CN102747048B - Mycobacterium tuberculosis candidate antigen and its application - Google Patents
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Abstract
The invention discloses a Mycobacterium tuberculosis candidate antigen and its application. The invention provides application of DXS2 protein in preparation of an accelerator that promotes secretion of IL-2 by a gamma delta T cell. The amino acid sequence of DXS2 protein is sequence 6 in a sequence table. The invention also provides application of the DXS2 protein in preparation of an accelerator that promotes value increase of the gamma delta T cell. The amino acid sequence of the DXS2 protein is sequence 6 in the sequence table. Experiments of the invention prove that, the protein and the possible gamma delta T cell recognized protein antigen discovered in the invention provides new ideas for development of novel tuberculosis vaccines or adjuvant components.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of tubercule bacillus candidate antigens and application thereof.
Background technology
Gamma delta T cells causes that owing to playing an important role in the tuberculosis immunity increasing concern, phosphoric acid antigen are considered to the major antigen of gamma delta T cells identification, but the gamma delta T cells of phosphoric acid antigen activation lacks the effective immunoprotection of tubercule bacillus.Comparatively speaking, the proteantigen of tubercule bacillus has more effective immanoprotection action.All the time, the investigator takes diverse ways to obtain the proteantigen of the tubercule bacillus of gamma delta T cells identification, early stage method is to process tubercule bacillus by different modes, activate the protein ingredient of gamma delta T cells by mass spectroscopy, the tuberculoprotein antigenic component that identifies roughly the activation gamma delta T cells concentrates in the scope of 10-14KD.But apply this kind of method and do not obtain specific tuberculosis antigen composition.The seminar of Alderson is with antiserum(antisera) and the reactive CD4 of tubercule bacillus of tuberculosis patient
+The T lymphocyte is probe, has found to occur with it the epitope of specific binding in the gene expression library by the screening tubercule bacillus, for obtaining effective tuberculosis subunit vaccine, lays a good foundation.But up to the present, take gamma delta T lymphocytes gets epitope research as probe angles has no report.
The tuberculosis patient peripheral blood gamma delta T cells obtained due to separation in vitro the survival time shorter, if there is no the hormesis of other cytokines and anti-gamma delta T CR antibody only can survive about 2 weeks, can not meet the needs in screening library, therefore if set up in vitro tuberculosis specificity gamma delta T cells system, as angling the probe of getting epitope, can meet the needs of the negre antigen epi-position of screening gamma delta T cells identification.Defective type T lymphoma cell line J.RT3-T3.5 cell exactly can meet the need, and this kind of clone is not expressed the TCR of function, but can accept the TCR chain of external source, is formed with the gamma delta T CR transfectional cell series of the homogenization of function.This kind of clone under the stimulation of exogenous antigen, Expression of Activated IL-2.
Therefore, secretion situation according to transfectional cell series IL-2, can judge that gamma delta T cells is to the identification of corresponding antigens and in the process of gamma delta T CR identification antigen, crucial recognition site (Xi XY, et al.2009.J.Bio.Chem.284:27449-27455.Xi XY, et al.2010.International Immunology 22:299-306.).But with gamma delta T CR transfectional cell, be probe, the research in screening library has no report.
Summary of the invention
The purpose of this invention is to provide a peptide species and application thereof, can be used as candidate's epitope peptide of tubercule bacillus proteantigen.
Polypeptide provided by the invention is the polypeptide with one of following amino acid residue sequence:
(a) polypeptide that the aminoacid sequence shown in sequence 5 forms in sequence table;
(b) aminoacid sequence shown in sequence in sequence table 5 is passed through to replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and has the identical function polypeptide derivative by (a).
The replacement of described one or several amino-acid residue and/or disappearance and/or be added to replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.
The gene of coding said polypeptide is also the scope of protection of the invention.
Described gene is the DNA molecular of following (1) or (2) or (3):
(1) DNA molecular shown in sequence 6 in sequence table;
(2) DNA molecular that the DNA sequence dna hybridization limited with (1) under stringent condition and coding have the polypeptide of identical function;
(3) DNA sequence dna limited with (1) at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have a DNA molecular that 99% homology and coding have the polypeptide of identical function.
The recombinant vectors that contains described gene, expression cassette, transgenic cell line or recombinant bacterium are also the scope of protection of the invention.
The application in the promotor of preparation promotion gamma delta T cells secretion IL-2 of described polypeptide or described gene or described recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium is also the scope of protection of the invention.
The application of described polypeptide in the value-added promotor of preparation promotion gamma delta T cells is also the scope of protection of the invention.
The application of described polypeptide in preparing tuberculosis vaccine is also the scope of protection of the invention.
The application of described polypeptide in preparing the anti-mycobacterium tuberculosis product is also the scope of protection of the invention.
Described product is medicine.
Of the present invention experimental results show that, the present invention has set up a kind of New Policy that screens the tuberculoprotein antigen of gamma delta T cells identification, with the external tuberculosis reaction gamma delta T CR transfectional cell of setting up, be that probe angles the tuberculosis antigen epi-position of getting gamma delta T CR identification in the phage random library, the result of functional verification shows that epi-position TP1 is the tuberculosis epi-position of possible gamma delta T cells identification, for development of new Vaccinum Calmette-Guerini or adjuvant composition provide new thinking.
The accompanying drawing explanation
Fig. 1 is for building tuberculosis specificity and non-specific gamma delta T CR transfectional cell
Fig. 2, for take transfectional cell as probe, screens the operating process in phage dodecapeptide storehouse
The binding ability that Fig. 3 is Dominant Epitopes peptide TP1 and tuberculosis specificity gamma delta T CR transfectional cell is identified
The binding ability that Fig. 4 is gamma delta T cells in Dominant Epitopes peptide TP1 and tuberculosis patient peripheral blood is identified
The Function Identification figure that Fig. 5 is DXS2 albumen
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The equal triplicate of experiment in following embodiment, results averaged.
