CN104450724A

CN104450724A - One group of nucleic acid aptamers capable of specifically identifying Beijing genotypes tuberculosis bacterial strain antigen and application of nucleic acid aptamers

Info

Publication number: CN104450724A
Application number: CN201410798757.XA
Authority: CN
Inventors: 章晓联; 唐晓磊
Original assignee: Wuhan University WHU
Current assignee: WUHAN SHUNKEDA BIOTECH Co.,Ltd.
Priority date: 2014-12-19
Filing date: 2014-12-19
Publication date: 2015-03-25
Anticipated expiration: 2034-12-19
Also published as: CN104450724B

Abstract

The invention discloses one group of nucleic acid aptamers capable of specifically bonding with tuberculosis mycobacterium Beijing genotype strain surface lipolysaccharide namely mannose modified mannosylated lipoarabinomannan (ManLAM) and application of the nucleic acid aptamers. The aptamers are specific to small-molecular single-chain DNA (ssDNA) of the ManLAM, and have a nucleotide sequence as shown in the SEQ ID No.1-5. The ssDNA has functions similar to those of a monoclonal antibody, and can be directly and specifically bonded with ManLAM. By adopting an enzyme-linked oligonucleotide assay (ELONA) method, the ManLAM antigen in serum of patients with pulmonary tuberculosis, extrapulmanory tuberculosis and the like can be quickly effectively detected in 2 hours, the ManLAM antigen in sputum of an active pulmonary tuberculosis patient can be directly identified, and the detection sensitivity on the Man LAM antigen can be 0.035mu g/mL. The aptamers are simple to prepare and low in cost, and can be used as an effective novel reagent for diagnosing the mycobacterium tuberculosis antigen of early and latent tuberculosis.

Description

The nucleic acid aptamer of one group-specific identification Beijing genotype mycobacterium tuberculosis strain antigen and application thereof

The present invention relates to biological technical field, be related specifically to utilize in Protocols in Molecular Biology SELEX technology (the Fas lignand system evolution technology of index concentration) preparation can with Beijing genotype mycobacterium tuberculosis strain surface lipolysaccharide---LAM (the Mannosylated lipoarabinomannan of mannose-modified, ManLAM) nucleic acid aptamer of high specific and high-affinity, can be used for the detection reagent preparing diagnosis Beijing genotype m tuberculosis infection.

Background technology

Tuberculosis infects disease caused by body by mycobacterium tuberculosis (M.tuberculosis), relates to the multiple internal organs of whole body.At present, the people in the whole world about 1/3 infected mycobacterium tuberculosis, 2,000,000 people are about had to die from tuberculosis infection (Dye C SS every year, Dolin P PV, Raviglione MC.Consensus statement.Globalburden of tuberculosis:estimated incidence, prevalence, and mortality by country.W.H.O.Global Surveillance and Monitoring Project.J Am Med Assoc.1999.282:677-686.), the health of the positive serious threat mankind of tuberculosis.The present situation of current China tuberculosis infection is: annual newly-increased tuberculosis sufferer reaches 1,300,000 more than, every year because the number of tuberculosis death has 150,000, tuberculosis epidemic situation presents the present situation that morbidity is high, mortality ratio is high, resistant rate is high, annual decline rate is low, and the epidemic situation was severe degree is only second to India (national tuberculosis epidemiological random sampling survey technical director group. the 4th national tuberculosis epidemiological random sampling survey report. Chinese tuberculosis and breathe magazine .2002.25 (1): 3-7.).

Mycobacterium tuberculosis kind lungy can be caused a lot.A class is had to have the Beijing genotype strain of similar genetic background in mycobacterium tuberculosis.This kind of bacterial strain widely distributed, all finds that there is the popular of Beijing genotype strain all over the world, and causes repeatedly eruption and prevalence, and it may be propagate rapider, pathogenic stronger and resistance to form an easier class bacterial strain that people propose Beijing genotype strain.And China is the country of a tuberculosis high incidence, it is also a high proportion of area of Beijing genotype strain.The investigation report of Beijing genotype strain distribution is thought, in northern China, Beijing genotype strain has comparative advantage, can 70% to 80% be reached, external even report Beijing area reaches 90% (van Soolingen D, Qian L, de Haas PE, etal.Predominance of a single genotype of Mycobacterium tuberculosis in countries ofeast Asia.J Clin Microbiol, 1995,33,3234-3238.), also have in various degree at other city Beijing genotype strains of China popular.Such as, Ningxia (67%), Shanghai (89%) (Li WM, Wang SM, Pei XY, et al. [DNA fingerprinting of Mycobacterium tuberculosis strains from Beijing, Guangdong and Ningxia] .Zhonghua Liu Xing Bing Xue Za Zhi, 2003,24,381-384.; Shen GM, Zha J, Xu L, et al. [Evaluation of the mycobacterial interspersed repetitiveunits typing as a practical approach in molecular epidemiology of Mycobacteriumtuberculosis] .Zhonghua Jie He He Hu Xi Za Zhi, 2005,28,292-296.).Thus, seem particularly important in the study on prevention meaning of China's Beijing genotype strain.And be a difficult problem urgently to be resolved hurrily to the diagnosis of m tuberculosis infection.

