CA125抗原核酸适体的筛选及鉴定
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摘要
目的建立一种基于核酸适体快速检测肿瘤血清标志物CA125抗原的方法。方法以CA125抗原为筛选靶标,CA199抗原为反筛选靶标,羧基化琼脂磁珠为筛选介质,利用指数级富集配体的系统进化技术筛选其高特异性核酸适体,利用生物信息学方法对核酸适体进行序列分析和二级结构预测,荧光定量法分析核酸适体的亲和力,ELONA法测定核酸适体的特异性。结果经过6轮消减SELEX筛选,次级ssDNA文库的亲和力逐渐提高,特异性次级ssDNA文库逐轮富集,经二代测序,获得3条拷贝数>1000的核酸序列(Apt-1、Apt-2、Apt-3),可特异性识别CA125抗原,测定Kd值,分别为41.02±4.9、19.06±9.0、10.02±5.0 nmol/L,Apt-3亲和力最高。结论功能性核酸适体Apt-3能高特异性识别CA125抗原,为肿瘤血清标志物CA125检测提供了一种新的、快速、有效的工具和手段。
Objective To establish a method for rapid detection of tumor serum marker CA125 antigen based on aptamer.Methods CA125 antigen was used as the screening target, CA199 antigen was used as the anti-screening target, and carboxylated agar magnetic beads were used as the screening medium. The phylogenetic technique of exponential enrichment ligand was used to screen the high specificity aptamer, and the bioinformatics method was used. The sequence analysis and secondary structure prediction of the aptamer were carried out, the affinity of the aptamer was analyzed by fluorescence quantification, and the specificity of the aptamer was determined by the ELONA method. Results After 6 rounds of SELEX screening, the affinity of the secondary ssDNA library was gradually increased, and the specific secondary ssDNA library was enriched round-by-round. After sequencing by the second generation, three nucleic acid sequences with copy number >1000were obtained(Apt-1, Apt-2, Apt-3), can specifically recognize CA125 antigen, and determine Kd values, which are 41.02±4.9nmol·L~(–1), 19.06±9.0 nmol·L~(–1), 10.02±5.0 nmol·L~(–1), Apt-3 the highest affinity. Conclusion The functional aptamer Apt-3 can highly recognize CA125 antigen, which provides a new, rapid and effective tool and means for the detection of tumor serum marker CA125.
Objective To establish a method for rapid detection of tumor serum marker CA125 antigen based on aptamer.Methods CA125 antigen was used as the screening target, CA199 antigen was used as the anti-screening target, and carboxylated agar magnetic beads were used as the screening medium. The phylogenetic technique of exponential enrichment ligand was used to screen the high specificity aptamer, and the bioinformatics method was used. The sequence analysis and secondary structure prediction of the aptamer were carried out, the affinity of the aptamer was analyzed by fluorescence quantification, and the specificity of the aptamer was determined by the ELONA method. Results After 6 rounds of SELEX screening, the affinity of the secondary ssDNA library was gradually increased, and the specific secondary ssDNA library was enriched round-by-round. After sequencing by the second generation, three nucleic acid sequences with copy number >1000were obtained(Apt-1, Apt-2, Apt-3), can specifically recognize CA125 antigen, and determine Kd values, which are 41.02±4.9nmol·L~(–1), 19.06±9.0 nmol·L~(–1), 10.02±5.0 nmol·L~(–1), Apt-3 the highest affinity. Conclusion The functional aptamer Apt-3 can highly recognize CA125 antigen, which provides a new, rapid and effective tool and means for the detection of tumor serum marker CA125.
引文
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[23]Mckeague M,De Girolamo A,Valenzano SA,et al.Comprehensive analytical comparison of strategies used for small molecule aptamer evaluation[J].Anal Chem,2015,87(17):8608-12.
[2]Huang Z,Zhai XM,Wang H,et al.Simultaneous quantitation of carbohydrate antigen 125 and carcinoembryonic antigen in human serum via time-resolved fluoroimmunoassay[J].Clinica Chimica Acta,2018,483(10):222-6.
[3]Kaaks R,Fortner RT,Huesing AA,et al.Tumor-associated autoantibodies as early detection markers for ovarian cancer?Aprospective evaluation[J].Int J Cancer,2018,143(3):515-26.
