CN1958809A - Method for detecting mycobacterium tuberculosis by using adaptor technique - Google Patents
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Abstract
This invention relates to a method for detecting Mycobacterium tuberculosis by utilizing gamete technique. This invention is aimed to solving the problems of low discovery rate and low curative rate of tuberculosis. The method comprises: utilizing gamete technique, performing bacteria culture or bioengineering method to obtain all kinds of targets of Mycobacterium tuberculosis, obtaining highly compatible specific gametes of the targets by gamete technique screening, and converting the gametes into report gametes for rapid and precise detection of corresponding targets. The method can further combine highly compatible gametes with pathogenetic factors to defunctionalize them, or develop new drugs by finding structural analogues of highly compatible gametes, thus can provides basis for diagnosis and clinical therapy of Mycobacterium tuberculosis.
Description
Technical field
The invention belongs to biomedical check field, relate to a kind of microorganism molecular Biological Detection method, particularly a kind of method of utilizing adaptive sub-technology for detection mycobacterium tuberculosis.
Background technology
Tuberculosis is the communicable disease of WHO emphasis control, is still the public health problem of serious harm people ' s health at present.According to pertinent data statistics, the whole world has 1/3 population to infect tubercule bacillus approximately, and existing tuberculosis patient is about 2,000 ten thousand, and annual neopathy 8,000,000～1,000 ten thousand people has 2,000,000 people to die from tuberculosis every year approximately, is the summation of other all transmissible disease death tolls.The tuberculosis epidemic situation of China is extremely severe, and existing tuberculosis infected students 500,000,000 accounts for 1/4 of the whole world, has every year 130000 people to die from tuberculosis, is other all kinds of transmissible diseases and parasitosis death toll summation 2 times.In addition, China still is the high resistance of tuberculosis country, and acquired total resistant rate is 46%, and acquired anti-multiple medicines rate reaches 17%.
The main problem that tuberculosis control at present exists is that patient's discovery rate is low, and curative ratio is low.Aspect diagnosis, existing inspection method all exists certain limitation, is difficult to reach diagnosis of tuberculosis fast and accurately.Aspect treatment, conventional chemotherapeutics is faced with great challenge, and existing medicine has been difficult to the anti-multiple medicines clinical strains that reply constantly occurs, and curative ratio is difficult to improve.Over year, the efficient antitubercular agent of Shang Wuxin comes out surplus in the of nearly 40.Therefore, strengthen the fundamental research of mycobacterium tuberculosis, seek quick, sensitive, easy, practical diagnosis of tuberculosis novel method, improve the recall rate of tuberculosis patient, and seek new methods of treatment or develop new antitubercular agent, be that present tuberculosis research needs the urgent problem that solves.
Discover, in the m tuberculosis infection process,, under the different modes of infection, can start and express different protein systems at different growing environments, growth phase.The early infection stage of mycobacterium tuberculosis can be secreted some small molecular weight antigens such as ESAT-6, CFP-10,10.4 etc., growth phase can discharge or secrete Ag85A, Ag85B, Ag85C, MPT53, MPT63, MPT64, MPT70, Mtb81 (88kd), MTC28, Mtb48, Rv2430c, MPT32,38kd, 19kd, and wherein some antigen is that toxic strain mycobacterium tuberculosis institute is peculiar.Theoretically, because secretion property antigen is to be produced and be discharged in the surrounding environment medium by the mycobacterium tuberculosis of living, therefore, the secretion antigen that detects above-mentioned multiple mycobacterium tuberculosis can significantly improve the positive rate of diagnosis of tuberculosis, also can determine mycobacterium tuberculosis especially viable bacteria existence and growing state thereof by detecting antigenic existence of secretion property and content.In addition, some membranin of mycobacterium tuberculosis plays an important role in the pathogenic course of tubercule bacillus, have and discover that Rv3872 (99aa) is the key factor in the ESAT-6-CFP-10 complex body movement system, knocking out Rv3872 can end the synthetic of complex body and secrete, therefore, above-mentioned albumen can be used as the screening target spot of new antitubercular agent, combines with its specificity and the molecule that suppresses its function might become new antitubercular agent.