Be divided three classes for the detection technique of mycobacterium tuberculosis in the world at present: bacteriological detection, immunology diagnosis, diagnosis of molecular biology.Bacteriological detection is a kind of traditional detection means, comprise Sputum smears acid-fast stain and Sputum culturing, widespread use clinically for many years, it is the standard diagnosis of generally acknowledged detection mycobacterium tuberculosis, Sputum smears acid-fast stain may fail to pinpoint a disease in diagnosis the patient of sputum smear negative, and Sputum culturing length consuming time, need 2-8 time-of-week, thus its use is also subject to great restriction (Martinez A, Balandrano S, Parissi A, et al.Evaluation of new external quality assessment guidelines involving randomblinded rechecking of acid-fast bacilli smears in a pilot project setting in Mexico.IntJ Tuberc Lung Dis.2005.9 (3): 301-305.).The method of immunology diagnosis mycobacterium tuberculosis has tuberculin test and T lymphocyte specific IFN-γ release test.Both detects the reaction of tuberculosis antigen specific T-cells, tuberculin test utilizes purified protein derivative (PPD) (purified protein derivative, the function of the delayed reaction of PPD) inducing to T cell detects, but most of protein ingredient is the common composition of various mycobacterium in PPD, especially inoculate after BCG with infection mycobacterium tuberculosis after, both is difficult to difference, and the method exists the drawback of poor specificity.So set up a kind of novel detection method, realize Mycobacterium tuberculosis early diagnosis, significant for control tuberculosis epidemic situation.Diagnosis of molecular biology mainly comprises DNA probe method, polymerase chain reaction (polymerase chain reaction, PCR), quantitative PCR, and the method for the XpertMTB/RIF of the PCR-based of developed recently, there is sensitive and that specificity is high advantage, wherein, the method of PCR is mainly for one section of conserved sequence in M. tuberculosis genes group, by the method energy Rapid Detection of Mycobacterium Tuberculosis of PCR, but there is certain false negative and false positive, as may because of the variation that detects and cause false negative, and cost is high, need your plant and instrument, be difficult to popularize in Endemic Area or poverty-stricken area, and viable bacteria or dead bacterium cannot be distinguished, therefore monitor after can not being used for chemotherapy.

Nucleic acid aptamer (aptamer) is a kind of artificial single stranded oligonucleotide part of bio-target molecule, is screened obtained by SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology from random single chain oligonucleotide library.The basic step of nucleic acid aptamer screening is as follows: design and synthesize random single chain oligonucleotide library; Library and target are hatched altogether, the oligonucleotide that can be combined with target specificity in library and target is made to form mixture, isolated complex wash-out oligonucleotide, with the oligonucleotide of wash-out for template carries out pcr amplification, be prepared into new strand random oligonucleotide library, start next round screening; Repeat number wheel hatches-is separated-increases, and the nucleotide sequence be combined with target high specific and high-affinity will to be stayed in library and by index concentration; After taking turns screening to last, clone, check order in the library of preparation, and the single strand oligonucleotide acid sequence that length and two terminal sequences conform to library is nucleic acid aptamer; Measure specificity and avidity that each nucleic acid aptamer is combined with target, select can high specific and high-affinity in conjunction with target person, namely can be applicable to the detection analysis of target.

The function of nucleic acid aptamer and antibody class seemingly, but have many advantages compared with antibody: 1. high specific and high-affinity: nucleic acid aptamer can tell the structural nuance of target molecule, and avidity can be better than native ligand; 2. target is extensive: the target that all can be used as screening nucleic acid aptamer from organic molecule, biomacromolecule to virion, intact cell etc.; 3. good stability: nucleic acid aptamer can be preserved for a long time, synthetic and without differences between batches, can transporting at normal temperatures; 4. reconstruct and modify easily: can mark accurately at the privileged site of nucleic acid aptamer, modify or Nucleotide replacement; 5. application advantage: nucleic acid aptamer non-immunogenicity, can Reusability in vivo; Molecule is little, is convenient to diagnostic imaging and treatment in body.Nucleic acid aptamer demonstrates good application prospect in the diagnosis of human diseases.In microorganism detection, particularly to the research of some pathogenic bacterias or virus, although the epi-position of unknown its internal structure, function and these materials, but also can it can be used as target material, the aptamer corresponding with it is screened by SELEX process, in order to detect target material, become the research and probe focus in this field at present.

Summary of the invention

The present invention is mainly devoted to the detection of the ManLAM antigen of the early stage and latent infection of mycobacterium tuberculosis, screen single stranded DNA (ssDNA) aptamer for Mycobacterium tuberculosis Beijing genotype strain surface specific ManLAM, and join oligonucleotide analysis method (Enzyme-linked oligonucleotide assay with enzyme, ELONA) detect the ManLAM antigen in serum and the tubercule bacillus in sputum, reach the object that diagnosis of tuberculosis infects.

Seminose adds LAM (the mannose-capped lipoarabinom-anan of cap, ManLAM) be Mycobacterial cell wall surface distinctive lipolysaccharide (the Nigou J that mycobacterium tuberculosis (comprising mycobacterium tuberculosis var bovis) and BCG etc. grow slowly, Gilleron M, Puzo G.Lipoarabinomannans:from structure to biosynthesis.Biochimie.2003,85 (1-2): 153-166.).But ManLAM its structure in different mycobacteriums still also exists difference (Rojas M, Barrera LF, Puzo G, GarciaLF.Differential induction of apoptosis by virulent Mycobacterium tuberculosis inresistant and susceptible murine macrophages:role of nitric oxide and mycobacterialproducts.J Immunol.1997, 159 (3): 1352-1361.), for the difference of ManLAM structure between different strain, the present invention designs and has filtered out the aptamer with the ManLAM specific binding on the Mycobacterium tuberculosis Beijing strain surface of Major Epidemic clinically.

Therefore, first object of the present invention be to provide one group can specifically, high-affinity in conjunction with the primary sequence of the ssDNA aptamer of Mycobacterium tuberculosis Beijing genotype strain ManLAM antigen (hereinafter referred to as ManLAM), as shown in SEQ ID No.1-5.

Second object of the present invention is to be provided in the aptamer that above-mentioned DNA aptamer 5 ' end or 3 ' end carry out vitamin H, FITC or digoxin chemically modified.

