CN101555630A - Phage random peptide library of mycobacterium tuberculosis - Google Patents

Abstract

The invention discloses a phage random peptide library of mycobacterium tuberculosis, which uses DNase I to digest an H37Rv gene group of the mycobacterium tuberculosis to obtain a 50-80bp gene fragment which is further connected on phage M13KE and then uses deer tuberculosis I gG for carrying out three rounds of panning to obtain the phage random peptide library of the mycobacterium tuberculosis under strict conditions. The cutaneous allergic reaction of mice, the detection test of specific antibody in immune mouse serum, the test on spleen lymphopoiesis of the immune mouse and the toxicity test of bovine mycobacterium tuberculosis BCG show that the bacterial load in lung of the mice has significant difference compared with a control group, thereby proving that the antigenicity of the phage random peptide library of the mycobacterium tuberculosis is superior to the bovine mycobacterium tuberculosis BCG, the toxicity thereof is lower than the bovine mycobacterium tuberculosis BCG and also laying a certain foundation for further panning of antigen-simulating epitope of the mycobecterium tuberculosis and the research and the development of novel diagnostic reagents and novel vaccines of tuberculosis.

Description

Phage random peptide library of mycobacterium tuberculosis

Technical field

The invention belongs to biology field, exactly is the structure of phage random peptide library of mycobacterium tuberculosis.

Background technology

Tuberculosis is the highest disease of the single pathogenic bacterium mortality ratio in the world today ^[1], account for the 7th of the total cause of the death in the world ^[2]Owing to the increase of Resistant strain, the therapy of multiple medication combined effect has been subjected to serious challenge in recent years, adds the problem of treatment cycle length and medical expense, presses for and takes proper prophylactic methods to contain increase lungy.Vaccine is the effective ways that controlling tuberculosis spreads.But get permission to be used to the vaccine bacille Calmette-Guerin vaccine (BCG) that prevents tuberculosis and generally use as at present unique; though 3,000,000,000 children are benefited; but it acts on unstable, and provide protection alters a great deal in different crowd, and only children is played specific effect.Therefore, the limitation of bacille Calmette-Guerin vaccine makes the development of prevention tuberculosis new generation vaccine become the urgent need of controlling tuberculosis epidemic situation.

The important epitope of determining cause of disease is the prerequisite of making effective vaccine and setting up accurately special diagnostic method.Epitope just is positioned at the antigenic determinant on antigen molecule surface, and it can be discerned corresponding antibody and react with it.The method of the simplest and the most direct at present the most effective definite epitope is exactly the phage peptide library panning technique.Its principle is that antibody is exposed in the peptide storehouse as acceptor, elutriation and analyze resulting can with the sequence of antibodies specific bonded recombinant phage peptide, just can obtain the information of antigenic determinant.Therefore, make up specific phage peptide storehouse and utilize the special epitope of its elutriation to become a big focus of scientific research circle in recent years.Be applied to the elutriation of epitope at present for the phage peptide library technology, report is a lot of both at home and abroad, but the structure of tubercule bacillus phage random peptide library and application phage peptide library technology elutriation negre antigen epi-position are not but appeared in the newspapers.

Summary of the invention

The purpose of this invention is to provide a kind of phage random peptide library of mycobacterium tuberculosis, prepare by following method:

A, extract mycobacterium tuberculosis H37Rv genome with art methods;

B, in H37Rv genome 12.0 μ l (DNA concentration 4.8 μ g/ μ l), DNase I0.5 μ l, 10 * Buffer1.5 μ l ratio preparation reaction system, 16 ℃ of digestion of temperature mycobacterium tuberculosis H37Rv genome; Reaction 30min; EDTA (0.25M) termination reaction that adds 1/10 volume gets random gene fragment 50-80bp;

The random dna fragment of C, the phage M13KE that gets dephosphorylation and flush endization is connected, and linked system is: M13KE8 μ l, random dna fragment 25 μ l, 10 * Buffer-24 μ l, T ₄Dna ligase (NEB) 3 μ l, cumulative volume are 40 μ l; Shock parameters is: 2mm shock by electricity the cup, voltage 2500v, resistance 200 Ω, electric capacity 25 μ F; Electric shock back record number is: voltage 2385v, and time 3.4ms connects 16h respectively at 16 ℃; Get phage random peptide library of mycobacterium tuberculosis, and usefulness TU (transducting unit, TU) value is weighed, and storage capacity is 62 * 100 * 100=6.2 * 10 ⁵/ ml;

Described phage random peptide library of mycobacterium tuberculosis carries out the three-wheel elutriation then with the amplification of conventional M13 method to phage:

First round elutriation

1) 96 hole enzyme plates, deer tuberculosis positive serum IgG solution (100 μ g/ml) is as target molecule solution;

2) bag quilt: every hole adds 1. solution of 150 μ l, adds along hole wall to make surperficial complete wetting;

3) in humidifier vessel, (the sealed plastics casing of wet gauze is housed) 4 ℃ spend the night;

4) blockade: outwell coating buffer, firmly clap and get rid of to remove residual solution; The liquid of blockading is filled it up with in every hole, 4 ℃ of effect 1h;

5) outwell the liquid of blockading, wash plate fast 6 times with TBST (Tris salt buffer+0.1%[v/v] Tween-20) damping fluid; The damping fluid that inclines is upside down in clean filter paper arsis and gets rid of to remove residual solution.This operation will be soon to avoid the plate drying.

