靶向磷脂酰肌醇蛋白聚糖1的DNA适配体筛选
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摘要
目的:筛选获得特异性结合磷脂酰肌醇蛋白聚糖1(GPC1)的单链DNA适配体。方法:合成全长81 nt,中间含39个随机序列的单链寡核苷酸(ss DNA)文库,运用指数富集配基系统进化(SELEX)技术筛选获得GPC1适配体;酶联免疫吸附分析法(ELISA)实验确定候选适配体与GPC1蛋白的亲和力,流式细胞术确定候选适配体与2种GPC1高表达细胞系(胰腺癌Panc-1细胞和工具细胞Luc-ZR-75-1~(GPC1))的结合特异性。结果:经过10轮SELEX筛选获得#9适配体,ELISA分析其亲和力为44±0.69 nmol/L,流式细胞术结果表明其与胰腺癌Panc-1细胞和Luc-ZR-75-1~(GPC1)细胞的阳性结合率分别为44.69%和51.44%。结论:筛选获得与GPC1蛋白有较高亲和力、与表达GPC1细胞系特异结合的ss DNA适配体。
Objective: To screen single stranded DNA aptamers with high specificity to glypican-1(GPC1).Methods: GPC1 aptamer was selected with the method systematic evolution of ligands by exponential enrichment(SELEX) based on a single stranded DNA(ss DNA) library with 81 nucleotides in length containing 39 random sequences. Affinity of candidate aptamers to GPC1 were measured by enzymed-linked immunosorbent assay(ELISA).The specific binding of candidate aptamers to GPC1 positive pancreatic cancer cell line Panc-1 and Luc-ZR-75-1~(GPC1) cell lines were tested by flow cytometry. Results: After 10 rounds of library screening, #9 aptamer(Apt#9)was screened out with a relative high affinity(K_d=44±0.69 nmol/L) to GPC1 as determined by ELISA. Flow cytometry analysis confirmed that Apt#9 was able to bind to GPC1-expressing Panc-1(44.69%) and Luc-ZR-75-1~(GPC1) cells(51.44%). Conclusion: A single stranded DNA aptamer targeting GPC1 was successfully screened, which has high affinity with GPC1 protein as well as good specificity to GPC1-expressing Panc-1 cell and Luc-ZR-75-1~(GPC1) cell lines.
Objective: To screen single stranded DNA aptamers with high specificity to glypican-1(GPC1).Methods: GPC1 aptamer was selected with the method systematic evolution of ligands by exponential enrichment(SELEX) based on a single stranded DNA(ss DNA) library with 81 nucleotides in length containing 39 random sequences. Affinity of candidate aptamers to GPC1 were measured by enzymed-linked immunosorbent assay(ELISA).The specific binding of candidate aptamers to GPC1 positive pancreatic cancer cell line Panc-1 and Luc-ZR-75-1~(GPC1) cell lines were tested by flow cytometry. Results: After 10 rounds of library screening, #9 aptamer(Apt#9)was screened out with a relative high affinity(K_d=44±0.69 nmol/L) to GPC1 as determined by ELISA. Flow cytometry analysis confirmed that Apt#9 was able to bind to GPC1-expressing Panc-1(44.69%) and Luc-ZR-75-1~(GPC1) cells(51.44%). Conclusion: A single stranded DNA aptamer targeting GPC1 was successfully screened, which has high affinity with GPC1 protein as well as good specificity to GPC1-expressing Panc-1 cell and Luc-ZR-75-1~(GPC1) cell lines.
引文
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[2]Shigdar S,Qian C,Lv L.et al.The use of sensitive chemical antibodies for diagnosis:detection of low levels of epcam in breast cancer[J].PLo S One,2013,8:e57613.
[3]Bibby D F,Gill A C,Kirby L,et al.Application of a novel in vitro selection technique to isolate and characterize high affinity DNA aptamers binding mammalian prion proteins[J].J Virol Methods,2008,151(1):107-115.
[4]Mosting R K,Mendonsa S D,Bowser M T,et al.Capillary electrophoresis-SELEXselectionofaptamerswithaffinityfor HIV-1reversetranscriptase[J].Anal Chem,2005,77(19):6107-6112.