The present invention also aims to provide above-mentioned DNA aptamer to detect the application in Mycobacterium tuberculosis Beijing genotype strain ManLAM antigen diagnose reagent and tuberculosis infection diagnostic reagent in preparation.

To achieve these goals, the present invention is by the following technical solutions:

The present invention obtains having the DNA aptamer of high specific with Mycobacterium tuberculosis ManLAM lipide-carbohydrate antigen, high-affinity by the structure, SELEX screening, pcr amplification, avidity mensuration, external different bacterium Binding experiment etc. of random single-stranded DNA banks and primer, and its nucleotide sequence is as shown in SEQ ID No.1-5.Confirm that this aptamer can detect ManLAM lipide-carbohydrate antigen in serum or body fluid delicately by immunological experiment, Accurate Diagnosis tuberculosis.

The ssDNA aptamer that the present invention screens and prepares with ManLAM lipolysaccharide is target, this aptamer energy high-affinity, with high specificity with this protein binding.This aptamer is directly applied to the detection of clinical serum sample and sputum sample by us, result shows, no matter be tuberculosis or the outer tuberculosis (tuberculosis of intestine bone tuberculosis, body cavity tuberculosis, meningeal tuberculosis, tuberculosis of epididymis, lymphoid tuberculosis) of lung in lung, use this aptamer all can effectively detect fast.Relative to the T-SPOT method of current diagnosis tuberculosis infection, the method is implemented more simple, without the need to being separated the peripheral blood lymphocytes (PBMC) of patient.Simultaneously, instant invention overcomes the serious problems that the acid-fast stain sensitivity of detection tuberculosis patient sputum bacterium is traditionally low, also overcome and only can detect tuberculosis patient Serum Antibody traditionally, and can not the limitation of detectable antigens, and can not the serious problems of diagnostic window phase early infection, the method cost is less.In addition, the function of nucleic acid aptamer and antibody class seemingly, therefore treat in preparation the using value that in the medicine of tuberculosis, also tool is potential.The ELONA method based on aptamer that the present invention adopts compares in table 1 from the different relative merits of clinical existing Diagnosis of Tuberculosis method.

Table 1 ELONA compares with clinical existing Diagnosis of Tuberculosis method

Accompanying drawing explanation

The extraction and isolation of Fig. 1 .ManLAM.

A), B) by the result of silver dye and staining for glycogen after capable for ManLAM raw sugar extract SDS-PAGE electrophoresis, ManLAM molecular weight is between 26kD-34kD; C) by between the result 26kD-34kD of silver dye after capable for the ManLAM of purifying SDS-PAGE electrophoresis; D) by between the result 26kD-34kD that identifies through immunoblotting (Western Blot) after capable for the ManLAM of purifying SDS-PAGE electrophoresis;

The spectrogram that Fig. 2 .ManLAM identifies through high performance liquid chromatograph (HPLC).

A) ManLAM is dissolved in moving phase liquid-mixing and makes 1mg/ml, adjust baseline with moving phase, from 190nm to 900nm, at interval of 1nm run-down, by the collection of illustrative plates that the magnitude value of gained absorbancy is drawn, have most high-selenium corn peak when 202nm.B) through the ManLAM of HPLC purification Identification, its retention time is about 6.3min;

Fig. 3. respectively screen the adaptive word bank of ssDNA of odd number wheel and the avidity of ManLAM.

The visible increase along with screening wheel number, the bonding force of aptamer increases gradually, the 13rd ssDNA storehouse of taking turns and ManLAM avidity the highest;

Fig. 4. compare the avidity of different aptamer and ManLAM in the 13rd ssDNA storehouse of taking turns.

Acquisition 36 single aptamers are separated in the 13rd ssDNA storehouse of taking turns, the numbering being numbered single aptamer of X-coordinate, as seen from the figure, 6,9,14,15, No. 25 all have higher bonding force with ManLAM, thus, by these 5 aptamers alternatively aptamer, and called after T6, T9, T14, T15, T25;

Fig. 5. detect candidate's aptamer to the dose-dependently of the bonding force of ManLAM.

Find that T6, T9, T14, T15, T25 and ManLAM all have good dose-dependently;

Fig. 6. compare the equilibrium dissociation constant K of 5 kinds of mono-clonal aptamers _d.

Bag, by the ManLAM of same concentrations purifying, detects with biotin labeled 5 kinds of aptamers (T6, T9, T14, T15, T25) of different concns, records its bonding force K _dvalue is respectively: 998 ± 207nM, 668 ± 159nM, 815 ± 144nM, 736 ± 207nM, 685 ± 131nM;

Fig. 7. compare 5 kinds of mono-clonal aptamers to the detection perform of clinical samples.

Bag is by clinical tuberculosis patient serum and Healthy Human Serum, biotin labeled 5 kinds of aptamers are used to detect, according to its ratio to the detection absorbancy of tuberculosis patient serum and the absorbancy of Healthy Human Serum, aptamer all demonstrates good differentiation tuberculosis patient and the ability of Healthy People;

Fig. 8. the specific detection of aptamer.

Biotin labeled aptamer and Mycobacterium tuberculosis Beijing genotype strain and Mycobacterium tuberculosis H37Rv, M. smegmatics, intestinal bacteria, bird mycobacterium, streptococcus aureus, faecalis, Bacillus proteus, streptococcus pneumoniae, klebsiella, surperficial staphylococcus, Pseudomonas aeruginosa, BCG, suis, Diplococcus gonorrhoeae is used to be combined, reflect the relative binding capacity of aptamer and different bacterium according to its absorbance, result display aptamer and Mycobacterium tuberculosis Beijing genotype strain bonding force are far above other bacterial strain;

Fig. 9. the sensitivity technique that aptamer detects.

