CN102839181B - Nucleic acid aptamer sequence of gastric cancer cells, and uses thereof - Google Patents
Nucleic acid aptamer sequence of gastric cancer cells, and uses thereof Download PDFInfo
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- CN102839181B CN102839181B CN201110172522.6A CN201110172522A CN102839181B CN 102839181 B CN102839181 B CN 102839181B CN 201110172522 A CN201110172522 A CN 201110172522A CN 102839181 B CN102839181 B CN 102839181B
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Abstract
The invention belongs to the technical field of cell biology, and relates to a nucleic acid aptamer sequence of gastric cancer cells, and uses thereof. The vitro SELEX screening technology of a nucleic acid aptamer is used, the gastric cancer cell lines HGC27 are used as a positive screening target, the gastric epithelial cell lines GES-1 are used as a negative screening target, and a nucleic acid aptamer specifically binding to the HGC27 is screened to obtain a characteristic sequence GGTTT specifically binding to the HGC27. The characteristic sequence is a basis of specific binding of the nucleic acid aptamer and the HGC27, and can be used for design of an anti-gastric cancer drug, preparing drugs or other products; and the nucleic acid aptamer containing the characteristic sequence can be used as an anti-gastric cancer molecular probe or drug target.
Description
Technical field
The invention belongs to cellular biological technique field, relate to a kind of aptamer sequence and purposes of stomach cancer cell.
Background technology
Cancer of the stomach is one of common malignant tumour of China, is also modal malignant tumor of digestive tract.According to statistics, cancer of the stomach occupies the second of all kinds of tumours in the morbidity of China; In the malignant tumour of all stomaches, gland cancer accounts for 95%.When most of Patients with Gastric Cancer are made a definite diagnosis clinically often in late period, its 5 years survival rates are no more than 10%, therefore early diagnosis is the key that improves cancer of the stomach survival rate, and the tumor markers of finding early diagnosis is for the diagnosis of cancer of the stomach and treat tool and be of great significance.
At present, known nucleic acid aptamers (aptamer) is oligonucleotide (DNA or the RNA) fragment of the energy specific combination metal ion, polypeptide, protein and even the whole cell that go out through in-vitro screening technology screening; Its specificity, as synantibody, has the avidity of strict recognition capability and height to combinative part.The in-vitro screening technology of aptamer is called as Fas lignand system evolution (Systematic evolution of ligand by exponential enrichment, SELEX) technology of index concentration; Described SELEX technical modelling natural evolution process, applies selective pressure (in conjunction with target) to random oligonucleotide library, elutriation and target high special binding fragment (as shown in Figure 1); Compare the aptamers (peptide aptamer) of the polypeptide classes such as antibody, the advantage of aptamer is quite obvious, as: prepare simple and fast, stable chemical nature, so far there are no, and report exists immunogenicity or toxicity, target molecule scope is wide, avidity is high, high specificity, be easy to transform modification.
The many advantages of aptamer makes it have purposes widely at aspects such as fundamental research, clinical detection, new drug developments.Aspect clinical detection, the technology that can detect with antigen antibody reaction at present nearly all can substitute with aptamer, as a kind of aptamers molecule sensor---the RiboReporter of Archemix company exploitation
tMthe protein signal detecting directly can be changed into optical signal and record and analyze, thereby realize the direct rapid detection to the high molecular weight protein in biological mixed solution (as serum, cell extract).Aspect new drug development, most typical example is first aptamer medicine Macugen that ratifies listing by U.S. FDA, it is the aptamer of anti-vascular endothelial cell growth factor (VEGF), is used to treat wet age-related macular degeneration (wet AMD).The example of another aptamer medicine is the AGR0100 being developed by Aptamera company, and preclinical test shows, AGRO100 all has restraining effect to multiple cancer cells.
At present, major part is engaged in the scientist of aptamer research and biotech company mainly for cardiovascular disorder in the world, and the Chinese common disease of less concern, as hepatitis, liver cancer, cancer of the stomach etc., therefore the present invention pays the utmost attention to aptamer and how to be applied to the major disease of above-mentioned serious threat China people ' s health, exploitation for the aptamer of stomach cancer cell as characteristic mark, provide powerful support for for the molecular diagnosis of cancer of the stomach and targeted therapy provide, there is important clinical value.