The acquisition of the epitope of the tubercule bacillus of embodiment 1, gamma delta T cells identification
1, build tuberculosis specificity and non-specific gamma delta T CR transfectional cell series
(by BJ Chest Science Hospital, obtained, the patient knows the inside story to get tuberculosis patient anticoagulant heparin fresh venous.) 5 milliliters, (Hyclone company, Catalog:SH30809) mix gently, above-mentioned mixed solution slowly added in the test tube that has added in advance the equal-volume lymphocyte separation medium to add equal-volume RPMI-1640 nutrient solution, avoid destroying interface, centrifugal 20 minutes of 500 * g; Get white interfacial layer in test tube and, in another test tube, add equal-volume RPMI-1640 nutrient solution (Hyclone company, Catalog:SH30809) cleaning secondary.Add Trizol after centrifugal, standing 5 minutes, add 200 μ l chloroforms, after high vibration, in room temperature (25 ℃), place 3 minutes, 12,000 * g, 4 ℃ are centrifugal 15 minutes.The careful upper strata water of drawing, be transferred to a new centrifuge tube, adds 500 μ l Virahols, turns upside down and mix, and room temperature is placed 10 minutes.12,000 * g, 4 ℃ centrifugal 15 minutes, abandon supernatant, add the ethanol of 1ml 75%, the vibration, 7,500 * g, 4 ℃ centrifugal 5 minutes, abandon supernatant.The drying at room temperature precipitation, be dissolved in the water that appropriate DEPC processes.Get RNA sample 12 μ l and add Oligo (dT)
15(500 μ g/ml) 1 μ l, 70 ℃ of heat denatured 5 minutes, after taking-up, be placed in immediately on ice, add successively after cooling 5 * Buffer, 5 μ l, dNTP (10mM) 5 μ l, RNA enzyme inhibitors 1 μ l, MMLV reversed transcriptive enzyme 1 μ l, cumulative volume is 25 μ l, hatches cDNA the first chain that carries out the synthetic peripheral blood PBMC of reverse transcription reaction in 60 minutes for 42 ℃.
According to the CDR3 region sequence of tuberculosis reaction gamma delta T cells, the synthetic γ 9 of design and δ 2 chain epimere bridging primers and hypomere bridging primer, concrete sequence is in Table 1.
The CDR3 sequence of table 1 Healthy People and tuberculosis patient advantage γ 9/ δ 2 chains
CDNA the first chain of peripheral blood PBMC of take is template, respectively with 5 '-CGGGGTACCATGCTGTCACTGCTCCACAC-3 ', 5 '-TTTTTTTGCCCAACTCCCACTCCCACAAGGCACAGTAGTA-3 ' and 5 '-CTCGAGTCATCATGATTTCTCTCCAT-3 ', GGAGTGGGAGTTGGGCAAAAAAATCAAGGTATTTGGTCCCGGAA-3 ' is primer, obtaining the fragment of 391bp and 586bp, is the epimere product of non-specific γ 9 chains and the hypomere product of non-specific γ 9 chains.
CDNA the first chain of peripheral blood PBMC of take is template, respectively with 5 '-CGGGGTACCATGCTGTCACTGCTCCACAC-3 ', 5 '-TTTTTTTGCC CAACTCCCACTCGCTTATTA CCAAGGCACA GTAGTA-3 ' and 5 '-CTCGAGTCATCATGATTTCTCTCCAT-3,5 '-GGAGGTAATAAGCGAGTTGGGCAAAAAAATCAAGGTATTTGGTCCCGGAA-3 ' is primer, obtaining the fragment of 397bp and 592bp, is the epimere product of specificity γ 9 chains and the hypomere product of specificity γ 9 chains.
CDNA the first chain of peripheral blood PBMC of take is template, respectively with 5 '-CGGGGTACCATGCAGAGGATCTCCTCCCTC-3 ', 5 '-ATCGGTTTCCCCTGTTACGT AGCTCCCTAC TGTGTCACAG GCACAGTAGT AAGACCCTTC-3 ', with 5 '-GCCTGTGACACA GTAGGGAGCT ACGTAAGCAC AGGGGAAACCGAT-3. ', 5 '-CCGCTCGAGTTAC AAGAAAAATAACTTGGCAGTC-3 ' is primer, obtaining the fragment of 387bp and 564bp, is the epimere product of non-specific δ 2 chains and the hypomere product of non-specific δ 2 chains.
CDNA the first chain of peripheral blood PBMC of take is template, respectively with 5 '-CGGGGTACCATGCAGAGGATCTCCTCCCTC-3 ', 5 '-GCTGACGAGGGTGTCACAGGCACAGTAGTAAG-3 ' and 5 '-CCGCTCGAGTTACAAGAAAAATAACTTGGCAGTC-3 ', 5 '-ACCCTCGTCAGCACCGATAAACTCATCTTTGG-3 ' is primer, obtaining the fragment of 372bp and 549bp, is the epimere product of specificity δ 2 chains and the hypomere product of specificity δ 2 chains.
Above PCR reaction conditions is 94 ℃ of denaturations 5 minutes, then by following parameter, carries out 30 circulations: 94 ℃ of sex change 30 seconds, and 60 ℃ of annealing 45 seconds, 72 ℃ are extended 1 minute, and after last loop ends, 72 ℃ are extended 10 minutes.After the DNA agarose gel electrophoresis detects the PCR product, cut glue with the PCR product and reclaim test kit recovery purpose fragment.
Do template with the epimere product of specificity γ 9 chains and the hypomere product of specificity γ 9 chains, with 5 '-CGGGGTACCATGCTGTCACTGCTCCACAC-3 ' and, 5 '-CTCGAGTCATCATGATTTCTCTCCAT-3 ' is primer, obtain about 900bp fragment with the amplification of bridging PCR method, be specificity γ 9 chain cDNA total lengths, through order-checking, its nucleotides sequence is classified the sequence 1 in sequence table as.
Do template with the epimere product of non-specific γ 9 chains and the hypomere product of non-specific γ 9 chains, with 5 '-CGGGGTACCATGCTGTCACTGCTCCACAC-3 ' and, 5 '-CTCGAGTCATCATGATTTCTCTCCAT-3 ' obtains about 900bp fragment for the amplification of primer bridging PCR method, be non-specific γ 9 chain cDNA total lengths, through order-checking, its nucleotides sequence is classified the sequence 2 in sequence table as.
Do template with the epimere product of specificity δ 2 chains and the hypomere product of specificity δ 2 chains, 5 '-CGGGGTACCATGCAGAGGATCTCCTCCCTC-3 ' and 5 '-CCGCTCGAGTTACAAGAAAAATAACTTGGCAGTC-3 ' is primer bridging PCR method, amplification obtains about 861bp fragment, be specificity δ 2 chain cDNA total lengths, through order-checking, its nucleotides sequence is classified the sequence 3 in sequence table as.