6) (titre is 1.50 * 10 with the phage display genome peptide storehouse of TBST damping fluid dilution to get 100 μ l ¹¹), joining on the enzyme plate of having blockaded, gentleness is shaken 10～60min;

7) topple over and remove, be inverted plate and get rid of at clean filter paper arsis and remove residual solution not in conjunction with phage;

8) wash plate 10 times by method described in 5. with the TBST damping fluid, change a clean filter paper at every turn to avoid crossed contamination;

9) (gentleness is shaken＞10min), elutriant is sucked in another clean Eppendorf tube, neutralizes with 15 μ lMTris-HCl to separate the bonded molecule with non-specific damping fluid (0.2M Glycine-HCl, 1mg/ml BSA);

10) conventional M13 method is measured the preceding titre of eluate amplification;

11) will measure remaining eluate amplification after the titre: eluate is joined in the 20ml ER2738 culture (logarithm early stage, OD ₆₀₀≈ 0.6), 37 ℃, 200rpm shaking culture 4.5h;

12) above-mentioned culture is changed in the centrifuge tube, 4 ℃ 10, the centrifugal 10min of 000rpm.Supernatant changes in another centrifuge tube, and is centrifugal again;

13) top 80% with supernatant changes in the fresh tube, adds the PEG/NaCl of 1/6 volume, and 4 ℃ of precipitations are spent the night;

14) 4 ℃ 10, the centrifugal 15min of 000rpm outwells supernatant liquor, and is of short duration more centrifugal, discards residual supernatant liquor;

15) throw out is resuspended among the 1ml TBS, and suspension changes in the Eppendorf tube, and 4 ℃ 10, the centrifugal 5min of 000rpm makes the residual cells precipitation;

16) supernatant changes in another fresh Eppendorf tube, with the PEG/NaCl redeposition of 1/6 volume.Hatch 15～60min on ice, 4 ℃ 10, the centrifugal 10min of 000rpm abandons supernatant, and is of short duration more centrifugal, inhales with micropipet and removes remaining supernatant;

17) throw out is resuspended in 200 μ lTBS, 0.02%NaN ₃In, 4 ℃ 10, the centrifugal 1min of 000rpm, supernatant changes in the fresh tube, and this is the eluate after the amplification.4 ℃ of storages;

18) measure amplification back eluate titre;

19) wrap again and prepared second by plate and use when taking turns elutriation;

Second takes turns elutriation

1) second add-on of taking turns the used phage of elutriation is 1.5 * 10 ¹¹Pfu;

2) second take turns elutriation: with the step in the eluate repetition first round screening of first round elutriation amplification, bag is that 50 μ g/ml (use 0.1M PH8.6NaHCO by the deer tuberculosis IgG concentration of usefulness ₃Dilute the deer tuberculosis IgG of 100 μ g/ml), the concentration with Tween-20 in cleaning step increases to 0.5% (v/v);

3) measure second titre of taking turns after the eluate amplification of elutriation gained;

Use when 4) wrapping again by a plate preparation third round elutriation;

The third round elutriation

Repeat step in the first round screening with second eluate of taking turns elutriation amplification, second add-on of taking turns the used phage of elutriation is 1.5 * 10 ¹¹Pfu, bag is that 25 μ g/ml (use 0.1M PH8.6NaHCO by the deer tuberculosis IgG concentration of usefulness ₃Dilute the deer tuberculosis IgG of 100 μ g/ml), the concentration with Tween-20 in cleaning step increases to 0.5% (v/v).

The present invention is under stringent condition, with DNaseI digestion mycobacterium tuberculosis H37Rv genome, obtains the 50-80bp gene fragment, is connected on the phage M13KE, with deer tuberculosis IgG, carries out the phage random peptide library of mycobacterium tuberculosis that the three-wheel elutriation obtains.

With through the positive colony mixed peptide storehouse of the mycobacterium tuberculosis phage display peptide library of elutriation to the skin allergic reaction of mouse, the result shows the left foot and the right crus of diaphragm thickness no significant difference (P＞0.05) of TBS control group and M13KE control group, and the thickness difference of bacille Calmette-Guerin vaccine (BCG) group and positive colony combined group is respectively 0.41mm and 0.34mm, the two difference is not remarkable, and is remarkable with TBS control group, M13KE control group comparing difference.

The detection of specific antibody test in immune serum, mice serum is surveyed the OD value by 1: 200 dilution back, the result shows that the antibody titers of antibody titers that experimental group produces and BCG control group is suitable, difference is not remarkable, with TBS control group and M13KE control group comparing difference remarkable (P＜0.05), prove that mycobacterium tuberculosis genome peptide of the present invention storehouse can stimulate mouse to produce specific immunity.

Test through the immune mouse spleen lymphocyte proliferation, the experimental mice splenic lymphocyte external after conA, LPS, PPD stimulate, cause that lymphocyte produces tangible proliferative response, and the SI value is higher than the BCG control group, TBS control group and M13KE control group mouse spleen lymphocyte under conA, LPS nonspecific stimulation has the propagation phenomenon, and lymphocyte does not have the propagation phenomenon under the differential stimulus of PPD.

The challenge test of mycobacterium tuberculosis var bovis BCG is used positive colony mixed peptide storehouse immunization experiment group and BCG control group through the mycobacterium tuberculosis phage display peptide library of elutriation, and mouse left side lung lotus bacterium amount is starkly lower than the TBS control group, and significant difference (P＜0.05); The HE coloration result shows TBS control group (as Figure 11) and M13KE control group (as Figure 12) pulmonary congestion alveolar structure disappearance tissue necrosis, the pulp fibers disposition transudate that apparent number does not wait and a small amount of lymphocyte and macrophages infiltration do not see that the tuberculosis granuloma forms; Visible big amount lymphocyte of experimental group and foam like cell soak into as (Figure 13); BCG control group alveolar septum and alveolar space have the tuberculosis granulation tissue that is made of epithelioid cell and new capillary vessel, cause alveolus wall gently to thicken to severe, alveolar space disappears and the visible tuberculosis granuloma of being made up of big amount lymphocyte and a small amount of epithelioid cell not of uniform size, do not see downright bad clear border, visible small amount of foam sample scavenger cell is as figure (14).

