CN101643499A - Binding peptide of mycobacterium tuberculosis and application thereof - Google Patents

Binding peptide of mycobacterium tuberculosis and application thereof Download PDF

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Publication number
CN101643499A
CN101643499A CN200910195820A CN200910195820A CN101643499A CN 101643499 A CN101643499 A CN 101643499A CN 200910195820 A CN200910195820 A CN 200910195820A CN 200910195820 A CN200910195820 A CN 200910195820A CN 101643499 A CN101643499 A CN 101643499A
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China
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binding peptide
mycobacterium tuberculosis
phage
tuberculosis
peptide
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杨华
胡忠义
张丽婷
崔振玲
王洁
陆俊梅
杨焕森
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Shanghai Pulmonary Hospital
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Shanghai Pulmonary Hospital
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Abstract

The invention belongs to the technical field of medicine, in particular to a binding peptide of mycobacterium tuberculosis and an application thereof. The amino acid sequence of the binding peptide isX1HX2GTX3H, wherein H represents histidine, G represents glycine, T represents threonine; X1-X3 represents any amino acid, optimal X1 is W or V, X2 is T, P or S, X3 is W or P, wherein W represents tryptophan, V represents valine, P represents praline and S represents serine. Experiments show that the specific binding of the binding peptide of mycobacterium tuberculosis to mycobacterium tuberculosis can be realized so that the binding peptide can be used to prepare the detection reagent of tuberculosis and the binding peptide is characterized by fastness, sensitiveness, simpleness and safety.

Description

A kind of binding peptide of mycobacterium tuberculosis and application thereof
Technical field
The invention belongs to medical technical field, be specifically related to a kind of binding peptide of mycobacterium tuberculosis, and this binding peptide is as the application of tuberculosis detection reagent.
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.
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.Therefore, strengthening the fundamental research of mycobacterium tuberculosis, seek quick, sensitive, easy, practical diagnosis of tuberculosis novel method, improve the recall rate of tuberculosis patient, is that present tuberculosis research needs the urgent problem that solves.
Phage display random peptide library technology is the method that allogenic polypeptide is showed in the phage particle surface, be a kind of high flux screening system of efficient molecular evolution research, in a plurality of fields such as immunology, cytobiology and drug development, obtained using very widely.The random peptide library technology can filter out the little peptide of high reactivity easy, effectively and reach and histocyte specificity bonded polypeptide by the affine screening enrichment of specificity respective ligand, need not predict its molecular structure information.Report is arranged with phage display random peptide library technology, separate having obtained and the efficient bonded small peptide of erythropoietin receptor, and confirm to have active preferably by a series of experiment in vitro.At the specific target molecule of tumour cell, and the random peptide library technology has also demonstrated special advantages in the screening process of the corresponding acceptor of cytokine etc.
The existing report of the application of phage display random peptide library technology aspect tuberculosis, have the scholar to utilize this technology screening to obtain the binding peptide of mycobacterium tuberculosis SigH (sigma factor) and Hsp16.3 (heat shock protein 16.3), and experiment in vitro all demonstrate active preferably.But do not appear in the newspapers as yet in the present home and abroad of research of using phage display random peptide library technology screening mycobacterium tuberculosis thalline binding peptide Chinese and English database retrievals such as (general) PubMed according to dimension.
Summary of the invention
The object of the present invention is to provide a kind of binding peptide of mycobacterium tuberculosis, also disclose of the application of this small peptide as the tuberculosis detection reagent.
A kind of binding peptide of mycobacterium tuberculosis, its aminoacid sequence general formula is: X 1HX 2GTX 3H, wherein H represents Histidine, and G represents glycine, and T represents Threonine, X 1～X 3Represent arbitrary amino acid.
The binding peptide of above-mentioned mycobacterium tuberculosis, preferred back X 1Be W or V, X 2Be T, P or S, X 3Be W or P, W representative color propylhomoserin wherein, V represents Xie Ansuan, and P represents proline(Pro), and S represents Serine.
