CN104450721B - The sequence and purposes of escherichia coli outer membrane protein TolC aptamers - Google Patents

Abstract

The present invention relates to the sequence and purposes of escherichia coli outer membrane protein TolC aptamers.The present invention expresses surface of E. coli albumen TolC using gene recombination plasmid, and the aptamer specifically bound with it group, the base sequence of sequencing analysis aptamers group are screened by SELEX processes.The sequence can as surface of E. coli albumen TolC specific binding probe, quickly detect marker and efflux pump inhibitor etc. for designing and preparing Escherichia coli.

Description

The sequence and purposes of escherichia coli outer membrane protein TolC aptamers

The present invention is 2 months 2013 Application No. 201310062950.2 filed in 28 days, entitled " large intestine bars The divisional application of the sequence and purposes of bacterial outer membrane protein TolC aptamers ".

Technical field

The invention belongs to molecular biology and medical microbial technical field, it is related to escherichia coli outer membrane protein TolC nucleic acid The sequence and purposes of aptamers.

Background technology

Escherichia coli (Escherichia coli) are the representative bacterium of Escherichia, be constitute human intestine in it is normal One of flora, is important conditioned pathogen again, easily invades parenteral tissue and organ by diversified forms and approach, makes Into various severe infections.With the wide clinical application of antibacterials, induction of the generation of a large amount of Pseudomonas antibody-resistant bacterium, and shape Into the bacterium to single antibiotic resistance to a variety of antibacterials simultaneously resistance, by the hair of low resistant rate to high resistant rate Exhibition, brings great difficulty to clinical treatment, causes higher case fatality rate.Current drug resistance machine both at home and abroad to Escherichia coli Reason expands in-depth study.Research shows that Active efflux-pump is the important mechanisms of bacillus coli multiple resistance.Outside Escherichia coli Row's pump includes：The class of RND, MFS, ABC, SMR, MATE five, wherein former three must constitute transhipment with outer membrane protein (OMP) TolC altogether Consubstantiality, could complete the outer row to multi-medicament, it can be seen that TolC is the important composition composition for constituting Escherichia coli efflux pump. TolC is the duct albumen being located on outer membrane, and upper end is open architecture, to provide the broad passage pumped out to outer row's substrate；Under Hold as enclosed construction.The change of the compound conformation of efflux pump three, can make duct periodically open or closed.The change of TolC structures The disintegration for weakening even three compounds of their triple interactions can be caused.In the research on escherichia coli outer membrane protein In report, many results have pointed out TolC albumen to play vital effect in the multidrug resistant mechanism of Escherichia coli.But Suppress drug resistance by changing the protein structure currently without report.

The research of aptamers (Aptamers) is a new hot fields, it be can with many target molecules (albumen, Medicine, inorganic or organic molecule) occur a class single-chain nucleic acid (DNA or RNA) for high affinity and specific binding.So far send out The method of existing high affine specific aptamers is part index concentration system expansion (systematic evolution of Ligands by exponential enrichment, SELEX).The high specific combined with ligand molecular due to aptamers with And high-affinity, it is widely used in medical research in terms of detection and drug research.Yuan etc. (Yuan L, et al, Anal Chem, 2007,79 (3)：1082) protein-chip is set up with aptamer technology using Plasmon Surface Resonance imaging, Detect the protein antibodies fibrin ferment adsorbed on the protein-chip, VEGF (VEGF) compound.(the Cao such as Cao X, et al, Nucleic Acids Res, 2009,37 (14)：What 4621-8) utilization was filtered out is directed to staphylococcus aureus Aptamer detects staphylococcus aureus.