A) bag is by the ManLAM of different concns (100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 3 μ g/ml, 1.5 μ g/ml, 0.75 μ g/ml, 0.37 μ g/ml, 0.18 μ g/ml), uses biotin labeled five kinds of aptamers combine with it and measure OD ₄₅₀nm.B) after ManLAM lipolysaccharide concentration being taken the logarithm with record OD value and carry out matching and obtain equation: y=0.3075x+0.4445;

Figure 10. aptamer is to the Serologic detection of active tuberculosis.

Figure 11. aptamer is to the Serologic detection of the outer tuberculosis of lung.

Figure 12. aptamer detects the sputum of active tuberculosis.

Embodiment

Following examples are used for further illustrating the present invention, but should not be construed as limitation of the present invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.

The screening of [embodiment 1] aptamer

1, extract, purifying identify Mycobacterium tuberculosis Beijing genotype strain surface lipolysaccharide ManLAM

(1) (taking mycobacterium tuberculosis substratum Middlebrook 7H9 4.79 grams is dissolved in 900ml deionized water to be incubated at 7H9 liquid nutrient medium, add glycerine 2ml to mix, 121 DEG C, sterilizing in 10 minutes, 9:1 adds enrichment liquid Middlebrook OADC by volume, for subsequent use) in OD about 1.8, deactivation 1 hour in 65 DEG C of water-baths, then in 10000rpm, 10min collected by centrifugation Mycobacterium tuberculosis Beijing genotype bacterium, add a small amount of PBS and wash 2 times, lyophilize;

(2) dried H37Rv is resuspended in the chloroform/methanol (v/v=2:1) of 10 times of volumes, degreasing 12 hours in 37 DEG C of water-baths;

(3) in 10000rpm, 10min centrifugal collecting precipitation, precipitation is resuspended in again in chloroform/methanol/water (v/v/v=10:10:1), degreasing 12 hours in 37 DEG C of water-baths;

(4) centrifugal in 10000rpm, 10min, collect the thalline after degrease, after nitrogen dries up organic solvent, after freezing in-80 DEG C, carry out drying;

(5) dried thalline is resuspended in again in the PBS of 10 times of volumes (containing DNase, RNase, N,O-Diacetylmuramidase, PMSF) 15 watts of ultrasonic broken bacterium 15 minutes are on ice placed in, 37 DEG C leave standstill 2 hours,, then add Tritonx-114 and make its final concentration be that 8% (v/v) is placed in 4 DEG C and spends the night;

(6) in the centrifugal 60min of 27000g, get upper phase and be placed in 37 DEG C of incubators to obvious layering;

(7) take off a layer liquid phase, the ice ethanol adding 95% of 9 times of volumes is placed in-20 DEG C of environment and spends the night to Precipitation;

(8) the centrifugal 5min of 5000rpm, collecting precipitation, adds the Proteinase K Solution of 2 times of volume 2mg/ml after lyophilize, in 60 DEG C of water-baths 2 hours, be then placed in 4 DEG C of dialysed overnight;

(9) raw sugar will be obtained after liquid in dialysis tubing again drying for next step separation and purification;

(10) raw sugar is separated (as Figure 1A, 1B) through the SDS-PAGE of 12% concentration, the adhesive tape of molecular weight between 26kD to 34kD scope is cut, gel is reclaimed grinding, 4 DEG C use deionized water to soak 12 hours, collected by centrifugation supernatant, use the small molecular weight impurities such as dialysis tubing dialysis removing SDS, lyophilize again, is the ManLAM lipolysaccharide of purifying;

(11) by the qualification of silver dye (Sylvain Pitarque et al.Tuberculosis, 2008,560-565) method, be summarized as follows:

11.1. be first 12%SDS-PAGE electrophoresis by capable for the sample of separation concentration;

11.2. PAGE glue is placed in the fixing oxidation of Periodic acid solution 22 DEG C 20 minutes containing 40% ethanol, 5% acetic acid, 7g/L, then uses deionized water rinsing 6 times, each 5 minutes;

11.3. with containing 4mL ammonium hydroxide, 6mL 0.1M sodium hydroxide, in 20% silver nitrate solution, room temperature silver dye 20 minutes;

11.4. by soak colour developing 10 minutes in concentration is 0.05g/L citric acid and 0.02% formaldehyde solution after clean distilled water rinses 6 times;

11.5. the acetic acid termination reaction of 1% is used, observations (as Fig. 1 C) on gel imaging instrument;

(12) by glycogen dyeing qualification: (Sergio A.Gradilone, Silvia E.Arranz, and Marcelo O.Cabada.Detection of Highly Glycosylated Proteins in Polyacrylamide Gels, Analytical Biochemistry, 1998,261:224-227)

12.1. SDS-PAGE glue is placed in 7.5% acetic acid (v/v) solution, 25 DEG C of fixing 10min;

12.2. glue is transferred in 1% Periodic acid (w/v) solution, is placed in 4 DEG C, 10min;

12.3. rinsed with deionized water SDS-PAGE glue 6 times are used under room temperature, each 5min;

12.4. glue is transferred in snow husband reagent, in 4 DEG C of effect 15min;

12.5. Sodium Metabisulfite (w/v) solution under room temperature, glue being placed in 0.5% washs 3 times, each 10min;

12.6. deionized water wash 6 times are again used, each 5min.

(13) the ManLAM lipolysaccharide of purifying is made immunoblotting assay (as Fig. 1 D):

13.1. by capable for the ManLAM lipolysaccharide of purifying SDS-PAGE electrophoresis;

13.2.200mA current stabilization goes to pvdf membrane in 2 hours, and TBST washs 3 times, each 5 minutes;

13.3. pvdf membrane to be soaked in 5%BSA 4 DEG C to close and spend the night, TBST washs 3 times, each 5 minutes;

13.4. use biotin labeled ConA (10 μ g/mL) 37 DEG C to act on 1 hour, TBST washs 3 times, each 5 minutes;

13.5. add HRP-streptavidin (1:2000) 37 DEG C effect 1 hour, TBST washs 3 times, each 5 minutes;

13.6. add chemical luminous substrate A, B liquid, detect.