[4]Liang C,Shi S,Meng Q,et al.MiR-29a,targeting caveolin 2expression,is responsible for limitation of pancreatic cancer metastasis in patients with normal level of serum CA125[J].Int JCancer,2018,12(4):482-9.
[5]Nishimoto-Kakiuchi A,Netsu S,Okabayashi S,et al.Spontaneous endometriosis in cynomolgus monkeys as a clinically relevant experimental model[J].Hum Reprod,2018,33(7):1228-36.
[6]Zhou H,Dong A,Xia H,et al.Associations between CA19-9 and CA125 levels and human epidermal growth factor receptor 2overexpression in patients with gastric cancer[J].Oncol Lett,2018,16(1):1079-86.
[7]Hamd-Ghadareh S,Salimi A,Parsa S,et al.Simultaneous biosensing of CA125 and CA15-3 tumor markers and imaging of OVCAR-3 and MCF-7 cells lines via bi-color FRET phenomenon using dual blue-green luminescent Carbon dots with single excitation wavelength[J].Int J Biol Macromol,2018,118(A):617-28.
[8]Zhang F,Wang JJ,Zheng XY,et al.Clinical value of jointly detection pleural fluid Midkine,pleural fluid adenosine deaminase,and pleural fluid carbohydrate antigen 125 in the identification of nonsmall cell lung cancer-associated malignant pleural effusion[J].J Clin Lab Anal,2018,32(8):e22576-83.
[9]Mckeague M,Mcconnell EM,Cruz-Toledo JA,et al.Analysis of in vitro aptamer selection parameters[J].J Mol Evol,2015,81(5/6,SI):150-61.
[10]Tuerk C,Gold L.Systematic evolution of ligands by exponential enrichment:RNA ligands to bacteriophage T4 DNApolymerase[J].Science,1990,249(4968):505-10.
[11]Wheeler LA,Trifonova R,Vrbanac VA,et al.Inhibition of HIVtransmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras[J].J Clin Invest,2011,121(6):2401-12.
[12]Fang XH,Sen A,Vicens M,et al.Synthetic DNA aptamers to detect protein molecular variants in a high-throughput fluorescence quenching assay[J].Chembiochem,2003,4(9):829-34.
[13]Cai D,Xu D,Zhang Q,et al.Classification of lung cancer using ensemble-based feature selection and machine learning methods[J].Molecular biolystems,2014,464(7291):1071-6.
[14]Gilboa E,McNamara J,Pastor F.Use of oligonucleotide aptamer ligands to modulate the function of immune receptors[J].J Ame Assoc Cancer Res,2013,43(19):1054-62.
[15]Hasegawa H,Sode K,Ikebukuro K.Selection of DNA aptamers against VEGF(165)using a protein competitor and the aptamer blotting method[J].Biotechnol Lett,2008,30(5):829-34.
[16]Ireson CR,Kelland LR.Discovery and development of anticancer aptamers[J].Mol Cancer Ther,2006,5(12):2957-62.
[17]Jun YP,Ye LC,Ju RC,et al.Gemcitabine-Incorporated G-QuadruplexAptamer for targeted drug delivery into pancreas cancer[J].Mol Ther Nucleic Acids,2018,12(8):543-53.
[18]Visintin I,Feng Z,Longton G,et al.Diagnostic markers for early detection of ovarian cancer[J].Clin Cancer Res,2008,14(4):1065-72.
[19]Yang JM,Dou BT,Yuan R,et al.Aptamer/protein proximity Binding-Triggered molecular machine for amplified electrochemical sensing of thrombin[J].Anal Chem,2017,89(9):5138-43.
[20]Li H,Arroyo-Curras N,Kang D,et al.Dual-Reporter drift correction to enhance the performance of electrochemical AptamerBased sensors in whole blood[J].J Am Chem Soc,2016,138(49):15809-12.
[21]葸玉琴,赵运旺,李东东,等.胃癌血清适配体的筛选及其鉴定[J].南京医科大学学报:自然科学版,2016,36(11):1306-12.
[22]Wu YX,Kwon YJ.Aptamers:the“evolution”of SELEX[J].Methods,2016,106(15):21-8.
[23]Mckeague M,De Girolamo A,Valenzano SA,et al.Comprehensive analytical comparison of strategies used for small molecule aptamer evaluation[J].Anal Chem,2015,87(17):8608-12.