Most of scientist for being engaged in aptamer research domestic and international at present and biotech company are mainly by aptamers Apply to viral infection, cancer, autoimmunity disease, the clinical treatment of vascular conditions, and as ideal clinical treatment New tool.Its therapy mechanism is mainly specific directly with pathology related protein by being folded into certain three-dimensional structure With reference to suppressing the activity of these protein, to reach therapeutic purposes.Feng H etc. (Feng H, et al, PLoS One, 2011, 6(11)：E27862) having filtered out can be combined with hepatitis type B virus high-affinity, the adaptation of viral interference P- ε compounds formation Body.But it there is no the aptamers of directed toward bacteria outer membrane protein to report or openly so far, therefore real be necessary that exploitation one kind can specificity With reference to escherichia coli outer membrane protein TolC aptamer.

The content of the invention

The present invention intends considering effectively to block outside Escherichia coli applied to change TolC protein structures by aptamer to arrange Activity is arranged outside pump.Exploitation can specifically bind escherichia coli outer membrane protein TolC aptamer, many to suppress Escherichia coli Weight resistance, which is provided, to be provided powerful support for, with important clinical value.

It is an object of the invention to provide it is a kind of can specific bond escherichia coli outer membrane protein TolC aptamer.This is fitted Part has following sequence：SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、 SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19 or SEQ ID NO：20.

Specifically, the SEQ ID NO：1 is：5’-TGCGTGTTTACGTGTCGATGTCCGGGTACCCTCGG-3’；

The SEQ ID NO：2 are：5’-CTCTTAGCCATTTTGGTATGCGTATTGCTCTACAG-3’；

The SEQ ID NO：3 are：5’-ATATATTGCTCTGATCGTACTCTTATTAGGTTAGT-3’；

The SEQ ID NO：4 are：5’-ATGACGCTGTGGTATTACGTCCTCCTGTATTTGCC-3’；

The SEQ ID NO：5 are：5’-TCTAAGTGGAATCTACCACCAACGTATTTTTCCTC-3’；

The SEQ ID NO：6 are：5’-TCCGCCTATTCGGGAGGGAATATTGCTGATTTGTA-3’；

The SEQ ID NO：7 are：5’-CTCTTCAGTTTTAGACAATGCACGTTTCAGCGGTG-3’；

The SEQ ID NO：8 are：5’-TCCAGGCTATAATTTCTTGGAACTCCCTCCGTTAG-3’；

The SEQ ID NO：9 are：5’-CTTTCGAAGGGACTTTAACGGGTATATCCGGTTTT-3’；

The SEQ ID NO：10 are：5’-ATTACATGCTCTGAGCTTTTTGTATGAGAAATGAT-3’；

The SEQ ID NO：11 are：5’-CGCTTAGCTTGTGATTTCATCTTTCACACAAGAAT-3’；

The SEQ ID NO：12 are：5’-CGGAGATCTGTTACACTTCGGTACGTCTTAGTTTG-3’；

The SEQ ID NO：13 are：5’-CTACCATGTTCAGTGGTTTTGGGATTTTCATACAT-3’；

The SEQ ID NO：14 are：5’-TGAGATCGGTGAGTTAGTATCTTTTATTCAGTTTT-3’；

The SEQ ID NO：15 are：5’-GCATTGCGTGACATGGCCTTGCTATCCCTGTTCGT-3’；

The SEQ ID NO：16 are：5’-TCCTAAAAGCTAGTTATAGTAAATTGAAAACTTAG-3’；

The SEQ ID NO：17 are：5’-TGGAGCTTAGATTTTGAAGCAGTTACATTTCCCGA-3’；

The SEQ ID NO：18 are：5’-TACTGCGAGCTTCATTTTTACTTGGTAGTGTTGTG-3’；

The SEQ ID NO：19 are：5’-CGAACGAATATAATTATGGCGTCCCCGGGGTTTCG-3’；

The SEQ ID NO：20 are：5’-CTAGTTATGACATTTGTGTCATTTATCCCACGCTG-3’

The escherichia coli outer membrane protein TolC nucleic acid aptamer sequences of the present invention, are made by following methods：

Using the external SELEX triage techniqueses of aptamer, using escherichia coli outer membrane protein TolC as Screening target, from Random oligo DNA library (the 5 '-GGGAGCTCAGAATAAACGCTCAA-N35- synthesized in vitro TTCGACATGAGGCCCGGATC-3 ') in filter out aptamer with TolC albumen specific bonds.