Do template with the epimere product of non-specific δ 2 chains and the hypomere product of non-specific δ 2 chains, 5 '-CGGGGTACCATGCAGAGGATCTCCTCCCTC-3 ' and 5 '-CCGCTCGAGTTACAAGAAAAATAACTTGGCAGTC-3 ' is primer bridging PCR method, amplification obtains about 876bp fragment, be non-specific δ 2 chain cDNA total lengths, through order-checking, its nucleotides sequence is classified the sequence 4 in sequence table as.
The PCR reaction conditions is the same.Cut glue with the PCR product after the total length amplification and reclaim the test kit recovery.
As shown in Figure 1A, A figure is that bridging PCR method builds tuberculosis specificity and non-specific total length γ 9 and δ 2 chains to above amplification.The 1st swimming lane is the non-specific TCR γ 9 chain epimere PCR products of tuberculosis, the 2nd swimming lane is the non-specific TCR γ 9 chain hypomere PCR products of tuberculosis, the 3rd swimming lane is the non-specific TCR γ 9 chain total length PCR products of tuberculosis, the 4th swimming lane is the non-specific TCR δ 2 chain epimere PCR products of tuberculosis, the 5th swimming lane is the non-specific TCR δ 2 chain hypomere PCR products of tuberculosis, and the 6th swimming lane is the non-specific TCR δ 2 chain total length PCR products of tuberculosis; The 7th swimming lane is tuberculosis specificity TCR γ 9 chain epimere PCR products, the 8th swimming lane is tuberculosis specificity TCR γ 9 chain hypomere PCR products, the 9th swimming lane is tuberculosis specificity TCR γ 9 chain total length PCR products, the 10th swimming lane is tuberculosis specificity TCR δ 2 chain epimere PCR products, the 11st swimming lane is tuberculosis specificity TCR δ 2 chain hypomere PCR products, and the 12nd swimming lane is tuberculosis specificity TCR δ 2 chain total length PCR products.
Respectively by special γ 9 chains of above-mentioned recovery, non-specific γ 9 chains, special δ 2 chains and non-specific δ 2 chain cDNA total lengths after Kpn I and Xho I double digestion, respectively with pREP7 (the Morita CT cut through same enzyme, Lee HK, Wang H etal.Structural features of nonpeptide prenyl prophosphates that determine their antigenicityfor human gamma delta T cells[J] .J Immunol, 2001; 167:36-41. the public can obtain from Pathogen Biology institute of the Chinese Academy of Medical Sciences.) and pREP9 (Morita CT, Lee HK, Wang H et al.Structural features ofnonpeptide prenyl prophosphates that determine their antigenicity for human gamma delta T cells[J] .JImmunol, 2001; 167:36-41. the public can obtain from Pathogen Biology institute of the Chinese Academy of Medical Sciences.) connect, be built into recombinant plasmid pREP7-γ 9 (SP), pREP7-γ 9 (non-SP), pREP9-δ 2 (SP) and pREP9-δ 2 (non-SP).Transform bacillus coli DH 5 alpha.The single bacterium colony of picking, prepare test kit upgrading grain with a small amount of plasmid, and enzyme is cut evaluation, send the order-checking of Nuo Sai company.
Result is to contain the sequence 1 in ordered list in recombinant plasmid pREP7-γ 9 (SP), and pREP7-γ 9 (SP) is the carrier obtained between the Kpn I of the sequence 1 insertion pREP7 carrier in sequence table and Xho I restriction enzyme site.To contain the recombinant bacterium called after DH5 α of pREP7-γ 9 (SP)/pREP7-γ 9 (SP).
Contain the sequence 2 in ordered list in recombinant plasmid pREP7-γ 9 (non-SP), and pREP7-γ 9 (non-SP) is the carrier obtained between the Kpn I of the sequence 2 insertion pREP7 carriers in sequence table and Xho I restriction enzyme site.To contain the recombinant bacterium called after DH5 α of pREP7-γ 9 (non-SP)/pREP7-γ 9 (non-SP).
Contain the sequence 3 in ordered list in recombinant plasmid pREP9-δ 2 (SP), and pREP9-δ 2 (SP) is the carrier obtained between the Kpn I of the sequence 3 insertion pREP9 carriers in sequence table and Xho I restriction enzyme site.To contain the recombinant bacterium called after DH5 α of pREP9-δ 2 (SP)/pREP9-δ 2 (SP).
Contain the sequence 4 in ordered list in recombinant plasmid pREP9-δ 2 (non-SP), and pREP9-δ 2 (non-SP) is the carrier obtained between the Kpn I of the sequence 4 insertion pREP9 carriers in sequence table and Xho I restriction enzyme site.To contain the recombinant bacterium called after DH5 α of pREP9-δ 2 (non-SP)/pREP9-δ 2 (non-SP).
But sequence 1-4 is synthetic all.
Plasmid small volume of reagent box extracts the plasmid of DH5 α/pREP7-γ 9 (SP), DH5 α/pREP7-γ 9 (non-SP), DH5 α/pREP9-δ 2 (SP) and DH5 α/pREP9-δ 2 (non-SP), concentration and the purity of Nanodrop instrument Detection and Extraction plasmid, result is that pREP7-γ 9 (SP), pREP7-γ 9 (non-SP), pREP9-δ 2 (SP) and pREP9-δ 2 (non-SP) concentration and purity are respectively 1.5 μ g/ μ l, 1.8 μ g/ μ l, 2.0 μ g/ μ l, 1.6 μ g/ μ l.
Collect 1.2 * 10
7J.RT3-T3.5 cell (ATCC, TIB 153), abandon supernatant liquor after centrifuge washing, RPMI-1640 perfect medium (the Hyclone company that adds 300 μ l, Catalog:SH30809) and 20 μ g pREP7-γ 9 (SP) plasmids and 20 μ g pREP9-δ 2 (SP) plasmids, after room temperature (25 ℃) is hatched 10 minutes, move in electric revolving cup and insert electroporation, it is 250V that electricity turns parameter, 975 μ F.After electricity turns, room temperature (25 ℃) is placed 10 minutes, then (Hyclone company Catalog:SH30809) carries out screening and culturing to move into the RPMI-1640 perfect medium that contains Liu Suanyan NEOMYCIN SULPHATE and homomycin.After surrounding, collect transfection tuberculosis specificity γ 9/ δ 2TCR chain cell.