The oligonucleotide aptamer technology is a kind of novel Protocols in Molecular Biology of development in recent years, be that (systematic evolution of ligands by exponentialenrichment, SELEX) screening obtains the phyletic evolution technology that adopts exponential enrichment part.The SELEX technology is a triage techniques of setting up the beginning of the nineties, be a kind of effective ways of studying nucleic acid construct and function, its ultimate principle is single stranded oligonucleotide library of external chemosynthesis, mix with the target material with it, form the mixture of target material nucleic acid, wash off not and target material bonded nucleic acid, separate and target material bonded nucleic acid molecule, with this nucleic acid molecule is that template is carried out pcr amplification, carries out the screening process of next round again.By multiple screening and amplification, some and the debond of target material or the DNA of low-affinity, middle avidity or RNA molecule are arranged by flush away with the target material, and be referred to as " adaptive son " promptly with the target material have the dna molecular of high-affinity or RNA molecule from very large with separating the hangar, and purity is carried out and is increased with the SELEX process, from pmol to nmol, that have even to μ mol, occupy the great majority (greater than 90%) in storehouse at last.This technology has big, the advantages such as the target molecule scope is wide, avidity is high, high specificity of storage capacity, be successfully applied to the screening of multiple target molecule, except that being used for nucleotide sequence, protein, amino acid, also can be used for dyestuff, medicine small molecules (as theophylline), somatomedin, peptide chain, steroid, carbohydrate, even can be used for complete cell, virus, spore and Protein virus etc.Adaptive son has high degree of specificity, by reverse SELEX technology, even can filter out its corresponding adaptive son under the situation of not knowing target character.Adaptive son makes disease controlled by occupying morbific target material epi-position, as the pharmacological agent of clinical disease, has manifested the potential application prospect.Existing research is by the SELEX technology, and the adaptive son that screens the respective target material is as antagonist, and the vascular endothelial growth factor when being used in tumor growth, thrombus generate the factor, some toxin proteins and somatomedin etc., to reach therapeutic purpose.Aspect microorganism detection, particularly to some unknown pathogenic bacterias or viral research, though do not know the epi-position of its internal structure, function and these materials, but with it as the target material, screen the adaptive son corresponding by the SELEX process with it, detect the target material, focus is explored in the research that has become this field.
Summary of the invention
The objective of the invention is at the patient's discovery rate that exists in the control of present tuberculosis lowly, problem such as curative ratio is low provides a kind of microorganism molecular biology method of inspection, particularly a kind of method of utilizing adaptive sub-technology for detection mycobacterium tuberculosis.
The present invention introduces a kind of new molecular biology method---adaptive sub-technology in the research field of mycobacterium tuberculosis.The phyletic evolution technology (SELEX technology) of the inventive method utilization index level enrichment part, various molecules with mycobacterium tuberculosis, comprise nucleotide sequence, protein, amino acid, the toxin factor, somatomedin, peptide chain, steroid, carbohydrate, or complete thalline is the target material, screening obtains the adaptive son of these target material molecules, for the diagnoses and treatment that further utilizes adaptive sub-technology to carry out mycobacterium tuberculosis provides foundation.
Described adaptive son is the single stranded oligonucleotide that screening obtains, it is from a kind of strand at random library, described strand at random library, can also can be RNA for DNA, feature is that two ends are fixed sequence program, be used to design primer, middle be stochastic sequence is used to provide and various target material bonded are abundant nucleic acid aglucon.
The present invention obtains the various target materials of above-mentioned mycobacterium tuberculosis by bacteriology cultivation or biological engineering method, utilize the screening process of SELEX technology by repeatedly, obtain the special adaptive son of high-affinity of above-mentioned target material, by being modified, adaptive son strengthens its stability then, the adaptive son that will obtain by fluorescein, vitamin H, radio isotope or colloid gold label method transfers the adaptive son of report to, be used for detecting corresponding target material the purpose of reach fast, accurately diagnosing from clinical blood, urine, tissue sample and culture supernatant; Or combine with virulence factor by the adaptive son of high-affinity that will obtain, make it lose the purpose that function reaches direct treatment; Or by the analog of searching with the adaptive son of these virulence factor high-affinity bonded, developing new drug is used for clinical treatment.