(14) length scanning instrument is used to carry out length scanning (Fig. 2 A) the ManLAM lipolysaccharide of purifying;

The ManLAM lipolysaccharide of purifying is mixed with 1mg/mL, use Beckman-Ku Erte length scanning instrument is every nm run-down from 190nm to 900nm, and the absorbancy measured is drawn collection of illustrative plates, determine the wavelength that its maximum absorption band is corresponding, the determined wavelength that this wavelength is identified as follow-up HPLC.

(15) the ManLAM lipolysaccharide of purifying is used HPLC qualification (as Fig. 2 B);

With Agilent 1260 high performance liquid chromatograph, use sephadex column (ZORBAX GF-250,9.4 × 250mm, 4 μm) with moving phase (0.2M NaCl, 0.25% Septochol, 1mM EDTA, 0.02%NaN3,10mM Tris (pH 8.0)) dissolve ManLAM lipolysaccharide, adjustment flow velocity is 1ml/min, is the monitoring of 202nm place at determined wavelength.

2, the aptamer of specific combination ManLAM lipolysaccharide is screened

(1) random single chain DNA (ssDNA) library and primer is built

Synthetic one segment length is about the random ssDNA sequence of 68nt: 5 '-GCGGAATTCAACAGTCCGAGCC-N ₃₀-GGGTCAATGCGTCATA-3 ' (68nt), N represent any one base in A, C, T or G.Upstream primer P1 is: 5 '-GCG gAATTCtAATACGACTCACTATAGGGAACAGTCCGAGCC-3 ', setting-out part is the DNA restriction enzyme site of EcoRI; Downstream primer P2 is: 5 '-GCG gGATCCtATGACGCATTGACCC-3 ', setting-out part is the DNA restriction enzyme site of BamHI.

(2) pcr amplification in double-stranded DNA (dsDNA) and strand (ssDNA) library and preservation

SsDNA amplified library is first preserve behind dsDNA library before taking turns screening by each, and the dsDNA library that takes a morsel goes out the ssDNA library of next round screening as template amplification.Response procedures is: 95 DEG C of 5min, 95 DEG C of 36s, 60 DEG C of 36s, 72 DEG C of 84s, 25 circulations, 72 DEG C of 5min.PCR primer is detected by the agarose gel electrophoresis of 3%.

(3) the Fas lignand system evolution technology (systematic evolution of ligands byexponential enrichment, SELEX) by index concentration screens

The method forms special space conformation based on small segment ssDNA molecular energy by local base pairing, and combine by hydrogen bond, hydrophobic bond, Van der Waals force etc. and target molecule high-affinity.The basic procedure of SELEX technology is: be separated the single stranded oligonucleotide with target molecule high-affinity, symmetrical PCR increases dsDNA library in a large number, then asymmetric PCR obtains ssDNA library, screen for next round, so after the some circulations combining-be separated-increase-combine, the oligonucleotide aptamer had with target molecule high-affinity can be obtained.

Take turns to the 17th from first to take turns, screen ssDNA input amount used and all successively decrease successively from 1000pmol to 150pmol, use and be prepended to 95 DEG C of water-baths rapid ice bath 5min after 10 minutes; The ManLAM lipolysaccharide of the Mycobacterium tuberculosis of purification is dissolved in screening damping fluid (Binding buffer:pH 7.4,20mM TrisHCl, 137mM NaCl, 5mM KCl, 2mM CaCl ₂, 1mM MgCl ₂) in bag by 96 hole elisa plates, hatch 2 hours for 37 DEG C; Add the ssDNA library of 40pmol after using screening damping fluid repetitive scrubbing 3 times, 37 DEG C hatch 1h after, with the washing of screening elutriant (20mM pH 7.4TrisHCl, 137mM NaCl, 5mMKCl, 2mM CaCl ₂, 1mM MgCl ₂, use 0.22 micron membrane filter filtration sterilization) and 3 times; The autoclaved distilled water of 100 μ l (dd H is added in screen holes ₂o), 95 DEG C are heated 10 minutes; By ddH in hole ₂o is drawn in new EP pipe, after PCR reaction, PCR primer is added the sodium-acetate (NaAc) of 3 moles of often liter of concentration of 1/10 volume and then adds the dehydrated alcohol of 2 times of volumes and be placed in-70 DEG C of precipitates overnight, reclaiming finally by gel.Along with the carrying out of screening, reduce the amount of the purifying ManLAM lipolysaccharide of bag quilt gradually, from the 10 every holes of μ g to the 0.5 every hole of μ g, thus further increase the binding specificity of aptamer to target material, be more conducive to the screening of the aptamer of high-affinity.