Adopting uses the same method proceeds to the J.RT3-T3.5 cell by pREP7-γ 9 (non-SP) and pREP9-δ 2 (non-SP), collects the non-specific γ 9/ δ 2TCR chain cell of transfection tuberculosis.
Counting 1 * 10
5Individual transfection tuberculosis specificity and non-specific γ 9/ δ 2TCR chain cell, with PBS (Hyclone, SH30256.01B) after washing 2 times, the anti-gamma delta T CR antibody that adds the anti-α β of 5 μ l fluorescein isothiocyanate (FITC) marks-TCR antibody and 5 μ l phycoerythrin (PE) marks, 4 ℃ of dyeing are after 1 hour, PBS (Hyclone, SH30256.01B) after washing cell 2 times, flow cytometer detects the cell of expressing the non-specific γ 9/ δ 2TCR of tuberculosis or tuberculosis specificity γ 9/ δ 2TCR, cultivate continuing after the positive cell sorting, make its purity reach 90%.
As shown in Figure 1B, 1 for after the non-specific γ 9/ δ 2TCR chain of transfection tuberculosis for concrete outcome, and through the surrounding screening, 36% transfectional cell is expressed gamma delta T CR.2 for after transfection tuberculosis specificity γ 9/ δ 2TCR chain, and through the surrounding screening, 38% transfectional cell is expressed gamma delta T CR.3 be airflow classification after, 88% transfectional cell is expressed the non-specific γ 9/ δ 2TCR of tuberculosis.4 be airflow classification after, 90% transfectional cell is expressed tuberculosis specificity γ 9/ δ 2TCR.
The function that tuberculosis specificity gamma delta T CR transfectional cell and the non-specific gamma delta T CR of tuberculosis transfectional cell are identified to mycobacterium tuberculosis (M.tuberculosis) H37Rv total protein to it is verified, specific as follows: 200ml mycobacterium tuberculosis (M.tuberculosis) H37Rv of surrounding is cultivated in centrifugal collection, and (Zhao et al.Screening and analysisof in vivo induced genes of Mycobacterium tuberculosis.Zhonghua Yi Xue Za Zhi.200888 (3): the 189-93. public can obtain from Pathogen Biology institute of the Chinese Academy of Medical Sciences.) thalline, add the 1ml protein lysate, place on ice 30 minutes, 12000 * g shifts supernatant to new 1.5ml centrifuge tube after centrifugal 30 minutes, obtain the H37Rv total protein, concentration and the purity of Nanodrop instrument Detection and Extraction H37Rv total protein.40 μ gH37Rv total proteins are coated in 24 orifice plates, and 37 ℃ of placements are spent the night, and PBS (Hyclone, SH30256.01B) washes away unconjugated albumen, in 24 orifice plates, add respectively 1 * 10
5Individual tuberculosis specificity gamma delta T CR transfectional cell, tuberculosis non-specific gamma delta T CR transfectional cell and blank (adding the RPMI-1640 perfect medium).Cultivate 24 hours for 37 ℃, collect the situation that supernatant detects its secretion IL-2.Specification sheets (Xin Bosheng company) according to test kit, to the standard substance and testing sample and the blank that add the IL-2 of doubling dilution in 96 orifice plates of coated good anti-IL-2 antibody, hatch 90 minutes for 37 ℃, after washing plate 3 times, add biotinylated IL-2 to detect antibody, hatch 60 minutes for 37 ℃.After washing plate 3 times, add the avidin of horseradish peroxidase-labeled, hatch 20 minutes for 37 ℃.After washing plate 3 times, add the developer tetramethyl benzidine, room temperature (25 ℃) lucifuge is hatched 15 minutes, adds stop buffer, detects the absorbance of OD450nm.According to the concentration of standard substance, calculate the IL-2 secretory volume of testing sample.
Result as shown in Figure 1 C, wherein the expression amount of the IL-2 of tuberculosis specificity gamma delta T CR transfectional cell (TB specificity) is 321pg/ml, the expression amount of the IL-2 of the non-specific gamma delta T CR of tuberculosis transfectional cell (TB is non-specific) is 84pg/ml, and the expression amount of the IL-2 of blank (contrast) is 33pg/ml.The mean value that this numerical value is three experiments.
Result proves, has successfully built tuberculosis specificity gamma delta T CR transfectional cell series, called after tester cell; Successfully built non-specific gamma delta T CR transfectional cell series, called after driver cell.
2. screen phage dodecapeptide storehouse
With tuberculosis specificity gamma delta T CR cell tester cell, the non-specific gamma delta T CR of tuberculosis cell driver cell is probe, the operating process in screening phage dodecapeptide storehouse, as shown in Figure 2 A.
The tuberculosis specificity gamma delta T CR transfectional cell tester cell of the above-mentioned acquisition of the centrifugal collection of difference, the non-specific gamma delta T CR of tuberculosis transfectional cell driver cell, RPMI-1640 nutrient solution (Hyclone company, Catalog:SH30809) after washing twice, cell, cell is resuspended in RPMI-1640 nutrient solution (Hyclone company, Catalog:SH30809), in, in 37 ℃, hatch 1 hour.Centrifugal collecting cell, be resuspended in cell in confining liquid respectively, hatches 1 hour for 37 ℃.To adding 20 μ l phage library stostes (BioLabs, Catalog:#E8110S) (approximately 3 * 10 in driver cell confining liquid
11Phage), hatch 1 hour for 37 ℃.Centrifugal 5 minutes precipitation driver cells of 800 * g, carefully join supernatant liquor in the confining liquid of tester cell, hatches 1 hour for 37 ℃.TBST (TBS (50mM Tris.HCl, pH 7.4and 150mM NaCl))+0.1%Tween-20) wash cell 3 times.Last cell is resuspended in TBS (50mMTris.HCl, pH 7.4 and 150mM NaCl), gets part TBS (50mM Tris.HCl, pH 7.4and 150mM NaCl) and measures phage titre.