Experiment shows, above-mentioned mycobacterium tuberculosis binding peptide can with mycobacterium tuberculosis generation specific combination, therefore, can be used to prepare the tuberculosis detection reagent, have quick, sensitive, easy, safe characteristics.
The present inventor is target molecule with the mycobacterium tuberculosis, screens bonded small peptide with it from the linear at random seven peptide storehouses (NewEngland Biolabs company) of phage display.Its aminoacid sequence of the small peptide that is screened is X 1HX 2GTX 3H.
Small peptide of the present invention can be by the preparation of methods such as routine techniques in the technology of the present invention field such as chemosynthesis.
Description of drawings
Fig. 1: the ELISA detected result of single phage.
Fig. 2: the affinity detected result of binding peptide and mycobacterium tuberculosis.
Fig. 3: the fluoroscopic examination result of binding peptide.
Embodiment
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1: the screening of mycobacterium tuberculosis binding peptide and sequential analysis
Experiment material
1. phage random peptide library test kit
The linear 7 peptide storehouse (Ph.D.-7 that phage surface capsid protein III shows TMPhage Display Peptide Library Kit) test kit is available from U.S. New England Biolabs company.The titre in peptide storehouse is 2 * 10 11Pfu/ml.Recipient bacterium E.coliER2738 genotype is F ' lacIq Δ (lacZ) M15proA+B+zzf::Tn 10 (TetR)/fhuA2 supE thi Δ (lac-proAB) Δs (hsdMS-mcrB) 5 (rk-m k-McrBC-).The full-automatic sequencing primer of DNA (96IIIsequencing primer): 5 '-CCCTCATAGTTAGCGTA ACG-3 '.
2. mycobacterium type strain
Mycobacterium tuberculosis H 37R V(ATCC95054) and M. smegmatics (ATCC19420), be Shanghai Pulmonary Hospital's strain storehouse and preserve bacterial strain, available from national DSMZ.
3. chemical reagent
BSA is available from U.S. Sigma company; The anti-M13 phage monoclonal antibody of HRP mark is available from Sweden Pharmacia company; Cyanogen bromide-activated sepharose (Sweden Amersharm company); Yeast extract, Tryptones, Tween-20, EDTA and PEG-8000 are available from traditional Chinese medicines group.
4. the preparation of related reagent
The LB substratum: yeast extract 5g, Tryptones 10g and NaCl 5g are dissolved in distilled water and are settled to 1L, autoclaving;
Item layer agarose: LB substratum+1g/L MgCl 26H 2The O+7g/L agarose, autoclaving;
Bottom LB flat board: LB substratum+15g/L agar powder, autoclaving is paved plate;
PEG/NaCl：20％(w/v)PEG-8000，2.5mol/L?NaCl。Autoclaving, room temperature storage.
TBS damping fluid: 50mmol/L Tris-HCl (pH 7.5), 150mmol/L NaCl.Autoclaving, room temperature storage.
Sodium iodide damping fluid: 10mmol/L Tris-HCl (pH 8.0), 1mmol/L EDTA, 4mol/L NaI.The room temperature lucifuge is stored.
5. key instrument
The desk-top high-speed refrigerated centrifuge of ROTINA38 (German Hettich company); Thermo MK3 microplate reader (U.S. THERMO company); HZQ-C airbath vibrator (east, Harbin connection electronic technology development corporation, Ltd.); Full-automatic high-pressure Sterilizers (Japanese Hirayama company); Full automatic gel imaging analysis instrument (Britain Syngene company); Ultrapure water separation system (Britain USF Elga company);
1.1 bacterial strain preparation
With mycobacterium tuberculosis type strain H 37R VTransferred species is to liquid nutrient medium (the Middlebrook 7H9 liquid nutrient medium that contains 10%OADC), and 37 ℃ are cultured to logarithmic phase, 80 ℃ of water-baths, deactivation 30min, centrifugal 10 minutes precipitations of 5000 * g thalline, twice of physiological saline washing, adjust turbidity to 5mg/L behind the mill bacterium, be used for wrapper sheet.