Antigenicity is better than mycobacterium tuberculosis var bovis BCG, and toxicity is lower than mycobacterium tuberculosis var bovis BCG, has also established certain basis for the research and development of the elutriation of further negre antigen mimic epitopes and novel diagnostic reagent of tuberculosis and new generation vaccine.

Description of drawings

Fig. 1 mycobacterium tuberculosis H37Rv of the present invention phage random peptide library makes up route map.

Fig. 2 mycobacterium tuberculosis H37Rv genome dna electrophoresis collection of illustrative plates, wherein: 1,2: tuberculosis group DNA; M:DNA Marker/ λ DNA/HindIII.

Fig. 3 random fragment reclaims electrophoresis result, and wherein 1: random fragment reclaims product; M:DNA Marker/DL2000.

The enzyme of Fig. 4 recombinant plasmid is cut evaluation, and wherein 1: recombinant plasmid; 2: the enzyme of recombinant plasmid is cut the result; M:DNAMarker/ λ DNA/HindIII.

The skin allergic reaction of Fig. 5 immune mouse is figure as a result, wherein

: left foot thickness,

: right crus of diaphragm thickness, 1:TBS control group, 2:M13KE control group, 3: bacille Calmette-Guerin vaccine (BCG) group, 4: genome peptide storehouse group.

The anti-PPD antibody horizontal of Fig. 6 immune serum, 1:TBS control group wherein, 2:M13KE control group, 3: bacille Calmette-Guerin vaccine (BCG) group, 4: genome peptide storehouse group.

Fig. 7 immune mouse spleen lymphocyte proliferation level, 1:TBS control group wherein, the 2:M13KE control group, 3: bacille Calmette-Guerin vaccine (BCG) group, 4: genome peptide storehouse group, ConA 5 μ g/ml,

LPS 10 μ g/ml,

PPD 5 μ g/ml.

Fig. 8 TBS control group lungs tissue slice.

Fig. 9 M13KE control group lungs tissue slice.

Figure 10 genome peptide storehouse group lungs tissue slice.

Figure 11 BCG control group lungs tissue slice.

Embodiment

The structure of embodiment 1 mycobacterium tuberculosis H37Rv phage random peptide library (making up route as shown in Figure 1):

A extracts mycobacterium tuberculosis H37Rv genome with art methods, and the mycobacterium tuberculosis genomic dna of extraction detects through 0.5% agarose gel electrophoresis and shows not have degraded, and no RNA is residual.The results are shown in Figure 2.

B.DNaseI digests genome

Get the genome that has extracted, measure genome concentration (260nm/280nm).Genome is carried out 3000 times of dilutions, survey A260nm, A280nm and calculation sample concentration with ultraviolet spectrophotometer.DNA concentration: OD260 * 50 * extension rate/1000=0.32 * 50 * 300/1000=4.8 μ g/ μ l.

The digestion reaction system:

Genome 12.0 μ l

DNase?I 0.5μl

10×Buffer 1.5μl

Cumulative volume 14 μ l

Totally 4 manage.To carry out on ice during the preparation reaction system.

16 ℃ of temperature of reaction, digestion mycobacterium tuberculosis H37Rv genome; Reaction 30min; Add EDTA (0.25M) termination reaction of 1/10 volume, get 5 μ l, carry out 1% agarose gel electrophoresis, the results are shown in Figure 3, gene fragment 50-80bp; All recovery segments are put into the 1.5mlEP pipe, add TE to 500 μ l, use the equal-volume chloroform: different the eleventh of the twelve Earthly Branches alcohol (24: 1) extracting, sodium-acetate/ice ethanol sedimentation spends the night.The centrifugal 15min of 12000rpm abandons supernatant.Precipitate the ethanol desalinization of soil by flooding or leaching with 70% ,-20 ℃ of preservations are dissolved in dry back with 50 μ l TE.

The processing of C purpose fragment flush end

Reaction system is:

H ₂O 12μl

10×NEBuffer-2 5μl

Dna fragmentation 30 μ l

dNTP(2.5mM) 10μl

Klenow enzyme (NEB) 3 μ l

BSA(100×) 0.6μl

Cumulative volume 60 μ l

Earlier dna segment is hatched 10min for 70 ℃, add H then successively ₂O, 10 * NEBuffer-2, BSA (100 *), dna fragmentation, dNTP (2.5mM), Klenow enzyme (NEB) are hatched 15min for 25 ℃.Put into 37 ℃ of water-baths, add T4DNA ligase enzyme 2 μ l, hatch 5min.Hatch 20min end reaction for 75 ℃ again, add TE to 500 μ l.Add equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, the centrifugal 10min of 12000rpm gets supernatant.The isopropanol precipitating that adds 0.6 times of volume spends the night.Abandon supernatant, be preheated to 65 ℃ distilled water dissolving.-20 ℃ of preservations.

The purpose fragment dephosphorylation of D flush endization

Reaction system:

H ₂O 12μl

10×NEBuffer 5μl

Dna fragmentation 30 μ l

CIAP 3μl

Cumulative volume 50 μ l

37 ℃ of reaction 4h.Reaction finishes the back and adds TE to 500 μ l, uses phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, the centrifugal 10min of 12000rpm.Get the isopropanol precipitating of resetting and adding 0.6 times of volume and spend the night, the centrifugal 15min of 12000rpm.Abandon supernatant, control is done, TE dissolving ,-20 ℃ of preservations.