The inventive method comprises the following steps:
1. the target material that seek from mycobacterium tuberculosis, preparation can be used for the SELEX technology screening comprises:
(1) the mycobacterium tuberculosis secretory antigen ESAT-6 of high specificity, CFP-10,10.4, Ag85A, Ag85B, Ag85C, MPT53, MPT63, MPT64, MPT70, Mtb81 (88kd), MTC28, Mtb48, Rv2430c, MPT32,38kd and/or 19kd;
(2) in pathogenic course, play the molecule of important function, as virulence factor, the control various functional proteins of virulence factor excretory (membranin, enzyme etc.), nucleotide sequence, amino acid, peptide chain, steroid and/or carbohydrate;
(3) thalline;
2. utilize the SELEX technology screening to obtain the adaptive son of target material;
(1) makes up the library of single stranded oligonucleotide at random that is fit to, comprise single stranded DNA and/or single stranded RNA;
(2) optimize the pcr amplification condition;
(3) asymmetric PCR method or vitamin H streptavidin magnesphere legal system are equipped with single-stranded DNA banks;
(4) with nitrocellulose filter, affine resin or microwell plate be the target screening substances process of separating medium;
3. set up the method for adaptive sub-technology for detection mycobacterium tuberculosis;
(1) modification of the adaptive son of high-affinity is primarily aimed at the modification of the adaptive son of RNA;
(2) adopt fluorescein, vitamin H, radio isotope or colloid gold label method will obtain adaptive son and transfer the adaptive son of report to;
(3) determine sample to be checked and negative control sample
Adopt the negative contrast of normal human serum, the sample that relates to derives from and needs examination, suspicious or at the human or animal's of morbid state body fluid or medium, comprise sputum, serum, urine or cerebrospinal fluid, or inoculum.
The present invention can further pass through to analyze the structure of adaptive son, and modifies increase stability, to resist intravital acid or alkali environment; With mycobacterium tuberculosis inoculation experiments animal, do not add adaptive son group as positive control, experimental group can add the adaptive son of different concns simultaneously, observes adaptive son and acts in the m tuberculosis infection process; Seek the analog of above-mentioned adaptive son, carry out external bacteriostatic experiment or experimentation on animals, the antitubercular agent that the screening exploitation is new.
The inventive method can significantly improve the tuberculosis patient positive rate, can be used for quick, the easy diagnosis lungy of people and animal, can be for the assessment of laboratory diagnosis lungy and antituberculosis therapy provide favourable foundation, and can be used for the adaptive son treatment and the new drug development of mycobacterium tuberculosis.The inventive method will be opened new approach for treatment lungy, also provide new research thinking for the exploitation antitubercular agent.
Description of drawings
Fig. 1 14 takes turns ssDNA that screening obtains and the affine curve between the MPT64.
Fig. 2 is 2% agarose gel electrophoretogram in synthetic ssDNA library.
Fig. 3 is 2% agarose gel electrophoretogram of the adaptive sublibrary of MPT64 antigen high-affinity ssDNA.
Fig. 4 is the secondary structure collection of illustrative plates of the adaptive sub-K11 of MPT64 albumen high-affinity.
Embodiment:
Embodiment 1
Can with the preparation method of mycobacterium tuberculosis secretory antigen ESAT-6, CFP-10, MPT53, the adaptive son of the protein bound high-affinity DNA of MPT63, MPT6, be example with MPT64, carry out according to the following step:
1. prepare and purifying MPT64 albumen.
According to a conventional method the proteic gene order of MPT64 is cloned in the carrier, transfection is to E.coli, induces, expression, purifying.The albumen of purifying is carried out SDS-PAGE analysis, quantitative, obtain antigenic pure product as screening target material.
Related carrier adopts pET21a, expresses back C end and contains pProEX-HTa, expresses back N end and contains 6 * His.
Related carrier is known in the art technology (commercial).
The technology used in the present invention measure can be avoided causing described proteic expression level low because of reasons such as gene constructed splicings in the biological engineering method, or the expressed proteins renaturation can not look like to be consistent after folding or approaching with tubercule bacillus excretory original protein structure, and directly the influence detection is with the affinity of adaptive son.
2. make up random single chain DNA (ssDNA) library and primer.