To the solid Russell medium, 37 ℃ are cultured to logarithmic phase with M. smegmatics type strain transferred species, and scrape and get slant culture, 80 ℃ of water-baths, deactivation 30min, physiological saline washing twice is adjusted turbidity to 5mg/L behind the mill bacterium, is used for wash-out.
1.2 seven peptide storehouses are screened
A. wrap quilt: 2mg/L H 37R VBacterium liquid bag is by 96 hole enzyme plates, 100 μ l/ holes, and 4 ℃ of bags are spent the night;
B. sealing: the coating buffer in the mobile hole, wrap by 300 μ l/ holes, hole 4 ℃ of sealing 2h then with 5mg/ml BSA sealing;
C. washing: mobile confining liquid, wash 6 times with TBST (0.1%Tween-20);
D. combination: dilute former generation phage library with TBST, every hole adds 100 μ l, and room temperature is shaken gently and hatched 60min;
E. clean: move unconjugated phage peptide library,, wash 10 times with TBST according to above-mentioned purging method;
F. M. smegmatics wash-out: act on 100 μ l M. smegmatics bacterium liquid (2mg/L), room temperature jog 60min draws supernatant, obtains the phage of shame dirt wash-out.
G. non-specific wash-out: with 100 μ l/ hole elution buffer (1mg/ml BSA, 0.2mol/L Glycine-HCl, pH 2.2) room temperature jog 10～15min wash-outs.
H. neutralization: the sucking-off elutriant, add and contain in the centrifuge tube of 15 μ l Tris-HCl (1mol/L, pH 9.1) neutralization buffer, make elutriant closely neutral;
I. the amplification of titer determination and phage: take out the 10 μ l neutralizers that previous step obtains, press 10: 1～10 with the LB substratum 4: 1 volume ratio is diluted neutralizer, carries out the mensuration of phage titre, and repeating step a-i carries out the next round screening behind remaining amplification, the purifying.
Annotate: for the specificity that obtains high-affinity in conjunction with phage, in next round, increase the concentration (can be increased to 0.5%) of Tween-20 among the washings TBST from 0.1%.
1.3 amplification of phage and purifying
The single bacterium colony of picking host bacterium ER2738 is inoculated in the LB substratum 37 ℃ of high vibration overnight incubation.
The bacterium that spends the night was inoculated in the 20ml LB substratum by 1: 100, added phage to be amplified, and 4.5h is cultivated in 37 ℃ of joltings;
Bacterium liquid is transferred in the 50ml centrifuge tube 4 ℃, the centrifugal 10min of 10000r/m; Supernatant is transferred in another centrifuge tube, and recentrifuge changes 80% of supernatant in another centrifuge tube over to, adds the PEG/NaCl of 1/6 volume, mixing, and 4 ℃ of precipitations are spent the night.4 ℃ then, the centrifugal 15min of 10000r/m abandon supernatant;
Precipitation is resuspended among the 1ml TBS, moves to then in the 1.5ml centrifuge tube, and 4 ℃, the centrifugal 5min of 10000r/m make the residual cells precipitation.
Supernatant moves in another 1.5ml centrifuge tube, adds the PEG/NaCl of 1/6 volume again, precipitates 1h in 4 ℃ behind the mixing.4 ℃ then, the centrifugal 10min of 10000r/m;
Precipitation is resuspended in and contains 0.02%NaN 3100 μ l TBS in, 4 ℃, the centrifugal lmin of 10000r/m.Get supernatant, 4 ℃ of storages are standby.
1.4 the mensuration of phage titre
The single bacterium colony of picking host bacterium ER2738 is inoculated in the LB substratum, and 37 ℃ are acutely shaken to logarithmic phase (OD600 ≈ 0.5);
37 ℃ of pre-temperature LB flat boards;
Top-agar melts the back branch and installs to sterile test tube in microwave oven, every pipe 3ml is incubated in 50 ℃ of water-baths;
With the LB substratum phage of wash-out is diluted by 10 times of gradient series;
The phage diluent of getting the different gradients of 10 μ l adds in the ER2738 bacterium liquid of 200 μ l logarithmic phases vortex mixing, incubated at room 5min respectively;
Behind rapid vortex in the above-mentioned mixed solution adding 3ml top-agar, bed board on the LB of 37 ℃ of preheatings flat board after the placement cooling, is inverted overnight incubation for 37 ℃ in the incubator immediately;
Calculate the plaque number: in flat board, select 100 plaque left and right sides persons to count, multiply by the number that extension rate promptly gets the contained phage of per 10 μ l stostes, represent with pfu/ μ l or pfu/ml usually.