The EM13KE preparing carriers

Ordinary method prepares e. coli jm109 thermal shock competent cell, after the room temperature dissolving, is put in immediately on ice; Get 0.5 μ l carrier M13KE and add in the competent cell mixing; Ice bath 30min; 42 ℃ of water-bath heat shock 1.5min are put in 5min on ice immediately; Add 800 μ lLB liquid nutrient mediums, place 37 ℃ of 200rpm jolting 2h; Get 200 μ l bacterium liquid and coat on the ready LB-Amp agar plate (containing X-gal/IPTG) 37 ℃ of incubator overnight incubation.Blue hickie screening: the positive clone of locus coeruleus.The single locus coeruleus of picking is in the LB liquid nutrient medium of 20ml, and 37 ℃, 200rpm jolting spend the night.Extract carrier M13KE with plasmid a small amount of extraction agent box, the working method by specification carries out.With Acc65I, EagI double digestion phage M13KE, reaction system is:

H ₂O 40μl

10×NEBuffer3 8μl

Carrier segments 20 μ l

Acc65I(NEB) 2μl

EagI(NEB) 2μl

BSA 0.8μl

Cumulative volume 80 μ l

37℃4h。After reaction finishes, reclaim test kit with gel and reclaim linearized vector.The recycling step by specification carries out, and the DNA of wash-out is recovered in the EP pipe ,-20 ℃ of preservations.The flush end reaction system that enzyme is cut carrier is:

H ₂O 12μl

10×NEBuffer-2 5μl

M13KE reclaims product 30 μ l

dNTP(2.5mM) 10μl

Klenow enzyme (NEB) 3 μ l

BSA(100×) 0.6μl

Cumulative volume 60 μ l

Earlier the carrier segment is hatched 10min for 70 ℃, add H then successively ₂O, 10 * NEBuffer-2, BSA (100 *), dna fragmentation, dNTP (2.5mM), Klenow enzyme (NEB) are hatched 15min for 25 ℃.Put into 37 ℃ of water-baths, add T4DNA ligase enzyme 2 μ l, hatch 5min.Hatch 20min end reaction for 75 ℃ again, add TE to 500 μ l.Add equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, the centrifugal 10min of 12000rpm gets supernatant.The isopropanol precipitating that adds 0.6 times of volume spends the night.Abandon supernatant, be preheated to 65 ℃ distilled water dissolving.-20 ℃ of preservations.The dephosphorylation reaction system:

H ₂O 12μl

10×NEBuffer 5μl

M13KE fragment 30 μ l

CIAP 3μl

Cumulative volume 50 μ l

37 ℃ of reaction 4h.Should finish the back and add TE to 500 μ l, use phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, the centrifugal 10min of 12000rpm.Get the isopropanol precipitating of resetting and adding 0.6 times of volume and spend the night, the centrifugal 15min of 12000rpm.Abandon supernatant, control is done, and is preheated to 65 ℃ distilled water dissolving.0.75% agarose gel electrophoresis detects.Obtain the segment of about 7200bp size.

The structure of F recombinant phage

The random dna fragment of connection carrier M13KE and flush endization

Genome peptide storehouse linked system is:

Connect 1 and connect 2 connections 3

M13KE 1μl 2μl 2μl

Random dna fragment 10 μ l 10 μ l 6 μ l

Water 6 μ l 5 μ l 9 μ l

10×Buffer-2 2μl 2μl 2μl

T4DNA ligase enzyme (NEB) 1 μ l 1 μ l 1 μ l

Cumulative volume 20 μ l

Carrier M13KE from disjunctor is:

From connecting 1 from connecting 2 from connecting 3

M13KE 1μl 2μl 2μl

Water 16 μ l 15 μ l 19 μ l

10×Buffer-2 2μl 2μl 2μl

T4DNA ligase enzyme (NEB) 1 μ l 1 μ l 1 μ l

Cumulative volume 20 μ l

Two groups respectively at 16 ℃ of connection 16h.Two groups add TE to 100 μ l respectively, add the equal-volume chloroform: primary isoamyl alcohol (24: 1) extracting, centrifuging and taking supernatant can be preserved a week for 4 ℃ in another centrifuge tube, are stored in-70 ℃ of refrigerators if prolonged preservation need add 7% DMSO.Reorganization is connected product and connects the intestinal bacteria ER that contrast joins the ordinary method preparation respectively certainly ₂₇₃₈In the competent cell, with the conversion of shocking by electricity of BTX SAFETY STAND 630B type electric shock conversion instrument.The conversion condition shock parameters is respectively: 2mm shock by electricity the cup, electric capacity is 25 μ F; Voltage is made as 2200v respectively; Resistance is made as 400 Ω respectively.Rapidly 1ml SOC substratum is joined after electric shock is finished in the electric shock cup, put upside down mixing, be transferred in the 1.5ml centrifuge tube, hatch 60min for 37 ℃.Getting after 10 times of the 10 μ l bacterium liquid dilutions coated plate observes and has locus coeruleus to produce explanation to transform successfully.

G recombinant vectors enzyme is cut evaluation

3 locus coeruleus branches that picking transforms on the rear plate are clipped in the 20ml LB liquid nutrient medium 37 ℃, 200rpm shaken overnight.Extract recombinant vectors with plasmid extraction kit.Plasmid Acc65I, EagI double digestion recombinant vectors;

Reaction system is:

H ₂O 40μl

10×NEBuffer-3 8μl

Carrier segments 20 μ l

Acc65I(NEB) 2μl

EagI(NEB) 2μl

BSA 0.8μl

Cumulative volume 80 μ l

37℃，4h。After reaction finishes, carry out 2.5% agarose gel electrophoresis detection.Observation obtains two bands about 7200bp and 80bp, illustrates that the external source segment successfully changes the M13KE carrier over to, sees Fig. 4.