[5]Clonis Y D.Affinity chromatography matures as bioinformatic andcombinatorial tools develop[J].J Chromatogr A,2006,1101(1-2):1-24.
[6]Shigdar S,Macdonald J,O'Connor M,et al.Aptamers as theranostic agents:modifications,serum stability and functionalisation[J].Sensors,2013,13:13624-13637.
[7]Ostendorf T,Kunter U,van Roeyen C,et al.The effects of platelet-derived growth factorantagonism in experimental glomerulonephritis are independent of thetransforming growth factor-beta system[J].J Am Soc Nephrol,2002,13(3):658-667.
[8]Jeyakumar A,Younis T.Trastuzumab for HER2-positive metastatic breast cancer:clinical and economic considerations[J].Clin Med Insights Oncol,2012,6:179-187.
[9]Bruno J G.A review of therapeutic aptamer conjugates with emphasis on new approaches[J].Pharmaceuticals(Basel),2013,6:340-357.
[10]Hedden L,O'Reilly S,Lohrisch C.et al.Assessing the real-world cost-effectiveness of adjuvant trastuzumab in HER-2/neu positive breast cancer[J].Oncologist,2012,17:164-200.
[11]Phillips J A,Lopez-Colon D,Zhu Z.et al.Applications of aptamers in cancer cell biology[J].Anal Chim Acta,2008,621:101-108.
[12]Meng L,Yang L,Zhao X,et al.Targeted delivery of chemotherapy agents using a liver cancer-specific aptamer[J].PLo S One,2012,7:e33434.
[13]Ruckman J,Green L S,Beeson J,et al.2′-fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor(VEGF165):inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain[J].J Biol Chem,1998,273:20556-20567.
[14]Deshpande P P,Biswas S,Torchilin V P.Current trends in the use of liposomes for tumor targeting[J].Nanomedicine(Lond),2013,8(9):doi:10.2217/nnm.13.118.
[15]Malam Y,Loizidou M,Seifalian A M.Liposomes and nanoparticles:nanosized vehicles for drug delivery in cancer[J].Trends Pharmacol Sci,2009,30:592-599.
[16]Kang H,O'Donoghue M B,Liu H.et al.A liposomebased nanostructure for aptamer directed delivery[J].Chem Commun,2010,46(2):249-251.
[17]Filmus J,Capurro M,Rast J.Glypicans[J].Genome Biol,2008,9(5):224.
[18]Matsuda K,Maruyama H,Guo F,et al.Glypican-1 is overexpressed in human breast cancer and modulates the mitogenic[J].Cancer Res,2001,61:5562-5569.
[19]Aikawa T,Whipple C A,Lopez M E,et al.Glypican-1 modulatesthe angiogenic and metastatic potential of human and mouse cancer cells[J].J Clin Invest,2008,118:89-99.
[20]Whipple C A,Lander A D,Korc M.Discovery of a novel molecule that regulates tumor growth and metastasis[J].Sci World J,2008,8:1250-1253.
[21]Kleeff J,Ishiwata T,Kumbasar A,et al.The cell-surface heparan sulfate proteoglycan glypican-1 regulates growth factor action in pancreatic carcinoma cells and is overexpressed in human pancreatic cancer[J].J Clin Invest,1998,102(9):1662-1673.
[22]Melo S A,Luecke L B,Kahlert C,et al.Glypican1identifies cancer exosomes and facilitates earlydetection of cancer[J].Nature,2015,523(7559):177-182.
[23]Ouellet E,Foley J H,Conway E M,et al.Hi-Fi SELEX:a high-fidelity digital-PCR based therapeutic aptamer discovery platform[J].Biotechnol Bioeng,2015,112(8):1506-1522.
[24]Jensen M A,Straus N.Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions[J].Genome Res,1993,3(3):186.
[25]Thompson J R,Marcelino L A,Polz M F.Heteroduplexes in mixed-template amplifications:formation,consequence and elimination by'reconditioning PCR'[J].Nucleic Acids Res,2002,30(9):2083-2088.
[26]Sriprakash K S,Lundh N,Mmo H,et al.The specificity of lambdaexonuclease.Interactions with singlestranded DNA[J].J Biol Chem,1975,250(14):5438-5445.