The phage of eluriating generally speaking, is with 10 1-10 4Doubly dilution, the phage after the amplification is with 10 8-10 11Doubly dilution.
1.5 the preparation of phage single-chain DNA, sequencing
The ER2738 bacterium that spends the night was inoculated in the LB substratum by 1: 100, and the 1ml/ pipe is sub-packed in the culture test tube, and the picking plaque adds wherein, and 4.5-5h are cultivated in 37 ℃ of joltings;
With the phage liquid after the 500 μ l amplification, the centrifugal 10min of 10000r/m gets the supernatant repeated centrifugation once;
Supernatant moves in another centrifuge tube, adds 200 μ lPEG/NaCl, mixing, and room temperature is placed 10min;
The centrifugal 10min of 10000r/m abandons supernatant, and is of short duration more centrifugal, exhausts supernatant;
With the abundant resuspended precipitation of 100 μ l iodide damping fluids, add 250 μ l ethanol, incubated at room temperature 10min.4 ℃, the centrifugal 10min of 10000r/m abandon supernatant, with 70% washing with alcohol precipitation, with 30 μ l TE dissolution precipitations, send associated companies to check order and are translated as aminoacid sequence.(with oppositely-96 far-end primer order-checkings of M13)
1.6 phage clone combines active ELISA and detects with bacterial strain
Bag quilt: H 37R VBacterium liquid (2mg/L) wraps respectively by 96 hole enzyme plates with M. smegmatics bacterium liquid (2mg/L), and other establishes the blank that only adds coating buffer, 100 μ l/ holes, and 4 ℃ of bags are spent the night;
Sealing: the coating buffer in the mobile hole, wrap by hole and 300 μ l/ holes, blank hole 4 ℃ of sealing 2h then with 5mg/ml BSA sealing;
Washing: mobile confining liquid, wash 6 times with TBST (0.1%Tween-20);
In conjunction with: dilute each single phage and the 1st with TBST and take turns that phage (negative control) to titre is 10 after the elutriation 8Pfu/ μ l, 100 μ/hole is added to 2 kinds of bags respectively by in hole and the blank hole, 37 ℃ of 1h;
Washing: move in conjunction with liquid, TBST washes plate 6 times;
Enzyme labelled antibody combination: in each reacting hole, add the anti-M13 phage antibody of the HRP mark of dilution in 1: 5000,100 μ l/ holes, 37 ℃ of lh;
Washing: mobile response liquid, TBST are washed plate 6 times;
Colour developing: add the tmb substrate colour developing, 2mol/L H 2SO 4Termination reaction;
Reading: spectrophotometer is read OD 450The results are shown in Figure 1.
The affinity of embodiment 2 binding peptides and mycobacterium tuberculosis detects
Experiment material
Bacterium liquid sample: mycobacterium tuberculosis H37RV (ATCC95054) preserves bacterial strain for Shanghai Pulmonary Hospital's strain storehouse.
2.1 small peptide is synthetic: according to the ELISA detected result of single phage, the small peptide (called after HP8) of selecting the single preferably phage H8 of detected result to show, it is external synthetic to entrust associated companies to carry out, simultaneously flag F ITC fluorescence.
2.2 small peptide dissolving: get a part of small peptide dry powder, accurately after the weighing, be dissolved as the solution of 10mg/ml with ultrapure water, 4 ℃ keep in Dark Place.