The screening so taken turns through 17, obtains 17 and takes turns dsDNA library, and take turns biotin labeled ssDNA library with the method for asymmetric PCR each odd number that increases, amplification system is as follows:

(4) join with enzyme the avidity that oligonucleotide analysis (Enzyme-linked oligonucleotide assay, ELONA) method detects each odd number wheel ssDNA library and target substance (ManLAM lipolysaccharide):

4.1 wrap after the dilution of ManLAM lipolysaccharide use screening damping fluid by microwell plate, the 1 every hole of μ g, and 4 DEG C are spent the night, and then wrap by 2 hours at 37 DEG C;

4.2 use 1%PBS-BSA closed porosity plate, and 37 DEG C act on 1 hour;

4.3 wash microwell plate 3 times with PBST, 5min/ time;

4.4, by each biotin labeled ssDNA storehouse of taking turns, measure its concentration respectively, for subsequent use.(before hatching, first 95 DEG C of 5min, are then placed on 5min on ice immediately);

4.5 take turns biotin labeled adaptive word bank and are added in microwell plate by each by 30pmol/ hole, often take turns adaptive word bank and do 3 multiple holes, 37 DEG C of effects 1 hour;

4.6 wash microwell plate 3 times with PBST, 5min/ time;

4.7 add HRP mark streptavidin (1:2000 dilution), 100 μ l/ holes, 37 DEG C act on 45 minutes;

4.8 wash microwell plate 3 times with PBST, 5min/ time;

4.9 add tmb substrate, act on 1min to 3min at 37 DEG C, observe colour-change, then stop with the 2M vitriol oil;

4.10 use microplate reader to read OD ₄₅₀nm value;

As shown in Figure 3 along with the increase of screening wheel number, the bonding force of aptamer increases gradually, the 13rd ssDNA storehouse of taking turns and ManLAM lipolysaccharide avidity the highest.

(5) join with enzyme the avidity that oligonucleotide analysis (Enzyme-linked oligonucleotide assay, ELONA) method detects each aptamer and target substance (ManLAM lipolysaccharide) in the 13rd ssDNA storehouse of taking turns:

5.1 take turns ssDNA pool by the highest for avidity the 13rd increases as dsDNA pool builds up in pUC19 carrier, is transformed into DH5 α bacterium, coats penbritin (50 μMs) resistance solid medium, cultivate 12 hours;

5.2 random pickings, 40 mono-clonal bacterium colonies, extract plasmid, cut qualification obtain 36 positive monoclonal aptamer plasmids through enzyme, and to 36 mono-clonal order-checkings.Increase 36 biotin labeled ssDNA of mono-clonal respectively, detects the avidity of each single aptamer and target protein;

As shown in Figure 4,6,9,14,15, No. 25 all have higher bonding force with ManLAM lipolysaccharide, thus, by these 5 aptamers alternatively aptamer, and called after T6, T9, T14, T15, T25.

(6) dose-dependently that each candidate's aptamer is combined with target protein is detected

6.1 bags are by the ManLAM lipolysaccharide of various dose (100 μ g/mL, 30 μ g/mL, 10 μ g/mL, 3 μ g/mL) in 96 hole microwell plates, and 37 DEG C of bags were by 1 hour, and PBST washs micropore 3 times;

6.21%BSA (200 μ L/ hole) closed porosity 37 DEG C closes 1 hour, and PBST washs micropore 3 times;

6.3 5 kinds of biotin labeled ssDNA that asymmetric PCR is obtained, after carrying out concentration determination, often kind of ssDNA drops into 30pmol respectively, hatches 1 hour for 37 DEG C, and PBST washs micropore 3 times;

6.4 streptavidins (1:2000 dilution) adding HRP mark, 100 μ l/ holes, 37 DEG C of effects 45 minutes, PBST washing micropore 3 times;

6.5 add tmb substrate, act on 1min to 3min at 37 DEG C, observe colour-change.Then stop with the 2M vitriol oil;

6.6 use microplate reader to read OD ₄₅₀nm value.

As shown in Figure 5, T6, T9, T14, T15, T25 and ManLAM lipolysaccharide all have good dose-dependently.

(7) the Dissociation equilibrium constant K that each candidate's aptamer is combined with target protein is detected _d:

7.1 use the capable asymmetric PCR of biotin labeling P1 primer, and increase 5 biotin labeled ssDNA of mono-clonal respectively;

7.2 bags are by ManLAM lipolysaccharide 20 μ g/mL in 96 hole microwell plates, and 37 DEG C of bags were by 1 hour, and PBST washs micropore 3 times;

7.31%BSA (200 μ l/ hole) closed porosity 37 DEG C closes 1 hour, and PBST washs micropore 3 times;

7.4 5 kinds of biotin labeled ssDNA that asymmetric PCR is obtained, 7 different concns (1 μM, 0.5 μM, 0.25 μM, 0.125 μM, 0.065 μM, 0.0325 μM, 0 μM) are not dropped into after often kind of ssDNA carries out concentration determination, hatch 1 hour for 37 DEG C, PBST washs micropore 3 times;

7.5 streptavidins (1:2000 dilution) adding HRP mark, 100 μ l/ holes, 37 DEG C of effects 1 hour, PBST washing micropore 3 times;

7.6 add tmb substrate, act on 3min to 5min at 37 DEG C, observe colour-change.Then stop with the 2M vitriol oil;

7.7 use microplate reader to read OD ₄₅₀nm value, uses its K of Graphpad 5.0 software the Fitting Calculation _dvalue;

As shown in Figure 6, its bonding force K is recorded _dvalue is respectively: 998 ± 207nM, 668 ± 159nM, 815 ± 144nM, 736 ± 207nM, 685 ± 131nM.

(8) 5 kinds of different mono-clonal aptamers are to the comparison of the detection perform of clinical samples:

8.1. wrap by tuberculosis patient serum and Healthy Human Serum in 96 hole microwell plates, 37 DEG C of bags were by 1 hour, and PBST washs micropore 3 times;

8.21%BSA (200 μ l/ hole) closed porosity 37 DEG C closes 1 hour, and PBST washs micropore 3 times;

8.3 5 kinds of biotin labeled ssDNA that asymmetric PCR is obtained, after carrying out concentration determination, often kind of ssDNA drops into 30pmol respectively, hatches 1 hour for 37 DEG C, and PBST washs micropore 3 times;

8.4 streptavidins (1:2000 dilution) adding HRP mark, 100 μ l/ holes, 37 DEG C of effects 45 minutes, PBST washing micropore 3 times;

8.5 add tmb substrate, act on 1min to 3min at 37 DEG C, observe colour-change, then stop with the 2M vitriol oil;

8.6 use microplate reader to read OD ₄₅₀nm value.