It is preferred that, the escherichia coli outer membrane protein TolC adopts purifying with the following method and obtained：

Using molecule clone technology, special primer, institute are designed according to escherichia coli outer membrane protein TolC genome sequence The sense primer Pa for stating special primer is 5 '-CGGGATCCATGAAGAAATTGCTCCCCATTCTT-3 ', and primer Pb is downstream 5 '-CCGCTCGAGGTTACGGAAAGGGTTATGACCGTT-3 ', using full-length cDNA as template, PCR amplifications obtain target gene Through Bam H I and Xho I double digestions, same processing PET-30a plasmids, then contain TolC genes with the connection of T4 ligases is obtained Recombinant plasmid；Recombinant plasmid transformed competence colibacillus cell BL21, chooses monoclonal from the flat board of conversion and is cultivated into fluid nutrient medium, IPTG induced expressions, through separation of solid and liquid, take precipitation to be dispelled with Tris-HCl solution, Ultrasonic Pulverization, then electrophoresis；Column chromatography for separation Protein purification.

In a specific embodiment, the preparation side of the escherichia coli outer membrane protein TolC nucleic acid aptamer sequences of the present invention Method is：

Using molecule clone technology, special primer is designed according to escherichia coli outer membrane protein TolC genome sequence, In one specific embodiment, the special primer is sense primer Pa, anti-sense primer Pb, using full-length cDNA as template, PCR amplifications. Target gene is obtained through Bam H I and Xho I double digestions, same processing PET-30a plasmids, then contained with the connection of T4 ligases is obtained There is the recombinant plasmid of TolC genes.

Recombinant plasmid transformed competence colibacillus cell BL21, monoclonal is chosen into 1.5ml LB fluid nutrient mediums from the flat board of conversion Culture, IPTG (0.5mM) induced expression, the bacterium solution for taking 1ml to induce, 12000rpm centrifuges 1min, abandons supernatant, and precipitation uses 50- 100 μ l 10mM Tris-HCl (pH8.0) solution dispel and (add the amount of buffer solution depending on biomass), Ultrasonic Pulverization (500W, 30 times, each 10s is spaced 15s).50 μ l2 × loading buffer are added, 100 DEG C are boiled 5min, electrophoresis.

Cross nickel post protein purification, protein peak, electrophoresis detection protein purification effect are collected respectively (purification result is shown in Fig. 2).

Followed by the SELEX in-vitro screening technologies of aptamers, to collect TolC after purification as Screening target, from external The random oligo DNA library (5 '-GGGAGCTCAGAATAAACGCTCAA-N35-TTCGACATGAGGCCCGGATC-3 ') of synthesis In filter out aptamer with TolC albumen specific bonds, by the sequence screened primer P1 (5 ' GGGAGCTCAGAATAAACGCTCAA-3 ') and primer P2 (5 '-GATCCGGGCCTCATGTCGAA-3 ') expanded.By PCR Amplified production takes 1 μ l after purification, is connected with carrier PGM-T in the presence of T4DNA ligases, then to be transformed into competence thin Born of the same parents, take proper volume be spread evenly across containing IPTG, x-gal, antibiotic (Amp) LA flat boards, after 12~16 hours with inoculation Single white colony in ring picking screening flat board is in the LB fluid nutrient mediums containing Amp, 37 DEG C of overnight incubations.Take bacterium solution lml in Hai Sheng works Bioisystech Co., Ltd (hereinafter referred to as Shanghai life work) sequencing is served in centrifuge tube, after sealer.

The sequence be selected from naturally occurring or artificial synthesized sequence, or any other source same sequence.

The present invention further provides the purposes of the nucleic acid aptamer sequence, for example the sequence is in medicine or product is prepared Application.In a specific embodiment, the present invention provides the sequence answering in Escherichia coli efflux pump inhibitor is prepared With.