Specific as follows: picking intestinal bacteria ER2738 original strain at first carries out the streak inoculation cultivation, in 37 ℃ of overnight incubation on LB-tsiklomitsin flat board.The mono-bacterium colony of picking intestinal bacteria ER2738, be inoculated in the 5mlLB liquid nutrient medium, and on 37 ℃ of shaking tables, shaking culture is spent the night, standby.With TBS (50mM Tris.HCl, pH 7.4and 150mMNaCl), phage is carried out to 1: 10 gradient dilution.The bacterium liquid shaken is carried out to packing by 200 μ l/ pipes, and every pipe adds the phage 10 μ l of a weaker concn, and room temperature (25 ℃) is hatched 5 minutes.In 37 ℃ of incubators, preheating LB/IPTG/X-gal flat board, standby., with microwave oven fusing top layer glue agarose, be sub-packed in test tube every pipe 3ml, the culture test tube of the corresponding use of an extent of dilution of phage simultaneously.The mixture of the phage of each concentration and bacterium is added respectively in the test tube that contains the top-agar carbohydrate gum, quick oscillation mixes, immediately by each the pipe in the mixture impouring be equipped with in the LB/IPTG/X-gal flat board of preheating, gently the turn plate so that top layer glue and bacterium be evenly distributed.It is fast that operational motion is wanted, in order to made it be uniformly distributed in whole agar plate before top layer glue is solidified.Cover plate, place 5 minutes in room temperature, agar is solidified, after upset in 37 ℃ of cultivations.Because the library phage comes from conventional cloning vector M13mp19, it contains lacZ α gene, and in the time of on being layered on containing the flat board of IPTG and X-gal, it is blue that plaque will be.The plaque and the filobactivirus of outside contamination will be white in color on same flat board.After 8～12 hours, blue plaque number is approximately to 100 flat board and carries out plaque counting.Calculate titre: the titre of locus coeruleus number * extension rate=10 μ l phage stostes.All the other are increased, and ER2738 overnight culture 200 μ l and 20ml LB substratum are added in the 250ml flask of sterilizing, and phage to be amplified is joined in above-mentioned flask, in 37 ℃ of lasting joltings, cultivate 4.5 hours.Culture is transferred in the 50ml centrifuge tube, in 4 ℃, centrifugal 10 minutes of 12,000 * g.Supernatant liquor is proceeded in another centrifuge tube, in 4 ℃, 12,000 * g recentrifuge 10 minutes.Get the supernatant liquor on centrifuge tube top 80%, proceed in new centrifuge tube, and add the PEG/NaCl of 1/6 volume.4 ℃ are spent the night.In 4 ℃, centrifugal 15 minutes of 12,000 * g, abandon supernatant, and recentrifuge 30 seconds, remove residual liquid with microsyringe.With the resuspended throw out of 1ml TBS (50mM Tris.HCl, pH 7.4 and 150mM NaCl), and transfer them in Eppendorf tube, in 4 ℃, centrifugal 5 minutes of 12,000 * g, make a small amount of bacterial precipitation mixed.Supernatant is proceeded in new centrifuge tube, with the PEG/NaCl of 1/6 volume, again precipitate phage, ice bath 1 hour.4 ℃, centrifugal 10 minutes of 12,000 * g, remove supernatant, centrifugal 30 seconds, with microsyringe, removes residual liquid.Precipitation is resuspended in 200 μ l TBS (50mM Tris.HCl, pH 7.4 and 150mMNaCl), is the good phage of purifying of amplification.With aforesaid method, it is carried out to titer determination, screen for next round.Carry out respectively several screenings of taking turns, so that further enrichment phagotope.Screening process just changes phage library into the phage suspension after last round of screening amplification, and amount is as the criterion with titre, and each add-on approaches 10 as far as possible
11Above.Washing lotion TBST second takes turns the Tween concentration of using 0.2% instead in addition, and third round is used 0.3% Tween concentration instead, and fourth round changes 0.4% Tween concentration into.Be followed successively by 75 minutes with the action time of driver cell, 90 minutes and 115 minutes, and be followed successively by 45 minutes with the action time of tester cell, 30 minutes and 15 minutes.Last phage of taking turns screening need not be increased, and is directly used in locus coeruleus and chooses the clone.Add the LB substratum that 15ml is fresh in clean triangular flask, then add the 150 μ l bacterium liquid that spends the night, mix.Above-mentioned mixing liquid is distributed into to test tube, every pipe 1ml.Open last clone's plate of taking turns the screening phage and use the rifle head by size to fit, independently blue single plaque is provoked, and carefully puts into test tube, and 37 ℃ are shaken bacterium 4.5 hours.Bacterium liquid is proceeded to 1.5ml EP pipe, centrifugal 10 minutes of 4 ℃ of 12,000 * g.Draw 500 μ l supernatant liquors and newly manage in one, add 100 μ l PEG/NaCl, 4 ℃ precipitate phage 2 hours.Centrifugal 10 minutes of 4 ℃ of 12,000 * g.The exhaustion supernatant liquor, precipitate resuspendedly with 100 μ l TBS (50mM Tris.HCl, pH 7.4 and 150mMNaCl), is the mono-clonal phage of purifying.
ELISA detect the phage screen and transfectional cell in conjunction with situation, specific as follows: collection tester cell, after TBS (50mM Tris.HCl, pH 7.4 and 150mM NaCl) solution is washed twice, with 1 * 10
4Individual/every porocyte number is fixed on the ELISA Sptting plate with 4% paraformaldehyde, and room temperature is placed 30 minutes.With 0.1%TBST, wash 3 times.Add 300 μ l containing 2%BSA or containing the PBS (Hyclone, SH30256.01B) of 5% skim-milk, sealing condition be 37 ℃ 2 hours.With 0.1%TBST, wash 3 times.Every hole adds respectively 1 * 10
8Individual phage, establish the blank group, and 37 ℃ are reacted 2 hours.With TBST, wash 3 times.Add the anti-M13 monoclonal antibody (TBST, dilution in 1: 5000) of HRP mark, 37 ℃ are reacted 1 hour.With TBST, wash 3 times.Add substrate nitrite ion colour developing 20 minutes, use 2M H
2SO
4Termination reaction.The microplate reader reading, measure wavelength 450nm, reference wavelength 630nm.
Reacting between the phage of the positive colony that obtains and transfectional cell, result is as shown in Fig. 2 B, and the phage clone of 2 times that is greater than the diver cell with the tester Cell binding is considered to the mono-clonal phage of positive purifying.Positive colony marks with arrow.