As shown in Figure 7, according to its ratio to the detection absorbancy of tuberculosis patient serum and the absorbancy of Healthy Human Serum, aptamer all demonstrates good differentiation tuberculosis patient and the ability of Healthy People.Its primary sequence is as shown in SEQ ID No.1-5.

[embodiment 2] ELONA (aptamer) method detection specificity

1, wrap by Beijing genotype mycobacterium tuberculosis, M. smegmatics, BCG, mycobacterium avium, bacillus coli DH 5 alpha, streptococcus aureus, sex change bacillus, faecalis, Klebsiella Pneumoniae, staphylococcus epidermidis, Pseudomonas aeruginosa, suis, Neisseria, H37Rv mycobacterium tuberculosis (10 ⁶cfu), be coated in 96 orifice plates respectively, be placed in 37 DEG C of wet boxes and wrap by 1 hour, PBST washs micropore 3 times;

2,1%BSA (200 μ l/ hole) closed porosity 37 DEG C closes 1 hour, and PBST washs micropore 3 times;

Above-mentioned five kinds of aptamers of the biotin labeled ManLAM 3, asymmetric PCR obtained, after carrying out concentration determination, drop into 30pmol, hatch 1 hour for 37 DEG C, PBST washs micropore 3 times;

4, add the streptavidin (1:2000 dilution) of HRP mark, 100 μ l/ holes, 37 DEG C act on 45 minutes, and PBST washs micropore 3 times;

5, add tmb substrate, at 37 DEG C, act on 1min to 3min, observe colour-change.Then stop with the 2M vitriol oil;

6, microplate reader is used to read OD ₄₅₀nm value.

As shown in Figure 8, according to aptamer and the relative binding capacity of different bacterium of its absorbance reflection screening, result show needle to the aptamer of ManLAM and Mycobacterium tuberculosis Beijing genotype strain bonding force far above other bacterial strain (p<0.01).

[embodiment 3] aptamer ELONA (aptamer) method detects the sensitivity of ManLAM lipolysaccharide

1, wrap by the ManLAM lipolysaccharide of different concns (100 μ g/mL, 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL, 3 μ g/mL, 1.5 μ g/mL, 0.75 μ g/mL, 0.37 μ g/mL, 0.18 μ g/mL) in 96 hole microwell plates, 37 DEG C of bags were by 1 hour, and PBST washs micropore 3 times;

3, by biotin labeled five kinds of aptamers that asymmetric PCR obtains, after carrying out concentration determination, drop into 30pmol, hatch 1 hour for 37 DEG C, PBST washs micropore 3 times;

6, microplate reader is used to read OD ₄₅₀nm value.

As shown in Figure 9, bag is by the ManLAM lipolysaccharide of different concns, detect with biotin labeled five kinds of aptamers, after concentration is taken the logarithm with record OD value and carry out matching and obtain equation: y=0.3075x+0.4445, corresponding for PBS group OD value is brought into and namely draws its detection sensitivity, namely can reach 0.035 μ g/mL to the detection sensitivity of ManLAM lipide-carbohydrate antigen.

ManLAM lipolysaccharide in [embodiment 4] ELONA (aptamer) method detected activity pulmonary tuberculosis serum

1, biotin labeled upstream primer P1 amplifies the aptamer of biotin labeled five kinds of ManLAM;

2, by active tuberculosis patients serum 102 parts and Healthy Human Serum 68 parts, be coated in 96 orifice plates respectively, be placed in 37 DEG C of wet boxes and wrap by 1 hour;

3,1%BSA (200 μ L/ hole) closed porosity 37 DEG C closes 1 hour, and PBST washs micropore 3 times;

4, PBST wash 3 times afterwards every hole add the biotin labeled aptamer that same concentrations is 300nM, hatch 1 hour in 37 DEG C;

5, PBST washes the streptavidin that horseradish peroxidase (HRP) that 3 times every hole adds the 1:1000 dilution of 100 μ l afterwards marks, and hatches 45 minutes for 37 DEG C;

6, PBST adds TMB nitrite ion and develops the color after washing 3 times, hatches 5 ~ 10 minutes for 37 DEG C;

7, after adding the sulfuric acid termination reaction of 2M, microplate reader detects;

8, the OD recorded ₄₅₀nm value.

As shown in Figure 10, bag is by tuberculosis patient serum (102 parts) and Healthy Human Serum (68 parts), detect with biotin labeled aptamer subsequently, find that there is between two groups significant difference (p<0.001).Carry out Graphpad 5.0 software analysis according to the OD450nm value recorded and carry out ROC fitting of a curve, draw Cut off value, thus distinguish positive and negative, it is 92.45% that Graphpad 5.0 software analysis goes out sensitivity; Specific degree is 77.45%.

[embodiment 5] ELONA (aptamer) method detects ManLAM lipolysaccharide in extrapulmonary tuberculosis human serum

2, by extrapulmonary tuberculosis human serum 62 parts (15 routine lymphoid tuberculosis patient (6 routine Neck lymphatic tuberculosis, 7 routine axillary lymph tuberculosis, 2 routine inguinal lymph tuberculosis), 14 routine bone tuberculosis patient (2 routine tuberculosis of spine, 9 tuberculosis of spine, 3 routine tuberculosiss of knee joint, 1 routine ankle arthrosis tuberculosis), 25 routine body cavity tuberculosis (9 routine abdominal cavity tuberculosis, 11 routine thoracic cavity tuberculosis, 5 routine encephalocoele tuberculosis), 8 other position tuberculosis of example (2 routine tuberculous esophagitis, 1 routine tuberculosis of epididymis, 4 routine tuberculosis of intestine, 1 routine tuberculous polyserositis)) and Healthy Human Serum 34 parts, be coated in 96 orifice plates respectively, being placed in 37 DEG C of wet boxes wraps by 1 hour,

4, every hole adds the biotin labeled aptamer that same concentrations is 300nM, hatches 1 hour in 37 DEG C;

8, the OD recorded ₄₅₀nm value.