Summary of the invention
The object of this invention is to provide a kind of aptamer sequence of stomach cancer cell, be specifically related to have in the aptamer of a kind of stomach cancer cell line HGC27 the characteristic sequence of specific combination HGC27.
In the present invention, the characteristic sequence that contains specific combination HGC27 in the aptamer of described stomach cancer cell line HGC27 (sequence 1), described characteristic sequence is GGTTT.
In the present invention, described stomach cancer cell is selected from stomach cancer cell line HGC27.
Further object of the present invention is to provide the purposes of described aptamer sequence.In the present invention, according to this sequential analysis, can further design medicine or the goods of anti-cancer of the stomach, and prepare medicine or other goods of anti-cancer of the stomach.
The present invention adopts in-vitro screening (SELEX) technology of aptamer, with stomach cancer cell line HGC27 for just sieving target, take gastric epithelial cell strain GES-1 as anti-sieve target, the aptamer of screening and HGC27 specific combination, make the characteristic sequence GGTTT with specific combination HGC27, No. 2, called after Huashan aptamers in the present invention, is called for short HSST02.
The present invention is achieved through the following technical solutions:
The in-vitro screening technology that adopts known aptamer is SELEX technology, with stomach cancer cell line HGC27(purchased from Shanghai cell bank) for just sieving target, take gastric epithelial cell strain GES-1(purchased from Shanghai cell bank) as anti-sieve target, from external synthetic random oligo DNA library
In (5 '-acgctcggatgccactacagNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNctcatggacgtgctggtgac-3 '), filter out the aptamer with HGC27 specific combination; By the sequence filtering out primer Aptamer_L(5 '-FAM-acgctcggatgccactacag-3 ') and Aptamer_R(5 '-biotin-gtcaccagcacgtccatgag-3 ') increasing and carrying out TA is cloned into pMD19-T carrier (purchased from Shanghai rich photo bio company), transforms DH5a bacterium (purchased from Beijing Tian Gen biotech firm); Choose white colony with primer RV-M(5'-GAGCGGATAACAATTTCACACAGG-3') and M13 (47) (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') carry out PCR and determine after positive colony, extracting plasmid and with M13 (47) (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') for sequencing primer carries out sequencing reaction, upper sequenator checks order; According to sequencing result analytical characteristic sequence.
In the present invention, described sequence is selected from the sequence of natural existence or synthetic, or the same sequence in any other source;
The sequence of described aptamer contains the whole identical sequence with the Nucleotide of described characteristic sequence, and all aptamers all contain described characteristic sequence.
The present invention with stomach cancer cell line HGC27 for just sieving target, take gastric epithelial cell strain GES-1 as anti-sieve target, through in-vitro screening technology, acquisition has the characteristic sequence GGTTT of the aptamer of specific combination HGC27, take this characteristic sequence as aptamer and the basis of HGC27 specific binding, be further used for anti-cancer of the stomach medicinal design, prepare medicine or other goods; The described aptamer containing this characteristic sequence, can be used as molecular probe or the drug target of anti-cancer of the stomach.
Accompanying drawing explanation
Fig. 1 is that the in-vitro screening technology of aptamer is the general flow figure of SELEX technology.
Embodiment
Embodiment 1: aptamer screening (takes turns)
First run oligo DNA library sequence: 5 '-acgctcggatgccactacagNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNctcatggacgtgctggtgac-3 '
Enrichment the primer:
Aptamer_L:5’-FAM-acgctcggatgccactacag-3’
Aptamer_R:5’-biotin-gtcaccagcacgtccatgag-3’
OD is measured in oligo DNA library
260after, centrifugal drying; With 300ul binding buffer liquid (containing 4.5g/L glucose, 5mM MgCl2,2mg/mL BSA, 0.2mg/mL yeast tRNA in PBS) dissolving library, get 250pmol(first round 10nmol), 95 ℃ of 5min, put rapidly on ice, centrifugal fast after a while.