The mono-clonal phage of getting positive purifying carry out the PCR reaction (primer sequence by test kit provide 5 '-TTATTCGCAATTCCTTTAGTG-3 ' and 5 '-GCCCTCATAGTTAGCGTAACG-3 '), result as shown in Figure 2 C, 1-9 is respectively the independent cloning of 9 representatives, and the fragment of amplification 150-180bp is containing the dodecapeptide Insert Fragment.
By above-mentioned, containing the dodecapeptide Insert Fragment, checked order, result is that the epitope peptide that the frequency of occurrences is higher is TP1.
The Function Identification of embodiment 2, epitope peptide TP1
1, candidate's epitope peptide stimulates the detection of tuberculosis specificity gamma delta T CR transfectional cell secretion IL-2
In 24 hole plastic culture plate holes, add respectively 500 μ l to include RPMI-1640 nutrient solution (the Hyclone company of 10 μ g, 20 μ g and 40 μ g candidate epitope peptide TP1 (the epitope peptide TP1 obtained by embodiment 1), Catalog:SH30809) be coated with, hatched 2 hours for 37 ℃.Abandon coating buffer, with the RPMI-1640 nutrient solution, (Hyclone company Catalog:SH30809) washes 3 times.Respectively by 1 * 10
6The tester cell, 1 * 10 of individual/ml
6The driver cell of individual/ml is added in 24 well culture plates that washed.24 hours, collect the tester cell conditioned medium liquid containing 10 μ g TP1, tester cell conditioned medium liquid containing 20 μ g TP1, tester cell conditioned medium liquid containing 40 μ g TP1, driver cell conditioned medium liquid containing 10 μ gTP1, driver cell conditioned medium liquid containing 20 μ g TP1, driver cell conditioned medium liquid containing 40 μ g TP1, containing the tester cell conditioned medium liquid after the blocking-up of 10 μ g TP1, containing the tester cell conditioned medium liquid after the blocking-up of 20 μ g TP1, containing the tester cell conditioned medium liquid after the blocking-up of 40 μ g TP1, the ELISA method detects the expression of (as aforementioned) IL-2.
Result as shown in Figure 3A, after wherein the tester cell of blocking-up refers to the tester cell is hatched to 2 hours altogether with gamma delta T CR blocking-up type antibody (Ebioscience, 16-9959) in advance, the cell obtained.
Expression amount containing the IL-2 of the tester cell of 10 μ g, 20 μ g, 40 μ g TP1 is respectively 385pg/ml, 425pg/ml, 554pg/ml;
Expression amount containing the IL-2 of the driver cell of 10 μ g, 20 μ g, 40 μ g TP1 is respectively 115pg/ml, 220pg/ml, 344pg/ml;
Expression amount containing the IL-2 of the block cell of 10 μ g, 20 μ g, 40 μ g TP1 is respectively 110pg/ml, 180pg/ml, 241pg/ml;
The tester cell is got to 1ml (approximately 1 * 10
6cells/ml), centrifugal 5 minutes of 3000 * g, abandon supernatant, with containing PBS (Hyclone, SH30256.01B) washing lotion is washed 3 times, centrifugal the same, (solute is biotinylated epitope peptide to the biotinylated epitope peptide TP1 solution that adds 10 μ l to be obtained by embodiment 1 containing 1 μ g/ μ l, solvent is PBS (Hyclone, SH30256.01B), hatch 1 hour for 4 ℃, after washing lotion centrifuge washing three times, streptavidin (the ebioscience that adds 2 μ l FITC marks, 11-4317), after 4 ℃ of lucifuges are hatched 30 minutes, washing lotion centrifuge washing three times, with 500 μ l PBS (Hyclone, SH30256.01B) after resuspended, fluorescence intensity according to FITC, flow cytometer detect tester cell and biotinylated epitope peptide TP1 in conjunction with per-cent.Take biotinylated control peptide (aminoacid sequence is ADAATRSSKMPK) as contrast.After TP1 peptide blocking-up refers to the tester cell is hatched to 2 hours altogether with gamma delta T CR blocking-up type antibody (Ebioscience, 16-9959) in advance, then with biotinylated epitope peptide TP1, be combined obtain in conjunction with per-cent.
As shown in Figure 3 B, control peptide, TP1 peptide and the blocking-up of TP1 peptide are respectively 3%, 32% and 12% with the combining ratio of tester cell to result.
Can find out, the combination of epitope peptide TP1 and transfectional cell is the most remarkable, and the combination of TP1 and tuberculosis specificity gamma delta T CR transfectional cell simultaneously can be blocked by gamma delta T CR antibody (Ebioscience, 16-9959).The combination that epitope peptide TP1 and transfectional cell are described is specific, can promote the tuberculosis specificity gamma delta T CR transfectional cell secretion IL-2 of specific combination with it.
2, in epitope peptide TP1 and tuberculosis patient peripheral blood gamma delta T cells in conjunction with situation
Collect respectively tuberculosis patient and normal people's peripheral blood mononuclear cell (from BJ Chest Science Hospital), with pan-gamma delta T CR antibody (Immunotech, Immu515), gamma delta T cells increases respectively.Immobilised 4 μ gpan-gamma delta T CR antibody are coated in 24 orifice plates, 37 ℃ hatch 2 hours after, add 1 * 10
7Individual peripheral blood mononuclear cell adds the IL-2 (BD company, 554603) of 200U/ml simultaneously, within every three days, changes nutrient solution and new cytokine.Proliferation time is 4 weeks.After 4 weeks, with the anti-α β of 5 μ l fluorescein isothiocyanate (FITC) marks-TCR antibody (BD, the purity that the anti-gamma delta T CR antibody (BD, 555717) of 347773) and 5 μ l phycoerythrin (PE) marks detects tuberculosis patient and normal people's gamma delta T cells is 80%.Get respectively the tuberculosis patient of the above-mentioned acquisition of 1ml and normal people's gamma delta T cells (approximately 1 * 10
6cells/ml), centrifugal 5 minutes of 3000 * g, abandon supernatant, with the PBS (Hyclone containing 1%BSA, SH30256.01B) washing lotion is washed 3 times, centrifugal the same, (solute is biotinylated epitope peptide to the biotinylated epitope peptide TP1 solution that adds 10 μ l to be obtained by embodiment 1 containing 1 μ g/ μ l, solvent is PBS (Hyclone, SH30256.01B), hatch 1 hour for 4 ℃, after washing lotion centrifuge washing three times, the streptavidin (ebioscience11-4317) that adds 2 μ l FITC marks, after 4 ℃ of lucifuges are hatched 30 minutes, washing lotion centrifuge washing three times, with 500 μ l PBS (Hyclone, SH30256.01B) after resuspended, the combining ratio of the biotinylated epitope peptide of flow cytometry analysis and tuberculosis patient and normal people's gamma delta T cells.