The mensuration of embodiment 2 storage capacity

Microwave oven melts top-layer agar, is divided into the 5ml equal portions in the sterilization test tube, each phage extent of dilution one pipe.Be stored in 45 ℃ standby.Bacterium liquid after the 10 μ l connection is diluted to 10 with the LB substratum respectively ^-2, 10 ^-3, 10 ^-4Three extent of dilution.The good bacterium liquid of above-mentioned 3 pipe dilutions is added respectively in the top-agar of 45 ℃ of pre-temperature, and mixing is poured on the LB/IPTG/Xgal flat board of 37 ℃ of pre-temperature immediately fast.Suitably tilt flat plate evenly spreads out top-layer agar.Treat the cooling of 5min rear plate, be inverted 37 ℃ of overnight incubation.Whether have locus coeruleus growth and counting locus coeruleus quantity, minimum growth colony number multiply by extension rate and is the TU value if observing each flat board.(transducting unit, TU) weigh, the formed colony number of promptly every microlitre peptide storehouse infection positive intestinal bacteria of F sex fimbria by value with TU for the storage capacity of phage peptide library.Be diluted to 10 ^-2Flat board on grown 62 locus coeruleus, 10 ^-3Flat board on grown 6 locus coeruleus, calculating storage capacity thus is 62 * 100 * 100=6.2 * 10 ⁵/ ml.

Embodiment 3 mycobacterium tuberculosis genome peptide storehouse elutriations

A the elutriation amplification of phage and the mensuration (conventional M13 method) of titre

1) get 20 μ lER2738 and add in the 200ml LB liquid nutrient medium, 37 ℃, 200rpm shaking culture are to OD ₆₀₀≈ 0.6 (approximately 7h).

2) from phage peptide library, get 10 μ l phage solutions and join 20ml 1. in the culture, 37 ℃, 200rpm shaking culture 4.5～5h.

3) culture is 2. changed in the Eppendorf tube, the centrifugal 30s of 8000rpm, supernatant changes in the fresh centrifuge tube, centrifugal again 30s, change in the fresh centrifuge tube with micropipet, this is phage storage liquid of amplification, can 4 ℃ stores several weeks and little to the titre influence, after prolonged preservation is used sterile glycerol dilution in 1: 1 ,-20 ℃ of storages.

4) get 20 μ l ER2738 (OD ₆₀₀≈ 0.6) join in the 20ml LB liquid nutrient medium, 37 ℃, 200rpm shaking culture are to mid-log phase (OD ₆₀₀≈ 0.5, approximately 3.5h).

5) melt top-layer agar, each sterilization test tube packing 3ml, 45 ℃ of preservations are standby.

6) 37 ℃ of preheating LB/IPTG/X-gal flat boards (5), flat board of each phage extent of dilution is standby.

7) with LB liquid nutrient medium dilution phage, be respectively * 1, * 10, * 100, * 1000, * 10000, each extent of dilution changes a fresh suction nozzle, and suggestion uses band filter core suction nozzle to avoid crossed contamination.

8) will be 4. culture be divided into 200 μ l equal portions and be loaded in the Eppendorf tube each extent of dilution one pipe.

9) every pipe adds the different dilution phages of 10 μ l, shakes mixing fast, room temperature incubation 1～5min.

10) 9. mixture adds in the top-layer agar pipe of 45 ℃ of pre-temperature, each pipe, and mixing is poured on the LB/IPTG/X-gal flat board of 37 ℃ of pre-temperature immediately fast, and suitably tilt flat plate evenly spreads out top-layer agar.

11) treat dull and stereotyped cooling 5min after, be inverted in 37 ℃ and cultivate 12h.

12) check flat board, count the spot number on the flat board that is less than 100 plaques.Multiply by plaque forming unit (pfu) titre that extension rate promptly obtains per 10 μ l phages with this number then.

First round elutriation

1) 96 hole enzyme plates, deer tuberculosis positive serum IgG solution (100 μ g/ml) is as target molecule solution.

2) bag quilt: every hole adds 1. solution of 150 μ l, adds along hole wall to make surperficial complete wetting (solution is spilt).

3) in humidifier vessel, (the sealed plastics casing of wet gauze is housed) 4 ℃ spend the night.

4) blockade: outwell coating buffer, firmly clap and get rid of to remove residual solution.The liquid of blockading is filled it up with in every hole, 4 ℃ of effect 1h.

5) outwell the liquid of blockading, wash plate fast 6 times with TBST (Tris salt buffer+0.1%[v/v] Tween-20) damping fluid.The damping fluid that inclines is upside down in clean filter paper arsis and gets rid of to remove residual solution.This operation will be soon to avoid the plate drying.

6) (titre is 1.50 * 10 with the phage display genome peptide storehouse of TBST damping fluid dilution to get 100 μ l ¹¹), joining on the enzyme plate of having blockaded, gentleness is shaken 10～60min.

7) topple over and remove, be inverted plate and get rid of at clean filter paper arsis and remove residual solution not in conjunction with phage.

8) wash plate 10 times by method described in 5. with the TBST damping fluid, change a clean filter paper at every turn to avoid crossed contamination.

9) (gentleness is shaken＞10min), elutriant is sucked in another clean Eppendorf tube, neutralizes with 15 μ lMTris-HCl to separate the bonded molecule with non-specific damping fluid (0.2M Glycine-HCl, 1mg/ml BSA).

10) conventional M13 method is measured the preceding titre of eluate amplification.

11) will measure remaining eluate amplification after the titre: eluate is joined in the 20ml ER2738 culture (logarithm early stage, OD ₆₀₀≈ 0.6), 37 ℃, 200rpm shaking culture 4.5h.

12) above-mentioned culture is changed in the centrifuge tube, 4 ℃ 10, the centrifugal 10min of 000rpm.Supernatant changes in another centrifuge tube, and is centrifugal again.

13) top 80% with supernatant changes in the fresh tube, adds the PEG/NaCl of 1/6 volume, and 4 ℃ of precipitations are spent the night.

14) 4 ℃ 10, the centrifugal 15min of 000rpm outwells supernatant liquor, and is of short duration more centrifugal, discards residual supernatant liquor.

15) throw out is resuspended among the 1ml TBS, and suspension changes in the Eppendorf tube, and 4 ℃ 10, the centrifugal 5min of 000rpm makes the residual cells precipitation.