The length that makes up sequence 1 is single stranded DNA (ssDNA) library of 78 bases: 5 '-GGGAGCTCAGAATAAACGCTCAA-N35-TTCGACATGAGGCCCGGATC-3 ', wherein N represents base A, G, C, among the T any one, the capacity in this library is about 1014～1015, makes up upstream primer: 5 '-GGGAGCTCAGAATAAACGCTCAA-3 ', structure downstream primer 5 '-TTCGACATGAGGCCCGGATC-3 '.Random single-stranded DNA banks and primer are synthetic by Synbiotics AB.
3. single-stranded DNA banks double chain DNA library synthesis.
With single-stranded DNA banks 0.1 μ g, upstream primer 10pmol, downstream primer 10pmol, 15mmol/L MgCl
2, 0.2mmol/LdNTP, the archaeal dna polymerase of 1 * dna polymerase reaction damping fluid and 1 activity unit adds distilled water, making cumulative volume is 20 μ l; Put into the PCR instrument then, carry out 94 ℃ of reactions pre-sex change in 5 minutes earlier, press following condition circulation 18 times then: 30 seconds, 72 ℃ reactions of 30 seconds, 65 ℃ reactions of 94 ℃ of reactions 30 seconds, last 72 ℃ were reacted 5 minutes, and obtained the pcr amplification product in double-stranded DNA library.
4, the pcr amplification product in the step 3 is reclaimed with phenol chloroform method purifying.
5, the asymmetric PCR legal system is equipped with single-stranded DNA banks.
The DNA that reclaims in the step 4 is template 0.1 μ g, upstream primer 0.1pmol, downstream primer 10pmol, 7.5mmol/L MgCl
2, 0.2mmol/LdNTP, the archaeal dna polymerase of 1 * dna polymerase reaction damping fluid and 1 activity unit adds distilled water, making cumulative volume is 20 μ l; Put into the PCR instrument then, carry out 94 ℃ of reactions pre-sex change in 5 minutes earlier, press following condition circulation 40 times then: 30 seconds, 72 ℃ reactions of 30 seconds, 65 ℃ reactions of 94 ℃ of reactions 30 seconds, last 72 ℃ were reacted 5 minutes, and obtained the pcr amplification product of single-stranded DNA banks.
6, the pcr amplification product in the step 5 is reclaimed with phenol chloroform method purifying, obtain the single-stranded DNA banks of purifying.
7, with 10 μ g mycobacterium tuberculosis MPT 64 albumen with the carbonic acid buffer bag of pH value 9.6 by on elisa plate, the blank hole is established in 37 ℃ of effects 3 hours simultaneously.Sample aperture and control wells were sealed 2 hours with 3% BSA.The single-stranded DNA banks 1ng of purifying in the step 6 is acted on 40 minutes for 37 ℃ with the blank hole earlier in SELEX binding buffer liquid, anti-sieve is removed and BSA bonded single stranded DNA, transfer to MPT64 albumen bag then and ℃ combined 40 minutes by hole and MPT64 protein 37, with SELEX dcq buffer liquid washing 6 times, add the SELEX elutriant again in 80 ℃ of effects 10 minutes, following and the MPT64 bonded ssDNA of wash-out through phenol-chloroform extracting, ethanol sedimentation, obtains the single stranded DNA of purifying.
8, repeating step 3～710 is taken turns, 10 take turns after, adaptive son and proteic affinity reach capacity, the template that is used for expanding double-stranded DNA in the step 3 is replaced with the every single stranded DNA that elutes in the screening of taking turns of step 7, wrap the proteic concentration of MPT64 of quilt simultaneously in the step 7 and be used for bonded single stranded DNA concentration and constantly reduce along with the increase of screening wheel number, when the 10th takes turns, the concentration of coating protein reaches every hole 0.05 μ g, accordingly the concentration in conjunction with single stranded DNA is every hole 0.02ng, obtain can with the single-strand DNA aptamer storehouse (see figure 3) of MPT64 albumen high-affinity.
Above-mentioned screening is along with the increase of screening wheel number, and the concentration of the concentration of corresponding reduction coating protein and bonded single stranded DNA helps sifting out of the adaptive son of albumen high-affinity, until occupy the overwhelming majority in the adaptive word bank of albumen affinity DNA.
9, increased according to step 3 in the high-affinity single-strand DNA aptamer storehouse that obtains in the step 8, obtain the pcr amplification product in double-stranded DNA library.
10, the purifying of pcr amplification product.