Result as shown in Figure 4 A, for flow cytometer detect gamma delta T cells in TP1 and tuberculosis patient peripheral blood in conjunction with situation, wherein, contrast is homotype contrast (adopting homotype antibody), can find out, the combination rate that the combination rate of TP1 and gamma delta T cells in normal people's (Healthy People contrasts) peripheral blood is the gamma delta T cells in 5%, TP1 and tuberculosis patient peripheral blood is 33%.
Can in addition, in order to prove TP1, follow naturally occurring gamma delta T cells combination, the biotinylated epitope peptide TP1 that 40 μ g control peptides (ADAATRSSKMPK) and 40 μ g are obtained by embodiment 1 be coated on respectively in 24 orifice plates, adds respectively 1 * 10
6Individual tuberculosis patient and normal people's peripheral blood mononuclear cell, (amplification condition is the same) increased, collecting cell after 2 weeks, with PBS washing lotion (Hyclone, SH30256.01B) wash 3 times, add 2 μ l containing FITC-α β TCR (BD, 347773) and PE-gamma delta T CR (BD, 555717) antibody, 4 ℃ of lucifuges are hatched 1 hour, after washing lotion centrifuge washing three times, with 500 μ l PBS (Hyclone, SH30256.01B), after resuspended, flow cytometer detection TP1 induces the Activation of gamma delta T cells in the tuberculosis patient peripheral blood.
Result as shown in Figure 4 B, for TP1 induces the Activation of gamma delta T cells in the tuberculosis patient peripheral blood.1 induces the increment ratio of gamma delta T cells in normal people's peripheral blood for control peptide, be 2%, 2 induce the increment ratio of gamma delta T cells in the tuberculosis patient peripheral blood for control peptide, be 5%, 3 induce the increment ratio of gamma delta T cells in normal people's peripheral blood for TP1, being 6%, 4 for TP1 induces the increment ratio of gamma delta T cells in the tuberculosis patient peripheral blood, is 18%.
Illustrate that TP1 can induce the increment of gamma delta T cells in the tuberculosis patient peripheral blood.
Acquisition and the functional verification thereof of the proteantigen DXS2 albumen of embodiment 2, gamma delta T cells identification
1, the acquisition of the proteantigen DXS2 albumen of gamma delta T cells identification
1) comparison
The aminoacid sequence of the epitope peptide TP1 that will be obtained by embodiment 1 (sequence 5) is analyzed, is compared with the BLAST analysis software of short sequence, inexact matching, with epitope peptide TP1 matching effect best be the 1-deoxidation-D xylulose-5 phosphate synthase 2 (1-deoxy-D-xylulose 5-phosphate synthase, DXS2) of branch tubercule bacillus M.tuberculosis 02_1987 and branch tubercule bacillus M.tuberculosis T46.
2) prokaryotic expression DXS2 albumen
Branch tubercule bacillus (M.tuberculosis) the H37Rv genomic dna of take is template,
With 5 '-CGGGGTACCATGTTCGACACCGGGCACC-3 '
5 '-CCGCTCGAGTTAATAACTATCGCCGGAATCA-3 ' is primer, and pcr amplification obtains the fragment of 1629bp.
Perhaps artificial synthesized sequence 7 is as template,
With 5 '-CGGGGTACCATGTTCGACACCGGGCACC-3 '
5 '-CCGCTCGAGTTAATAACTATCGCCGGAATCA-3 ' is primer, and pcr amplification obtains the fragment of 1629bp equally.
Above-mentioned PCR product is cut through KpnI and XhoI enzyme, and the enzyme obtained is cut the connection of product and prokaryotic expression carrier PET30a (Novagen, the 69909-3) large fragment of cutting through same enzyme, obtain connecting product, to connect product and transform e. coli bl21, picking list spot, extracting plasmid in a small amount.
Plasmid, through KpnI and XhoI double digestion, is obtained to the positive plasmid of 1629bp, this positive plasmid is sent to order-checking, result containing the sequence 6 in ordered list, is the carrier obtained between the KpnI by sequence 6 insertion PET30a and XhoI restriction enzyme site for this plasmid.Unnamed gene shown in sequence 6 is dxs2, and the albumen of its coding is DXS2, and its aminoacid sequence is the sequence 7 in sequence table, and the direction of insertion of sequence verification goal gene dxs2 and reading frame all correct.By this plasmid called after PET30a-dxs2.Clone's called after DH5 α/PET30a-dxs2 that will contain this plasmid.
BL21/PET30a-dxs2 thalline list spot is seeded to 37 ℃ of incubated overnight in 2ml LB.By 1: 50 dilution proportion bacterium that spends the night, the 1ml bacterium is joined in the 300ml culturing bottle containing the 50mlLB substratum, it is to add the IPTG inductor to continue 37 ℃ of concussions to final concentration 0.8mM at 0.8 o'clock to cultivate 3 hours that 37 ℃ of concussions are cultured to the OD600 value.Get respectively thalline 1ml, centrifugal 12000 * g, 30 seconds results precipitations, resuspended with 100 μ l 1%SDS, mix 70 ℃ of 10min.Centrifugal 12000g * 1min, get supernatant liquor as sample, and antibody (Sigma, H1029) the western blot that does SDS-PAGE and anti-his label detects the expression of DXS2 albumen.