Another object of the present invention is to provide the nucleic acid aptamer sequence prepare detection TolC albumen probe or Application in target spot.

The present invention also provides application of the aptamers in Escherichia coli efflux pump inhibitor is prepared.

The present invention also provides application of the aptamers in the probe or target spot of detection TolC albumen is prepared.

Another object of the present invention is to provide row's activity or reduction Escherichia coli outside a kind of suppression Escherichia coli efflux pump The method arranged outside efflux pump, including give the aptamers described in effective dose.

In the present invention, the aptamers that described nucleotide sequence is constituted can be tied with escherichia coli outer membrane protein TolC specificity Close, design and preparation available for efflux pump inhibitor medicine.The aptamers that described nucleotide sequence is constituted can also be used as inspection Survey the probe or target spot of TolC albumen.Nucleic acid aptamer sequence provided by the present invention and the Escherichia coli outer membrane egg as target White TolC has very strong compatibility, thus the sequence has the advantages that high specificity；In addition, the sequence can be a large amount of Rapidly synthesize in vitro, and preparation method is simple, thus be easier to obtain.

Brief description of the drawings

Fig. 1 is that aptamers suppress the Escherichia coli efflux pump mechanism of action；

Fig. 2 is that escherichia coli outer membrane protein TolC purifies electrophoretogram；

Brief description of the drawings：

Fig. 2 abscissas are represented：Swimming lane 1 is TolC supernatants；Swimming 2 is that TolC is precipitated；Swimming lane M is albumen marker；Fig. 2 is vertical to be sat Mark is represented：The molecular weight of protein, unit kD.

Embodiment

Coordinate the preferred embodiments of the present invention below, the present invention is expanded on further and reaches predetermined goal of the invention and is taken Technological means.

The present invention intends considering effectively to block outside Escherichia coli applied to change TolC protein structures by aptamer to arrange Activity is arranged outside pump, obstruction principle is as shown in Figure 1.Exploitation, for escherichia coli outer membrane protein TolC aptamer, will be suppression Bacillus coli multiple resistance processed is provided and provided powerful support for, with important clinical value.

Embodiment 1：Outer membrane protein TolC gene magnification

Using molecule clone technology, according to escherichia coli outer membrane protein TolC genome sequence design special primer (on Primer Pa is swum, downstream primer Pb), using full-length cDNA as template, PCR amplifications.PCR reaction conditions：94 DEG C, pre-degeneration 5min, Then with 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 3O circulation, last 72 DEG C of extensions 10min are carried out.PCR Product is through 1wt% agarose gel electrophoresis, gel extraction purpose fragment.

Embodiment 2：The structure of recombinant expression plasmid

(1) target gene is obtained through Bam H I and Xho I double digestions, and digestion system is as follows：25 μ l plasmids, 2 μ l Bam H I, 2 μ l Xho I, 8 μ l 10 × Buffer, 43 μ l ddH₂O, 37 DEG C of reaction overnights of mixture are pure with quick glue reclaim kit Change digestion products.

(2) PET-30a plasmids are through Bam H I and Xho I double digestions, and digestion system is as follows：5 μ l carriers (pET-30a) matter Grain, 2 μ l BamH I, 2 μ l Xho I, 8 μ l 10 × Buffer, 63 μ l ddH₂O, 37 DEG C of reaction overnights of mixture, uses quick glue QIAquick Gel Extraction Kit purifies digestion products.

(3) with T4 ligases connection escherichia coli outer membrane protein TolC gene and PET-30a carriers, restructuring matter is obtained Grain.Linked system is as follows：1ul carriers (pET30a), 3ul PCR recovery products, 4ul ligases (Takara, DNA ligation Kit Ver2.0), mix, more than 30min is reacted at room temperature.