16) supernatant changes in another fresh Eppendorf tube, with the PEG/NaCl redeposition of 1/6 volume.Hatch 15～60min on ice, 4 ℃ 10, the centrifugal 10min of 000rpm abandons supernatant, and is of short duration more centrifugal, inhales with micropipet and removes remaining supernatant.

17) throw out is resuspended in 200 μ lTBS, 0.02%NaN ₃In, 4 ℃ 10, the centrifugal 1min of 000rpm, supernatant changes in the fresh tube, and this is the eluate after the amplification.4 ℃ of storages.

18) measure amplification back eluate titre.

19) wrap again and prepared second by plate and use when taking turns elutriation.

Second takes turns elutriation

1) second add-on of taking turns the used phage of elutriation is 1.5 * 10 ¹¹Pfu.

2) second take turns elutriation: with the step in the eluate repetition first round screening of first round elutriation amplification, bag is that 50 μ g/ml (use 0.1M PH8.6NaHCO by the deer tuberculosis IgG concentration of usefulness ₃Dilute the deer tuberculosis IgG of 100 μ g/ml), the concentration with Tween-20 in cleaning step increases to 0.5% (v/v).

3) measure second titre of taking turns after the eluate amplification of elutriation gained.

Use when 4) wrapping again by a plate preparation third round elutriation.

The third round elutriation

1) carry out the third round elutriation: the step in screening with the second eluate repetition first round of taking turns the elutriation amplification, second add-on of taking turns the used phage of elutriation is 1.5 * 10 ¹¹Pfu, bag is that 25 μ g/ml (use 0.1M PH8.6NaHCO by the deer tuberculosis IgG concentration of usefulness ₃Dilute the deer tuberculosis IgG of 100 μ g/ml), the concentration with Tween-20 in cleaning step increases to 0.5% (v/v).

2) obtained enrichment through the three-wheel screening positive clone.

Grouping of experimental example 1 animal and immunization

Get body weight 18～22g, the female BALB/c small white mouse in 6～8 ages in week, totally 40, be divided into four groups (10 every group) at random, be respectively TBS control group, M13KE control group, bacille Calmette-Guerin vaccine group, genome peptide storehouse group.

The TBS control group: every mouse abdominal injection TBS (Tris salt buffer pH8.4) 100 μ l, inject once after two weeks again.

The M13KE control group: every mouse abdominal injection M13KE 100 μ l comprise 1 * 10 ¹²Pfu (plaque forming unit) injects once after two weeks again.

BCG group: other respectively organize initial immunity before 5 weeks (, carry out BCG and inoculate.After BCG diluted with the card thin liquid, 0.1ml comprised 5 * 10 ⁵CFU (colony-forming unit)/only, subcutaneous injection.

Experimental group: after the mycobacterium tuberculosis genome peptide storehouse amplification that makes up among the embodiment 2, mouse peritoneal is injected 100 μ l (10 ¹²Pfu), after two weeks, inject once again.

Each organize mouse behind booster immunization around the at left side foot rear solid end subcutaneous injection 20 μ lPPD (200 μ g/ml), simultaneously in contrast at right side foot intradermal injection 20 μ lTBS; Inject the variation in thickness of observing the foot pad after 48 hours; Compare with its thickness of vernier caliper measurement;

Then, get 5 eyeball of mouse and extract and get blood, separation of serum for every group; In order to detecting specific antibody in the immune serum: disconnected again neck is put to death, and puts in the 75% alcohol cylinder behind the sterilization 3min, and the aseptic spleen of getting carries out the test of immune mouse spleen lymphocyte proliferation.

Every group of remaining 5 mouse carry out the challenge test of mycobacterium tuberculosis var bovis BCG.

BCG causes weak mycobacterium tuberculosis var bovis, and is harmless to the mankind, reaches 1 * 10 but work as intranasal inoculation dosage ⁶Can cause mouse invasion during CFU, thus this experimental selection Mycobacterium bovis vaccine strain BCG as attack the poison carry out the immune protective analysis with bacterial strain.

The skin allergic reaction of experimental example 2 immune mouses

Each organize mouse behind booster immunization around the at left side foot rear solid end subcutaneous injection 20 μ lPPD (200 μ g/ml), simultaneously in contrast at right side foot intradermal injection 20 μ lTBS; Inject the variation in thickness of observing the foot pad after 48 hours; Compare with its thickness of vernier caliper measurement;

Four groups of right side of mice rear solid ends subcutaneous injection, 20 μ lPPD measured transformation reactions, measurement result such as Fig. 5 in back 48 hours in injection.The result shows the left foot and the right crus of diaphragm thickness no significant difference (P＞0.05) of TBS control group and M13KE control group, and the thickness difference of bacille Calmette-Guerin vaccine (BCG) group and positive colony combined group is respectively 0.41mm and 0.34mm, the two difference is not remarkable, and is remarkable with TBS control group, M13KE control group comparing difference.

The detection of specific antibody in experimental example 3 immune serums

(1) respectively organize behind the mouse booster immunization the in the experimental example 1 around eyeball get blood, isolating serum is diluted to 45 μ g/ml (determining through trial test), coated elisa plate with the carbonate coating buffer of pH9.6 with PPD (bovine tuberculin purified protein derivative), every hole 100 μ l, 4 ℃ of bags are spent the night.

(2) discard coating buffer, with PBST washing 3 times, each 3min.

(3) every hole adds 1%BSA100 μ l, in 37 ℃ of sealing 1.5h.Discard coating buffer, wash the same.

(4) with PBS mice serum is done the dilution of different multiples (1: 2,1: 4,1: 10,1: 20), every hole 100 μ l.37 ℃ of incubation 1.5h.Discard serum, wash the same.

(5) every hole adds 1: 1000 the anti-mouse IgG two anti-100 μ l of HRP mark rabbit, 37 ℃ of incubation 1.5h.Discard two and resist, wash the same.