Use binding buffer liquid to separate from intestinal bacteria, wash inclusion body, remove impurity albumen, then dissolve inclusion body with the binding buffer liquid containing 6M urea.10000 * g collects thalline in centrifugal 10 minutes.Abandon supernatant, remove substratum as far as possible.Carry out ultrasonication, resuspended bacterium liquid, shear degradation nucleic acid.Centrifugal 15 minutes of 5000 * g, inclusion body and cell debris are arranged in precipitation.Other soluble proteins partly are arranged in supernatant.Remove supernatant, in every 100ml culture, add 5ml damping fluid ratio, add the resuspended precipitation of binding buffer liquid containing 6M urea.Ice bath is hatched 1 hour, thoroughly dissolves inclusion body.12000 * g removes insoluble composition in centrifugal 30 minutes.Use 0.45um membrane filtration supernatant before crossing His prepacked column (GE, 17-5319) purifying.The preparation of prepacked column and balance, take off the prepacked column lid, and all preservation damping fluids in sucking-off top, remove under post and lock running-on as far as possible.The all damping fluids of 10ml binding buffer liquid balance resin relief flow to end as far as possible.Treat that binding buffer liquid drains off, carry out loading (being appropriate off-the-shelf bacterial lysate).10ml binding buffer liquid is washed post.10ml rinsing damping fluid is washed post.5ml elution buffer elution of bound albumen, be DXS2.
As shown in Figure 5A, the DXS2 molecular weight of albumen of SDS-PAGE electrophoresis prokaryotic expression is at 60KD, with the prediction in the same size for result.Carry out western blot detection by the monoclonal antibody (Sigma, H1029) of anti-his, stripe size correct (Fig. 5 B).
Synthetic NM_032514 (Homo sapiens microtubule-associated protein 1 light chain 3alpha, LC3), adopt above-mentioned method expression and purification to go out reference protein.
3, the Function Identification of the proteantigen DXS2 albumen of gamma delta T cells identification
To adding respectively in 24 hole plastic culture plate holes, 500 μ l include 0,20,40,60,80,100pmol is by reference protein (Homo sapiens microtubule-associated protein 1 light chain 3 alpha of above-mentioned acquisition, LC3.NM_032514) PMI-1640 nutrient solution (Hyclone company, Catalog:SH30809) and 500 μ l include 0,20,40,60,80,100pmol is by PMI-1640 nutrient solution (the Hyclone company of the DXS2 of above-mentioned acquisition, Catalog:SH30809) be coated with, hatched 2 hours for 37 ℃.Abandon coating buffer, with the RPMI-1640 nutrient solution, (Hyclone company Catalog:SH30809) washes 3 times.Respectively by 1 * 10
6The tester cell, 1 * 10 obtained by embodiment 1 of individual/ml
6The driver cell obtained by embodiment 1 of individual/ml is added in 24 well culture plates that washed.24 hours, driver cell (DXS2) supernatant liquor that collect tester cell (reference protein) supernatant liquor contain the different concns reference protein, driver cell (reference protein) supernatant liquor that contains the different concns reference protein, tester cell (DXS2) supernatant liquor that contains different concns DXS2, contains different concns DXS2.
The ELISA method detects the expression (method is the same) of IL-2, result as shown in Figure 5 C,
Containing 0,20,40,60,80, the expression amount of the IL-2 of reference protein tester cell (specificity reference protein) supernatant liquor of 100pmol is respectively 12pg/ml, 16pg/ml, 18pg/ml, 19pg/ml, 18pg/ml, 20pg/ml;
Containing 0,20,40,60,80, the expression amount of the IL-2 of driver cell (non-specific reference protein) supernatant liquor of the reference protein of 100pmol is respectively 12pg/ml, 16pg/ml, 18.5pg/ml, 19.5pg/ml, 18pg/ml, 21pg/ml;
Containing 0,20,40,60,80, the expression amount of the IL-2 of tester cell (specificity DXS2) supernatant liquor of the DXS2 of 100pmol is respectively 21pg/ml, 59pg/ml, 81pg/ml, 135pg/ml, 168pg/ml, 182pg/ml;
Containing 0,20,40,60,80, the expression amount of the IL-2 of the driver cell (non-specific DXS2) of the DXS2 of 100pmol is respectively 19pg/ml, 22pg/ml, 24pg/ml, 26pg/ml, 26pg/ml, 28pg/ml.
The combination that DXS2 and transfectional cell are described is specific, can promote the gamma delta T cells secretion IL-2 of specific combination with it.
In addition, in order to prove DXS2, can follow naturally occurring gamma delta T cells combination, 40pmol reference protein and DXS2 albumen are coated in 24 orifice plates, add respectively 1 * 10
6individual tuberculosis patient and normal people's peripheral blood is (all from BJ Chest Science Hospital, the patient knows the inside story) mononuclearcell, (amplification condition is the same) increased, collecting cell after 2 weeks, with the PBS washing lotion (Hyclone contained, SH30256.01B) wash 3 times, add 2 μ l containing FITC-α β TCR (BD, 347773) and PE-gamma delta T CR (BD, 555717) antibody, 4 ℃ of lucifuges are hatched 1 hour, after washing lotion centrifuge washing three times, with 500 μ l PBS (Hyclone, SH30256.01B) after resuspended, the Activation of gamma delta T cells in the protein induced tuberculosis patient peripheral blood of flow cytometer detection DXS2.
Result as shown in Figure 5 D, increment situation for gamma delta T cells in DXS2 protein activation tuberculosis patient peripheral blood, 1 induces the increment ratio of normal people's peripheral blood gamma delta T cells for reference protein, being 4%, 2 for reference protein, to induce the increment ratio of tuberculosis patient peripheral blood gamma delta T cells, is 8%, the 3 increment ratios that are the protein induced normal people's peripheral blood of DXS2 gamma delta T cells, being 8%, the 4 increment ratio that is the protein induced tuberculosis patient peripheral blood of DXS2 gamma delta T cells, is 20%.
Illustrate that DXS2 albumen can induce the increment of gamma delta T cells in the tuberculosis patient peripheral blood.
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| A.C.Brown et al.The Nonmevalonate Pathway of Isoprenoid Biosynthesis in Mycobacterium tuberculosis Is Essential and Transcriptionally Regulated by Dxs.《JOURNAL OF BACTERIOLOGY》.2010,第192卷(第9期), |
| Deciphering the biolody of Mycobacterium tuberculosis from the complete genome sequence;S.T.Cole et al;《Nature》;19980611;第393卷(第6685期);537-544 * |
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| The Nonmevalonate Pathway of Isoprenoid Biosynthesis in Mycobacterium tuberculosis Is Essential and Transcriptionally Regulated by Dxs;A.C.Brown et al;《JOURNAL OF BACTERIOLOGY》;20100219;第192卷(第9期);2424-2433 * |
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