(4) recombinant plasmid transformed competence colibacillus cell BL21.The competent cell (BL21) that -80 DEG C of temperature are preserved is taken out, is put Thawed slow on ice.It is respectively 42 DEG C and 37 DEG C to adjust 2 water-bath to water temperatures.Competent cell BL21 is added into connection production Thing, is placed 30 minutes on ice.42 DEG C of heat shocks 90 seconds.Put back on ice, the LB culture mediums that 800ul non-resistants are added after 2 minutes (block that Mycin) (giving birth to work purchased from Shanghai).It is put into 37 DEG C of shaking tables 45 minutes, 8000rpm is centrifuged 3 minutes, is abandoned most of supernatant, is stayed about 100-150ul is resuspended, and selection has the LB flat boards of corresponding resistant, coated plate.Dry, overnight incubation is inverted in 37 DEG C of incubators.

Embodiment 3：Expression of the TolC albumen in Escherichia coli

Monoclonal is chosen into 1.5ml LB fluid nutrient mediums from the flat board of conversion, 37 DEG C, 200rpm cultures.By the bacterium of culture Liquid is transferred to the mixing of 500mL LB fluid nutrient mediums, 37 DEG C, 200rpm, culture to OD=0.6-0.8, IPTG (0.5mM) induction 4h.With the big concentrator bowls of 400m, 6000rpm centrifuges 5min, takes supernatant.Precipitation 20-30ml 10mM Tris-HCl (pH8.0) Solution is dispelled, Ultrasonic Pulverization (500W, 30 times, each 10s is spaced 15s).Bacteria suspension after taking 100 μ l ultrasonic, 12000rpm, 10min is centrifuged, TolC supernatants TolC precipitations are obtained respectively, takes 50 μ lTolC supernatants to be managed to another EP, TolC is precipitated with 50 μ l 10mM Tris-HCl pH8.0) solution dispels, and adds 50 μ l2 × loading buffer, 100 DEG C are boiled 5min, electrophoresis.Electrophoresis Figure is as shown in Figure 2.As a result being shown in the i.e. TolC supernatants TolC of swimming lane 1 and 2 precipitations at 52kD has the albumen of an obvious thickening Matter band (as shown in Figure 2) M is the conventional standard molecular weight marker of electrophoretic techniques, purpose band and Marker that band position The identical molecular weight for being assured that purpose band is put, as a result shows that destination protein has expression in supernatant precipitation.

Embodiment 4：Protein purification

Nickel post is washed with pure water to pH7.0, rear nickel of hanging is to pH2-3.Pure water washes post to pH7.0, uses 100mlTris-HCl (10mM, pH8.0) solution equilibria nickel post, then with 10mMTris-HCl (pH8.0) solution equilibria nickel of 50ml sodium chloride containing 0.5M Post.Sample Outer membrane protein TolC is diluted to the 10mM Tris-HCl pH8.0 that 0.5M sodium chloride is used after loading, end of the sample) it is molten Liquid washes post, respectively with imidazoles containing 15mM, 60mM imidazoles, 300mM imidazoles 10mM Tris-HCl (pH8.0) (chlorination containing 0.5M Sodium) solution elution, collect the protein peak of 300mM imidazoles elution.

Embodiment 5：Aptamer screening (wheel)

Shanghai life work synthesizes the ssDNA pool of 78 nt length, and upstream is 23 nt primer binding site, downstream For 20 nt primer binding site, centre is 35 nt random sequence, and storage capacity is 4³⁵.SsDNA sequence is：

It is used in 5 '-GGGAGCTCAGAATAAACGCTCAA-N35-TTCGACATGAGGCCCGGATC-3 ' screening processes PCR amplification DNA primer be respectively：Primer P1, primer P2.

(1) TolC albumen is dissolved in after 200 μ lPBS and added in 96 hole elisa plates, and 4 DEG C overnight.

(2) albumen coating hole is washed after 6 times with PBS, is added 200 μ l3%BSA (giving birth to work purchased from Shanghai), is incubated 2 hours. Meanwhile, 200 μ l3%BSA37 DEG C are added in the hole elisa plate of blank 96 and are incubated 2 hours.