Anti-sieve: the library of 250pmol is sucked to GES-1 growth coverage and reach in 70～80% diameter 6cm culture dish, supply 1mL with binding buffer liquid; 4 ℃ of shaking table vibration 1h; Get supernatant liquor.
Just sieve: the anti-supernatant liquor that sieves is added during HGC27 growth coverage reaches in 70～80% diameter 6cm culture dish to 4 ℃ of shaking tables vibration 1h; Abandon supernatant liquor; With lavation buffer solution (in PBS containing 4.5g/L glucose, 5mM MgCl2) washing HGC27 cell three times; Scrape HGC27 cell with cell, with 1mL lavation buffer solution, HGC27 cell is transferred in EP pipe, 95 ℃ of 5min, put rapidly on ice.After turning cold, the centrifugal 5min of 10000rpm; Supernatant is transferred in a clean EP pipe.
Enrichment: configuration PCR system (cumulative volume 1000ul): H
2o740ul, 10 × PCR damping fluid 100ul, dNTP80ul, primer mixture 25ul(H
2o:100uM Aptamer_L:100uM Aptamer_R=4:0.5:0.5), DNA profiling (just sieving supernatant) 50ul, Taq HS enzyme 5ul.
Increase on PCR instrument by following condition: after 94 ℃ of heating denaturation 2min, 94 ℃ of sex change 30s, 58 ℃ of annealing 20s, 72 ℃ are extended 20s, 20 circulations, 72 ℃ are extended 4min, 4 ℃ of insulation <1h.
Purifying: MilliQ washing one time for purification column, PBS washes twice, add the Streptavidin Sepharose of 150ul GE after PBS wash twice, add PCR product, shaking table vibration 20min, emits liquid, and PBS washes after twice, add after 0.5mL NaOH leaves standstill 2-3min and emit liquid, desalting column on relief liquor adds 1mL MilliQ water elution in the time that desalting column almost flows to end until liquid, start to connect elutriant 1mL simultaneously, this elutriant be enrichment oligo DNA library, can carry out next round screening.
Embodiment 2: aptamer TA clone
In Eppendorf tube, prepare following solution, full dose is 5ul:pMD19-T Vector1ul, aptamers PCR product 1ul, dH2O3ul; Add the Solution I of 5ul; 16 ℃ are reacted 30 minutes.Full dose (10ul) is added in 100ul DH5a competent cell, places 30 minutes in ice; 42 ℃ are heated after 45 seconds, then in ice, place 1 minute; Add 890ul LB substratum, 37 ℃ of shaking culture 60 minutes; On the LB Agar Plating that contains X-Gal, IPTG, Amp, cultivate, form single bacterium colony.
Embodiment 3: aptamer clone's bacterium colony PCR and order-checking
PMD19-T bacterium colony PCR and sequencing primer:
RV-M:5'-GAGCGGATAACAATTTCACACAGG-3'
M13(-47):5'-CGCCAGGGTTTTCCCAGTCACGAC-3'
Join in 4ml LB (Amp50ng/L) liquid nutrient medium with sterilizing toothpick picking white mono-clonal bacterium colony, 180rpm, 37 ℃ are shaken bacterium spend the night (<16h); Get 1ul bacterium liquid and make pcr template, carry out pcr amplification; Using water as negative control.
Configuration PCR system: bacterium liquid 1ul, primer RV-M (10uM) 0.5ul, primer M13 (47) 0.5ul, 2 × Taq Mix7.5ul, the total system 15ul of water 5.5ul(); By following condition, on PCR instrument, increase: 95 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 45s, totally 33 circulations; 72 ℃ of total elongation 10min.
After reaction finishes, get 3-6ul bacterium liquid PCR product and carry out agarose gel electrophoresis, 140V electrophoresis 20min; If there is bright object size strip, negative control does not have band, shows the positive clone of this bacterium liquid.
Row plasmid extraction subsequently: get the fresh bacterium liquid of 4ml incubated overnight, collect thalline, carry out plasmid extraction according to the plasmid extraction kit operation instructions of Omega; Finally be dissolved in water; Adopt Nanodrop to carry out concentration and purity testing to the plasmid extracting.