2.3 fluoroscopic examination: with physiological saline washing back H 37R VGet 10 μ l and drip on slide, other gets 10 μ l physiological saline and drips on slide as blank.Uviolizing is 4hrs fixedly.Get 15 μ l fluorescent mark binding peptides (1 μ g/ml, the dilution of 1 * PBS damping fluid) and be covered on the bacterium liquid lucifuge room temperature effect 1hrs in the wet box of sealing.PBS damping fluid rinsing 1min, distilled water rinsing 1min, the affinity detected result of fluorescence microscope binding peptide and mycobacterium tuberculosis is with observation by light microscope result (Fig. 2) in contrast.As seen from the figure, binding peptide can combine with mycobacterium tuberculosis generation specificity.
Embodiment 3 binding peptides are as the application of mycobacterium tuberculosis detection reagent
Experiment material
Bacterium liquid sample: mycobacterium tuberculosis clinical separation strain and M. smegmatics are Shanghai Pulmonary Hospital's strain storehouse and preserve bacterial strain; 3 kinds of non-branch bacillus (Pseudomonas aeruginosa, streptococcus aureus, Candida albicans) are the isolating clinical strains of clinical laboratory of Shanghai Pulmonary Hospital.
3.1 small peptide is synthetic: with embodiment 2.
3.2 small peptide dissolving: with embodiment 2.
3.3 fluoroscopic examination: with physiological saline washing back mycobacterium tuberculosis, M. smegmatics, Pseudomonas aeruginosa, streptococcus aureus, Candida albicans bacterium liquid (being 5mg/L) are respectively got 10 μ l and are dripped on slide, and other gets 10 μ l physiological saline and drips on slide as blank.Uviolizing is 4hrs fixedly.Respectively get 15 μ l fluorescent mark binding peptides (1 μ g/ml, the dilution of 1 * PBS damping fluid) and be covered on every kind of bacterium liquid lucifuge room temperature effect 1hrs in the wet box of sealing.PBS damping fluid rinsing 1min, distilled water rinsing 1min, the fluoroscopic examination result (Fig. 3) of microscopic examination binding peptide.As seen from the figure, binding peptide only combines with mycobacterium tuberculosis, produces fluorescence, and with other 4 kinds of equal debonds of bacterium, demonstrate better specificity.

Claims (3)

1. the binding peptide of a mycobacterium tuberculosis is characterized in that having following aminoacid sequence:
X 1HX 2GTX 3H, wherein H represents Histidine, and G represents glycine, and T represents Threonine, X 1～X 3Represent arbitrary amino acid.
2. binding peptide according to claim 1 is characterized in that described X 1Be W or V, X 2Be T, P or S, X 3Be W or P, W representative color propylhomoserin wherein, V represents Xie Ansuan, and P represents proline(Pro), and S represents Serine.
3. the application of binding peptide as claimed in claim 1 in preparation tuberculosis detection reagent.
CN200910195820A 2009-09-17 2009-09-17 Binding peptide of mycobacterium tuberculosis and application thereof Pending CN101643499A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102120758A (en) * 2010-12-22 2011-07-13 上海市肺科医院 Mycobacterium tuberculosis antibody binding peptide and application thereof
CN110734474A (en) * 2019-11-29 2020-01-31 中山大学 Screening method and application of antibacterial peptides
CN111285920A (en) * 2020-03-10 2020-06-16 宁夏大学 Amino acid sequence and nucleotide sequence of specific binding mycobacterium tuberculosis and application thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102120758A (en) * 2010-12-22 2011-07-13 上海市肺科医院 Mycobacterium tuberculosis antibody binding peptide and application thereof
CN102120758B (en) * 2010-12-22 2013-01-23 上海市肺科医院 Mycobacterium tuberculosis antibody binding peptide and application thereof
CN110734474A (en) * 2019-11-29 2020-01-31 中山大学 Screening method and application of antibacterial peptides
CN110734474B (en) * 2019-11-29 2021-10-19 中山大学 Screening method and application of antibacterial peptide
CN111285920A (en) * 2020-03-10 2020-06-16 宁夏大学 Amino acid sequence and nucleotide sequence of specific binding mycobacterium tuberculosis and application thereof

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Application publication date: 20100210