As shown in Figure 11, bag is by reactivity extrapulmonary tuberculosis human serum (62 parts) and Healthy Human Serum (34 parts), detect with biotin labeled five kinds of aptamers subsequently, find that there is between two groups significant difference (p<0.001).Carry out Graphpad 5.0 software analysis according to the OD450nm value recorded and carry out ROC fitting of a curve, draw Cut off value, thus distinguish positive and negative, it is 90.32% that Graphpad 5.0 software analysis goes out sensitivity, and specific degree is 94.12%.

[embodiment 6] ELONA (aptamer) method detects the ManLAM lipolysaccharide in pulmonary tuberculosis patient sputum

1, biotin labeled upstream primer P1 amplifies five kinds of aptamers of biotin labeled ManLAM;

2, sputum sample is added 3% sodium hydroxide of 1/2 sputum volume, use pasteur pipet fully to mix until viscosity reduces, then add the hydrochloric acid of isopyknic 0.8M;

3, sputum is coated in 96 orifice plates respectively, is placed in 37 DEG C of wet boxes and wraps by 1 hour, PBST washs micropore 3 times;

4,1%BSA (200 μ l/ hole) closed porosity 37 DEG C closes 1 hour, and PBST washs micropore 3 times;

5, every hole adds biotin labeled five kinds of ManLAM aptamers that same concentrations is 300nM, hatches 1 hour in 37 DEG C;

6, add the streptavidin (1:2000 dilution) of HRP mark, 100 μ l/ holes, 37 DEG C act on 45 minutes, and PBST washs micropore 3 times;

7, PBST adds TMB nitrite ion and develops the color after washing 3 times, hatches 5 ~ 10 minutes for 37 DEG C;

8, after adding the sulfuric acid termination reaction of 2M, microplate reader detects;

9, the OD recorded ₄₅₀nm value.

As shown in Figure 12, bag is by pulmonary tuberculosis patient sputum (16 parts) and Healthy People sputum (12 parts), detect with biotin labeled ManLAM aptamer subsequently, find that there is between two groups significant difference (p<0.001).

[embodiment 7] ELONA (aptamer) method compares with the detection of T-Spot method to 98 routine tuberculars

2,98 tuberculosis patient serum and 31 parts of Healthy Human Serums are coated in 96 microwell plates respectively, are placed in 37 DEG C of wet boxes and wrap by 1 hour;

8, the OD recorded ₄₅₀nm value, uses Graphpad 5.0 computed in software to go out cut-off value, judges that 98 parts of tuberculosis patient detected results are as negative or positive according to this.

9, use Shanghai Foxing Changzheng medical science Co., Ltd's T-Spot test kit to detect to the blood PBMC (peripheral blood mononuclear cell) with a tuberculosis patient sample, operate according to test kit specification sheets and judge that 98 parts of tuberculosis patient detected results are as negative or positive.

The results are shown in as shown in Table 2, use two kinds of methods to detect to 98 routine tuberculosis patients, it is 85.7% that result shows two kinds of method coincidence rates.

Table 2 ELONA (aptamer) method and T-Spot method comparing Diagnosis of Tuberculosis

sequence table

<110> Wuhan University

The nucleic acid aptamer of <120> mono-group-specific identification Beijing genotype mycobacterium tuberculosis strain antigen and application thereof

<130>

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 30

<212> DNA

<213> artificial sequence

<400> 1

tattcagtga gaggggatga gggggggaga 30

<210> 2

<211> 30

<212> DNA

<213> artificial sequence

<400> 2

ttctgatgga gagatggaga gtgagagaga 30

<210> 3

<211> 30

<212> DNA

<213> artificial sequence

<400> 3

cttcgtgttg tttgtgtgtg ttgtgtgatg 30

<210> 4

<211> 30

<212> DNA

<213> artificial sequence

<400> 4

ccttgtgttt tgtttgcctg tgattaggtt 30

<210> 5

<211> 30

<212> DNA

<213> artificial sequence

<400> 5

aggtggtggt tgtttgtggt gtgttttggt 30

<210> 6

<211> 68

<212> DNA

<213> artificial sequence

<220>

<221> misc_feature

<222> (23)..(52)

<223> n is a,c,g,or t

<400> 6

gcggaattca acagtccgag ccnnnnnnnn nnnnnnnnnn nnnnnnnnnn nngggtcaat 60

gcgtcata 68

<210> 7

<211> 42

<212> DNA

<213> artificial sequence

<400> 7

gcggaattct aatacgactc actataggga acagtccgag cc 42

<210> 8

<211> 25

<212> DNA

<213> artificial sequence

<400> 8

gcgggatcct atgacgcatt gaccc 25

Claims

1. the nucleic acid aptamer of the LAM of one group of specific recognition Mycobacterium tuberculosis Beijing genotype strain surface mannose-modified, its nucleotide sequence is as shown in SEQ ID No.1-5.

2. the nucleic acid aptamer of the LAM of Mycobacterium tuberculosis Beijing genotype strain surface according to claim 1 mannose-modified, is characterized in that its 5 ' end or 3 ' end can carry out vitamin H, FITC or digoxin chemically modified.

3. the application of nucleic acid aptamer in the LAM antigen detecting agent preparing Mycobacterium tuberculosis Beijing genotype strain surface mannose-modified and tuberculosis infection diagnostic reagent of the LAM of Mycobacterium tuberculosis Beijing genotype strain surface according to claim 1 mannose-modified.