With 2% sepharose that contains concentration 0.5 μ g/ml ethidium bromide, the pcr amplification product in the double-stranded DNA library in the step 9 is carried out electrophoresis, behind the electrophoresis sepharose is seated on the fluoroscopic examination plate, the pcr amplification product that will be the double-stranded DNA library of orange band downcuts, the DNA purifying that provides with TaKaRa company reclaims the test kit purifying, obtains the double-stranded DNA of purifying.
11, the double-stranded DNA that step 10 is obtained, the pMD18-T Simple Vector test kit that provides with TaKaRa company is connected on the pMD18-T carrier, is transformed into bacillus coli DH 5 alpha, ammonia benzyl resistance screening, the single growth bacterium colony of picking checks order.
12, with the adaptive son biotin labeling of the pairing DNA of each single growth bacterium colony, amount is 0.1 μ g, with the MPT64 albumen of every hole 10 μ g bag quilt in SELEX binding buffer liquid 37 ℃ combine 40 minutes, with SELEX dcq buffer liquid washing 6 times, the horseradish peroxidase mark Streptavidin that adds 1: 1000,37 ℃, act on 30 minutes, with PBST damping fluid washing 4 times, flush away is not gone up DNA vitamin H bonded enzyme mark streptavidin with MPT64, add the effect in 20 minutes of tetramethyl benzidine color development at room temperature then, add the reaction of 2M vitriol oil color development stopping, 450nm enzyme connection instrument is measured the OD value, choose the highest adaptive son of DNA of OD value, this adaptive son be can with the adaptive son of the protein bound DNA of MPT64, should be adaptive the son order-checking obtain sequence 2, and carry out the secondary structure collection of illustrative plates (see figure 4) that structural analysis obtains this adaptive son.
The present invention tests used SELEX binding buffer liquid: 20mmol/L Hepes pH 7.35,120mmol/LNaCl, 5mmol/L KCl, 1mmol/L CaCl
2, 1mmol/L MgCl
2SELEX dcq buffer liquid is: 0.05% Tween20+SELEX binding buffer liquid; The SELEX elution buffer is: 20mmol/LTris-HCl, and the 4mol/L guanidinium isothiocyanate, 1mmol/L DTT, pH 8.3; PBST dcq buffer liquid is: 0.05%Tween20,13.7mmol/L NaCl, 0.27mmol/L KCl, 0.43mmol/L Na
2HPO
4.7H
2O, 0.14mmol/L KH
2PO
4
The present invention tests used phenol chloroform extract: with the phenol of 25ml, and the chloroform of 24ml, the primary isoamyl alcohol of 1ml mixes.
To modify by the adaptive son that said process obtains and strengthen its stability, carry out mark with fluorescein, vitamin H, radio isotope or Radioactive colloidal gold then and transfer the adaptive son of report to, just can adopt euzymelinked immunosorbent assay (ELISA) from clinical blood, urine, tissue sample and culture supernatant, to detect corresponding target material, thereby reach the purpose that detects mycobacterium tuberculosis.
Replace antibody with the antigenic adaptive filial generation that the present invention obtains, carry out the detection of mycobacterium tuberculosis, have many superiority: because adaptive son and antigenic avidity are better than the avidity between the antigen-antibody, thereby adaptive son combines with strong specificity between tuberculosis antigen, almost can avoid non-specific binding fully, even and the albumen of less immunogenic can obtain the adaptive son of high specific too; The screening of adaptive son is shorter than the antibody screening time, preparation easily, good stability, and easy mark; Compare with antibody, the adaptive sub-molecule of tuberculosis antigen is less, not only can detect the mycobacterium tuberculosis surface antigen, also than being easier to enter the inner internal antigens that detects of mycobacterium tuberculosis thalline.Therefore, utilize the special adaptive son of SELEX technology screening mycobacterium tuberculosis, will provide a kind of new research thinking for diagnosis lungy.