(6) every hole adds 100 μ l substrate solutions, room temperature lucifuge colour developing, treat the substrate colour developing after, every hole adds stop buffer 50 μ l, measures the absorbance value (OD of 490nm on enzyme-linked immunosorbent assay instrument ₄₉₀).

(7) dilute 100 times as negative control with PBS control group mice serum, OD ₄₉₀Below 0.05, OD _{490 experimental group}/ OD _{490 Negative control group}〉=2.1 is positive;

Each group mice serum is surveyed OD value result such as Fig. 6 by 1: 200 dilution back, the antibody titers that experimental group produces is suitable with the antibody titers of BCG control group, difference is not remarkable, with TBS control group and M13KE control group comparing difference remarkable (P＜0.05), prove that mycobacterium tuberculosis genome peptide of the present invention storehouse can stimulate mouse to produce specific immunity.

The test of experimental example 4 immune mouse spleen lymphocyte proliferations

(1) experimental example 1 interrupts the mouse that neck is put to death, and puts in the 75% alcohol cylinder behind the sterilization 3min, and the aseptic spleen of getting cuts off fatty tissue on the spleen, prepares splenocyte suspension with RPMI1640 (contain 10% calf serum, 10000U/ml is two anti-), and spleens cell number is transferred to 1 * 10 ⁷Individual/ml;

(2) get 96 porocyte culture plates, every hole adds splenocyte suspension 100 μ l.

(3) first first three hole of row adds complete RPMI1640 substratum 100 μ l as negative control then; All the other nine holes add the every hole 100 μ l of ConA (10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml) respectively, and each concentration is established three multiple holes.

(4) second first three hole of row add complete RPMI1640 substratum 100 μ l as negative control; All the other nine holes add the every hole 100 μ l of LPS (20 μ g/ml, 10 μ g/ml, 5 μ g/ml) respectively, and each concentration is established three multiple holes.

(5) the 3rd first three hole of row add complete RPMI1640 substratum 100 μ l as negative control; All the other nine holes add the every hole 100 μ l of PPD (20 μ g/ml, 10 μ g/ml, 5 μ g/ml) respectively, and each concentration is established three multiple holes.Cultivated 40 hours under 37 ℃, 5%CO2 condition.

(6) every hole adds 20 μ l MTT, continues to cultivate 4 hours.The careful suction goes supernatant, every hole to add 100 μ lDMSO, vibration 10min, and enzyme-linked immunosorbent assay instrument is surveyed OD ₅₇₀, the calculating stimulation index (stimulation index, SI), SI=OD _{570 experimental group}/ OD _{570 Control group}

The experimental mice splenic lymphocyte external after conA, LPS, PPD stimulate, cause that lymphocyte produces tangible proliferative response, and the SI value is higher than the BCG control group, TBS control group and M13KE control group mouse spleen lymphocyte under conA, LPS nonspecific stimulation has the propagation phenomenon, lymphocyte does not have the propagation phenomenon under the differential stimulus of PPD, sees Fig. 7.

The challenge test of experimental example 5 mycobacterium tuberculosis var bovis BCG

Around the booster immunization, and after the skin allergic reaction test, by scraping Mycobacterium bovis BCG on the modified Russell medium, adjusting colony number with 1 * PBS-0.05%Tween80 is 5 * 10 ⁷CFU/ml, getting every group of residue mouse intranasal inoculation 20 μ l is 1 * 10 ⁶The BCG of CPU (every nostril 10 μ l).

Take off neck and put to death attacking mouse after malicious three weeks, take lungs, carry out immune protective analysis and pathological change analysis respectively.

A, immune protective analysis

The mouse nasal cavity is attacked poison 1 * 10 ⁶Get the lung grinding behind the CPU BCG 3 weeks and dilute 10 respectively ^-1, 10 ^-2, 10 ^-3, modified Russell medium is cultivated, and colony number experimental group is as shown in Table 2 measured with BCG control group mice left side lung lotus bacterium and is starkly lower than the TBS control group, and significant difference (P＜0.05).

Table 2 mouse lung is organized lotus bacterium amount (CPU/mg)

After Paraformaldehyde 96 through 4% is fixing, routine paraffin wax Bao Li, section, hematoxylin-eosin staining, preparation histopathologic slide,

Concrete steps are as follows:

(1) lungs is cut into size and is the fritter of 0.5cm～1.0cm, drop into fixedly 6h-24h of stationary liquid immediately.

(2) tissue to be checked is handled through lower concentration to the alcohol of high density, removes moisture in the clean tissue.Displace alcohol with dimethylbenzene then, tissue block becomes transparent after immersing dimethylbenzene gradually.Again tissue block is put in the dissolved paraffin, paraffin is immersed organize and replace out dimethylbenzene.At last tissue block is embedded in the paraffin.

(3) with slicing machine tissue is cut into the thick thin slice of 5 μ m, and, puts in the incubator and dry on its attached slide glass that scribbles poly-lysine.

(4) section is put into dimethylbenzene paraffin is purified, through the alcohol entry, dye respectively with h and E more then.

(5) section after the dyeing is through dehydration of alcohol, and dimethylbenzene is transparent.In section, drip an amount of natural gum again, cover glass be placed on the natural gum, to be dried after, observe down in mirror.

The HE coloration result shows TBS control group (as Fig. 8) and M13KE control group (as Fig. 9) pulmonary congestion alveolar structure disappearance tissue necrosis, the pulp fibers disposition transudate that apparent number does not wait and a small amount of lymphocyte and macrophages infiltration do not see that the tuberculosis granuloma forms; Visible big amount lymphocyte of experimental group and foam like cell soak into (as Figure 10); BCG control group alveolar septum and alveolar space have the tuberculosis granulation tissue that is made of epithelioid cell and new capillary vessel, cause alveolus wall gently to thicken to severe, alveolar space disappears and the visible tuberculosis granuloma of being made up of big amount lymphocyte and a small amount of epithelioid cell not of uniform size, do not see downright bad clear border, visible small amount of foam sample scavenger cell (as Figure 11).