Sequencing reaction: BDT v3.10.5ul, plasmid 100ng, primer M13 (47) 0.5ul, adds water to 5ul.After 96 ℃ of heating denaturation 1min, 96 ℃ of sex change 10s, 50 ℃ of annealing 10s, 60 ℃ are extended 4min, 33 circulations, 4 ℃ of insulations.After reaction finishes by 5ul check order product+1.25ul EDTA(125mM, pH8.0)+15ul dehydrated alcohol seals, shakes 4 times, room temperature is placed 15min, and 3860rpm immediately, tips upside down on paper handkerchief after the centrifugal 40min of room temperature (25 ℃) immediately, centrifugal to 900rpm, stop at once; Add 60ul70% ethanol, (not needing concussion) seals; 3860rpm under room temperature subsequently, centrifugal 15min.Tip upside down on immediately on paper handkerchief, centrifugal to 900rpm, stop at once.Lucifuge drying at room temperature 15min, adds 10ul Hi-Di, seals lid; 95 ℃ of sex change 4min, leave standstill 4min at once on ice, of short duration centrifugal, upper 3730xl sequenator.Sequencing result is found all to have same characteristic sequence GGTTT in 18 clones.Therefore, obtained the characteristic sequence with specific combination HGC27 stomach cancer cell line, this section of characteristic sequence is GGTTT.
SEQUENCE LISTING
<110> Huashan Hospital Affiliated To Fudan Univ
Aptamer sequence and the purposes of a <120> stomach cancer cell
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 80
<212> DNA
<213> Artificial
<400> 1
acgctcggat gccactacag ccgttagcgc ggcgaggagt ttgggtttgg ttgggttggc 60
ctcatggacg tgctggtgac 80
Claims (2)
1. an aptamer sequence for stomach cancer cell, is characterized in that, described aptamer sequence is made up of the sequence shown in SEQ.ID.No.1.
2. the purposes of the aptamer sequence of stomach cancer cell claimed in claim 1 in the molecular probe of preparation detection cancer of the stomach.
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| CN103642810B (en) * | 2013-11-20 | 2016-01-20 | 中山大学附属第三医院 | Application in a kind of aptamer and screening method, the before detection strain of row adenocarcinoma cell |
| CN104561014B (en) * | 2015-01-12 | 2018-04-17 | 兰州大学 | Early stage sdenocarcinoma of stomach primary cell single-strand DNA aptamer and preparation method |
| CN105891478A (en) * | 2015-11-29 | 2016-08-24 | 卢美珍 | Stomach disease detection kit |
| CN105802973B (en) * | 2016-03-31 | 2019-04-30 | 浙江大学 | It is a kind of target myeloid differentiation antigens c D33 albumen aptamer and application |
| CN105671050B (en) * | 2016-03-31 | 2018-12-04 | 浙江大学 | A kind of efficient fast adaptation body screening technique |
| CN106754935B (en) * | 2016-11-30 | 2019-04-30 | 吴冬 | The aptamer WYZ-5 and its screening technique of ovarian mucinous cancer cell 3AO and application |
| CN106754936B (en) * | 2016-11-30 | 2019-06-07 | 吴冬 | The aptamer WYZ-2 and its screening technique of ovarian mucinous cancer cell 3AO and application |
| CN108929873B (en) * | 2018-07-10 | 2021-10-29 | 安徽省昂普拓迈生物科技有限责任公司 | Aptamer specifically binding to metastatic gastric cancer cells and application thereof |
| CN113308475B (en) * | 2021-06-04 | 2022-10-04 | 燕山大学 | Aptamer for specifically recognizing gastric adenocarcinoma serum and application thereof |
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| CN1611509A (en) * | 2003-10-31 | 2005-05-04 | 中国人民解放军军事医学科学院毒物药物研究所 | Nucleic acid adaptor for specific identifying human cerebral acetylcholinesterase and its use |
Non-Patent Citations (4)
| Title |
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| Development of DNA aptamers using Cell-SELEX;Sefah et al;《Nature protocols》;20100603;第5卷(第6期);全文 * |
| Sefah et al.Development of DNA aptamers using Cell-SELEX.《Nature protocols》.2010,第5卷(第6期),全文. |
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