(3) ssDNA pool (first run consumption is 800pmol) is dissolved in 200 μ l 1 × SHCMK, 95 DEG C of denaturation 5min. 10min on ice is immediately placed on, it is rapidly decreased to room temperature, it is to avoid ssDNA renaturation is into double-strand.

(4) ssDNA is added in the blank ELISA holes that PBS washs 6 times, 37 DEG C are incubated 2 hours.

(5) supernatant be transferred to PBS wash 6 times albumen coating hole in, 37 DEG C be incubated 2 hours after PBS wash 6 times.

(6) the enzyme mark hole for combining ssDNA is resuspended with 200 μ l elution buffers, 95 DEG C of heating 5min, is taken supernatant, is passed through Ethanol precipitation DNA, is dissolved in Millipore water, the template screened as next round.

(8) 5 μ lPCR products are taken to be splined on 5wt% agaroses (giving birth to work purchased from Shanghai), whether observation pillar location is correct. After the first run, the amount in ssDNA level storehouse of input is gradually decreased, and 12 circulations are so repeated.

Embodiment 6：Aptamer is cloned

The amplification and purifying of 12nd wheel screening product：The ssDNA sequences that 12nd wheel screening is obtained, use primer P1, primer P2 is expanded.After 100 μ lPCR amplifications system add 500pl combination liquid BB (20mmol/L Hepes (pH7.35), 120mmol/L NaCl, 5mmol/L KCl, 1mmol/L CaCl2,1mmol/LMgCl2,1%BSA), fully mix.By upper one Resulting solution is walked to add in adsorption column EC (adsorption column is put into collecting pipe), room temperature placement lmin, 12000rpm centrifugation 30~ 60s, outwells the waste liquid in collecting pipe.Add 700pmol rinsing WB (with reference to liquid BB+0.05%Tween20), 12000rpm centrifugations 30s, discards waste liquid.500pmol rinsing liquids WB, 12000rpm centrifugation 30s is added, waste liquid is discarded.Adsorption column EC is put back into empty receipts In collector, 12000rpm centrifugations 2min.Adsorption column EC is taken out, is put into a clean centrifuge tube, in the pars intermedia of adsorbed film Position plus the elution buffer EB of 20pmol65 DEG C of water-bath preheating, room temperature place 2min, 12,000rpm centrifugation lmin.By what is obtained Solution is rejoined in adsorption column, and lmin is centrifuged again.

The clone of PCR purified products：1 μ l amplified productions are taken, are connected with carrier PGM-T in the presence of T4DNA ligases, Competent cell is transformed into again, and taking proper volume to be spread evenly across containing IPTG, x-gal, antibiotic (Amp), (they divide Gou Ziyu Shanghai life work) LA flat boards, be inverted culture dish, " indigo plant-hickie screen " carried out within 12-16 hour in 37 DEG C of cultures.

Sequencing：With the single white colony in oese picking screening flat board in the LB fluid nutrient mediums containing Amp, 37 DEG C of trainings Support overnight.Bacterium solution lml is taken to serve the raw work sequencing in sea in centrifuge tube, after sealer.Sequencing result finds wherein have 20 nucleic acid to fit Ligand sequence has very strong compatibility with escherichia coli outer membrane protein TolC, and 20 sequences are respectively：

