙志浩.串珠镰孢霉D-泛解酸内酯水解酶基因的克隆及在大肠杆菌中的表达[J].生物工程学报,2005,21(3):390-395.
[3]赵征,文玲英,金岩,等.ADAM28基因原核表达载体的构建和在大肠杆菌中的融合表达[J].牙体牙髓牙周病学杂志,2005,15(5):246-250.
[4]梁毅.结构生物学[M].第一版.北京:科学出版社,2005,27-31.
[1]刘丰伟.体外筛选铜绿假单胞菌适体的研究及初步应用[D].福州:福建医科大学,2007.
[2]邬芳玉.SELEX筛选AFP-L3核酸适配子方法的建立[D].南昌:南昌大学医学院,2006.
[3] Kubik MF,Stephens AW,Schneider D,et al.High-affinity RNA ligands to humanα-thrombin[J].Nucleic Acids Res,1994,22:2619-2626.
[4] Lin Y,Padmapriya A,Morden KM,et al.Peptide conjugation to an in vitro-selection DNA ligand improves enzyme inhibition[J].Proc Natl Acad Sci USA,1995, 92:11044-11048.
[5] Jun Sheng Lin,Kenneth P.McNatty.Aptamer-Based Regionally Protected PCR for Protein Detection[J].Clinical Chemistry,2009,55(9):1686-1693.