SEQUENCE LISTING sequence table
＜110〉Shanghai Pulmonary Hospital
＜120〉a kind of method of utilizing adaptive sub-technology for detection mycobacterium tuberculosis
<130>11
<160>1
<170>PatentIn?version?3.1
<210>1
<211>78
<212>DNA
＜213〉mycobacterium tuberculosis
<400>1
gggagctcag?aataaacgct?caatgggagc?tgatgtcgca?tgggttttga?tcacatgatt 60
cgacatgagg?cccggatc 78
Claims (7)
1, a kind of method of utilizing adaptive sub-technology for detection mycobacterium tuberculosis is characterized in that the various molecules with mycobacterium tuberculosis are target molecule, utilizes adaptive sub-technology screening to obtain the adaptive son of described target molecule, detects mycobacterium tuberculosis, comprises the steps:
(1) the target material that from mycobacterium tuberculosis, seek, preparation can be used for adaptive sub-technology screening;
(2) utilize adaptive sub-technology screening to obtain the adaptive son of target material;
1) makes up single stranded oligonucleotide library at random;
2) optimize the pcr amplification condition;
3) asymmetric PCR method or vitamin H streptavidin magnesphere legal system are equipped with single-stranded DNA banks;
4) with nitrocellulose filter, affine resin or microwell plate be the target screening substances process of separating medium;
(3) set up the method for adaptive sub-technology for detection mycobacterium tuberculosis;
1) modification of the adaptive son of high-affinity;
2) adopt fluorescein, vitamin H, radio isotope or colloid gold label method will obtain adaptive son and transfer the adaptive son of report to;
3) determine sample to be checked and negative control sample.
2, by the described method of utilizing adaptive sub-technology for detection mycobacterium tuberculosis of claim 1, the mycobacterium tuberculosis target material that it is characterized in that step (1) is the mycobacterium tuberculosis secretory antigen of high specificity or molecule or the thalline that plays important function in pathogenic course.
3, by claim 1 or the 2 described methods of utilizing adaptive sub-technology for detection mycobacterium tuberculosis, the mycobacterium tuberculosis target material that it is characterized in that step (1), wherein said secretion antigen is ESAT-6, CFP-10,10.4, Ag85A, Ag85B, Ag85C, MPT53, MPT63, MPT64, MPT70, Mtb81,88kd, MTC28, Mtb48, Rv2430c, MPT32,38kd and/or 19kd; The described molecule that plays important function in pathogenic course is selected from virulence factor, control virulence factor excretory functional protein, nucleotide sequence, amino acid, peptide chain, steroid and/or carbohydrate.
4, by the described method of utilizing adaptive sub-technology for detection mycobacterium tuberculosis of claim 1, it is characterized in that described adaptive son is the single stranded oligonucleotide that screening obtains, it is from a kind of strand at random library.
5, by the described method of utilizing adaptive sub-technology for detection mycobacterium tuberculosis of claim 4, it is characterized in that described strand at random library, be that two ends are that fixed sequence program is used to design primer, middle be stochastic sequence is used to provide and various target material bonded are abundant nucleic acid aglucon.
6, by the described method of utilizing adaptive sub-technology for detection mycobacterium tuberculosis of claim 1, it is characterized in that the library of single stranded oligonucleotide at random of described step (2), comprise single stranded DNA and/or single stranded RNA.
7, by the described method of utilizing adaptive sub-technology for detection mycobacterium tuberculosis of claim 1, the sample to be checked that it is characterized in that described step (3) is to need examination, suspicious or at the human or animal's of morbid state body fluid or medium, comprise sputum, serum, urine or cerebrospinal fluid, or inoculum; Described negative control sample adopts normal human serum.
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| CN102884192A (en) * | 2009-11-23 | 2013-01-16 | 国家健康与医学研究院 | Aptamers directed against the matrix protein-1 of type a influenza viruses and uses thereof |
| CN102010865B (en) * | 2010-09-29 | 2012-08-15 | 江南大学 | Nucleic acid aptamer capable of specifically recognizing Listeria monocytogenes, screening method and application thereof |
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| CN102367476A (en) * | 2011-10-14 | 2012-03-07 | 太湖瑞晶生物科技有限公司 | Detection method for slow virus infected cell |
| CN104946655A (en) * | 2015-07-01 | 2015-09-30 | 上海市肺科医院 | DNA aptamer of mycobacterium tuberculosis standard strain H37Rv and preparation method thereof |
| CN106047882A (en) * | 2016-06-01 | 2016-10-26 | 湖南大学 | Aptamer group in specific binding with mycobacterium tuberculosis and application of aptamer group |
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Application publication date: 20070509 |