5’-TGCGTGTTTACGTGTCGATGTCCGGGTACCCTCGG-3’(SEQ ID NO：1)；

5’-CTCTTAGCCATTTTGGTATGCGTATTGCTCTACAG-3’(SEQ ID NO：2)；

5’-ATATATTGCTCTGATCGTACTCTTATTAGGTTAGT-3’(SEQ ID NO：3)；

5’-ATGACGCTGTGGTATTACGTCCTCCTGTATTTGCC-3’(SEQ ID NO：4)；

5’-TCTAAGTGGAATCTACCACCAACGTATTTTTCCTC-3’(SEQ ID NO：5)；

5’-TCCGCCTATTCGGGAGGGAATATTGCTGATTTGTA-3’(SEQ ID NO：6)；

5’-CTCTTCAGTTTTAGACAATGCACGTTTCAGCGGTG-3’(SEQ ID NO：7)；

5’-TCCAGGCTATAATTTCTTGGAACTCCCTCCGTTAG-3’(SEQ ID NO：8)；

5’-CTTTCGAAGGGACTTTAACGGGTATATCCGGTTTT-3’(SEQ ID NO：9)；

5’-ATTACATGCTCTGAGCTTTTTGTATGAGAAATGAT-3’(SEQ ID NO：10)；

5’-CGCTTAGCTTGTGATTTCATCTTTCACACAAGAAT-3’(SEQ ID NO：11)；

5’-CGGAGATCTGTTACACTTCGGTACGTCTTAGTTTG-3’(SEQ ID NO：12)；

5’-CTACCATGTTCAGTGGTTTTGGGATTTTCATACAT-3’(SEQ ID NO：13)；

5’-TGAGATCGGTGAGTTAGTATCTTTTATTCAGTTTT-3’(SEQ ID NO：14)；

5’-GCATTGCGTGACATGGCCTTGCTATCCCTGTTCGT-3’(SEQ ID NO：15)；

5’-TCCTAAAAGCTAGTTATAGTAAATTGAAAACTTAG-3’(SEQ ID NO：16)；

5’-TGGAGCTTAGATTTTGAAGCAGTTACATTTCCCGA-3’(SEQ ID NO：17)；

5’-TACTGCGAGCTTCATTTTTACTTGGTAGTGTTGTG-3’(SEQ ID NO：18)；

5’-CGAACGAATATAATTATGGCGTCCCCGGGGTTTCG-3’(SEQ ID NO：19)；

5’-CTAGTTATGACATTTGTGTCATTTATCCCACGCTG-3’(SEQ ID NO：20)；

Described above is only the preferred embodiments of the present invention, not does any formal limitation to the present invention, though So the present invention is disclosed above with preferred embodiment, but is not limited to the present invention, any to be familiar with this professional technology people Member, in the range of technical solution of the present invention is not departed from, when the technology contents using the disclosure above make a little change or repair The equivalent embodiment for equivalent variations is adornd, as long as being the content without departing from technical solution of the present invention, the technology according to the present invention is real Any simple modification, equivalent variations and modification that confrontation above example is made, still fall within the scope of technical solution of the present invention It is interior.

Claims (3)

1. specific bond escherichia coli outer membrane protein TolC aptamer, the aptamer such as SEQ ID NO：11 institutes Show sequence.

2. the aptamers as described in claim 1, it is characterised in that the escherichia coli outer membrane protein TolC is adopted with the following method Purifying is obtained：

Using molecule clone technology, special primer, the spy are designed according to escherichia coli outer membrane protein TolC genome sequence The sense primer Pa of different primer be 5 '-CGGGATCCATGAAGAAATTGCTCCCCATTCTT-3 ', downstream primer Pb be 5 '- CCGCTCGAGGTTACGGAAAGGGTTATGACCGTT-3 ', using full-length cDNA as template, PCR amplifications obtain target gene warp Bam H I and Xho I double digestions, same processing PET-30a plasmids, then connect the obtained weight containing TolC genes with T4 ligases Group plasmid；Recombinant plasmid transformed competence colibacillus cell BL21, chooses monoclonal from the flat board of conversion and is cultivated into fluid nutrient medium, IPTG Induced expression, through separation of solid and liquid, takes precipitation through Ultrasonic Pulverization, then electrophoresis；Purified again using column chromatography outside the Escherichia coli Memebrane protein TolC.

3. the aptamers as described in claim 1 or 2, it is characterised in that the sequence is artificial synthesized sequence, or it is any its The same sequence that he originates.