Claims (2)

1, phage random peptide library of mycobacterium tuberculosis is characterized in that, is prepared by following method:

A, extract mycobacterium tuberculosis H37Rv genome with art methods;

B, in H37Rv genome 12.0 μ l (DNA concentration 4.8 μ g/ μ l), DNaseI0.5 μ l, 10 * Buffer1.5 μ l ratio preparation reaction system, 16 ℃ of digestion of temperature mycobacterium tuberculosis H37Rv genome; Reaction 30min; EDTA (0.25M) termination reaction that adds 1/10 volume gets random gene fragment 50-80bp;

The random dna fragment of C, the phage M13KE that gets dephosphorylation and flush endization is connected, and linked system is: M13KE8 μ l, random dna fragment 25 μ l, 10 * Buffer-24 μ l, T ₄Dna ligase (NEB) 3 μ l, cumulative volume are 40 μ l; Shock parameters is: 2mm shock by electricity the cup, voltage 2500v, resistance 200 Ω, electric capacity 25 μ F; Electric shock back record number is: voltage 2385v, and time 3.4ms connects 16h respectively at 16 ℃; Get phage random peptide library of mycobacterium tuberculosis, and usefulness TU (transducting unit, TU) value is weighed, and storage capacity is 62 * 100 * 100=6.2 * 10 ⁵/ ml.

2, phage random peptide library of mycobacterium tuberculosis according to claim 1 is characterized in that, with the amplification of conventional M13 method to phage, carries out the three-wheel elutriation then:

First round elutriation

1) 96 hole enzyme plates, deer tuberculosis positive serum IgG solution (100 μ g/ml) is as target molecule solution;

2) bag quilt: every hole adds 1. solution of 150 μ l, adds along hole wall to make surperficial complete wetting;

3) in humidifier vessel 4 ℃ spend the night;

4) blockade: outwell coating buffer, firmly clap and get rid of to remove residual solution; The liquid of blockading is filled it up with in every hole, 4 ℃ of effect 1h;

5) outwell the liquid of blockading, wash plate fast 6 times with the TBST damping fluid; The damping fluid that inclines is upside down in clean filter paper arsis and gets rid of to remove residual solution;

6) (titre is 1.50 * 10 with the phage display genome peptide storehouse of TBST damping fluid dilution to get 100 μ l ¹¹), joining on the enzyme plate of having blockaded, gentleness is shaken 10～60min;

7) topple over and remove, be inverted plate and get rid of at clean filter paper arsis and remove residual solution not in conjunction with phage;

8) wash plate 10 times by method described in 5. with the TBST damping fluid, change a clean filter paper at every turn to avoid crossed contamination;

9) (gentleness is shaken＞10min), elutriant is sucked in another clean Eppendorf tube, neutralizes with 15 μ lMTris-HCl to separate the bonded molecule with non-specific damping fluid (0.2M Glycine-HCl, 1mg/ml BSA);

10) conventional M13 method is measured the preceding titre of eluate amplification;

11) will measure remaining eluate amplification after the titre: eluate is joined in the 20ml ER2738 culture (logarithm early stage, OD ₆₀₀≈ 0.6), 37 ℃, 200rpm shaking culture 4.5h;

12) above-mentioned culture is changed in the centrifuge tube, 4 ℃ 10, the centrifugal 10min of 000rpm, supernatant change in another centrifuge tube, and be centrifugal again;

13) top 80% with supernatant changes in the fresh tube, adds the PEG/NaCl of 1/6 volume, and 4 ℃ of precipitations are spent the night;

14) 4 ℃ 10, the centrifugal 15min of 000rpm outwells supernatant liquor, and is of short duration more centrifugal, discards residual supernatant liquor;

15) throw out is resuspended among the 1ml TBS, and suspension changes in the Eppendorf tube, and 4 ℃ 10, the centrifugal 5min of 000rpm makes the residual cells precipitation;

16) supernatant changes in another fresh Eppendorf tube, with the PEG/NaCl redeposition of 1/6 volume; Hatch 15～60min on ice, 4 ℃ 10, the centrifugal 10min of 000rpm abandons supernatant, and is of short duration more centrifugal, inhales with micropipet and removes remaining supernatant;

17) throw out is resuspended in 200 μ lTBS, 0.02%NaN ₃In, 4 ℃ 10, the centrifugal 1min of 000rpm, supernatant changes in the fresh tube, and this is the eluate after the amplification; 4 ℃ of storages;

18) measure amplification back eluate titre;

19) wrap again and prepared second by plate and use when taking turns elutriation;

Second takes turns elutriation

1) second add-on of taking turns the used phage of elutriation is 1.5 * 10 ¹¹Pfu;

2) second take turns elutriation: with the step in the eluate repetition first round screening of first round elutriation amplification, bag is that 50 μ g/ml (use 0.1M PH8.6NaHCO by the deer tuberculosis IgG concentration of usefulness ₃Dilute the deer tuberculosis IgG of 100 μ g/ml), the concentration with Tween-20 in cleaning step increases to 0.5% (v/v);

3) measure second titre of taking turns after the eluate amplification of elutriation gained;

Use when 4) wrapping again by a plate preparation third round elutriation;

The third round elutriation

Repeat step in the first round screening with second eluate of taking turns elutriation amplification, second add-on of taking turns the used phage of elutriation is 1.5 * 10 ¹¹Pfu, bag is that 25 μ g/ml (use 0.1M PH8.6NaHCO by the deer tuberculosis IgG concentration of usefulness ₃Dilute the deer tuberculosis IgG of 100 μ g/ml), the concentration with Tween-20 in cleaning step increases to 0.5% (v/v).

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