CN101311189B - Antigen epitope for exciting protective immunity against tubercle bacillus of human body and uses thereof - Google Patents

Abstract

The invention relates to the field of molecular immunology. In recent years, tuberculosis revives in the global range. The emergence of drug-resistant strain always invalidates antibiotic therapy and the normally used tuberculosis vaccine-BCG vaccine also has limitations. The invention aims at screening the molecular simulation peptide of epitope which can activate the protective immune response of human anti-mycobacterium tuberculosis, studying the protective immune response mechanism of the tuberculosis and further developing a novel concatenated poly epitope tuberculosis vaccine. The invention provides the molecular simulation peptide of the epitope which can activate the protective immune response of human anti-mycobacterium tuberculosis; the amino acid sequence of the peptide is shown as SEQ ID NO:14. The invention further provides a screening method and the function of the peptide. Therefore, the molecular simulation peptide of the invention can more effectively prevent and control the generation and the development of the tuberculosis.

Description

Epitope of the protective immunological reaction of exciting human anti-tubercle bacillus and uses thereof

The application is that the application number of submitting on June 6th, 2006 is 200610027311.2, and denomination of invention is divided an application for the patent application of " epitope of the protective immunological reaction of exciting human anti-tubercle bacillus and uses thereof ".

Technical field:

The present invention relates to the molecular immunology field, be specifically related to can exciting human anti-tubercle molecular mimic peptide and the screening method and the purposes of epitope of protective immunological reaction of bacillus.

Background technology:

(Tuberculosis's tuberculosis in the world TB) revives again in recent years.According to World Health Organization statistics, the whole world have approximately about 2,000,000,000 people of 1/3 population infected tubercule bacillus (Mycobacterium tuberculosis, Mtb), annual New Development patient is about 9,000,000, annual death toll is up to 3,000,000.The World Health Organization has proposed " the global tuberculosis emergency state " (global emergency).This shows that tuberculosis is difficult to simple assessment to impact and the infringement that human health and economy cause.Tuberculosis in China's popular characteristics is: high morbidity, and high resistant rate, high mortality, high infection rate and low degradation rate have had a strong impact on quality of life, society and the national construction and the Economic development of individual and family.The strategy of controlling tuberculosis mainly is directly to use microbiotic at present.Yet, need unite and use multiple microbiotic to reach more than 6 months at least, and because the extensive appearance of Mtb Resistant strain often causes antibiotic therapy invalid.Chang Yong Vaccinum Calmette-Guerini---bacille Calmette-Guerin vaccine (BCG) has significant limitation now: (1) is also unsatisfactory to the effect of adult's tuberculosis; (2) can in the individuality of hypoimmunity, cause serious disease even death; (3) can cause the tuerculoderma positive, make it lose diagnostic significance in mycobacterium tuberculosis infection.Therefore, press for development of new protectiveness Vaccinum Calmette-Guerini efficiently, exploitation detects, treats and prevent novel method lungy, with the generation and the PD of effective controlling tuberculosis, the application prospect that this research field is significant and wide.

Tubercule bacillus (Mtb) is a kind of intracellular pathogen, and body depends on identification and destruction to infected cell to its control.Studies show that in mouse model, the restricted CD4+T cell of MHC II class, the restricted CD8+T cell of MHC I class, IFN-γ, the restricted T cell of TNF-alpha, gamma delta T cells and CD1 has positive protective effect in vivo.In human body, identified other CD8+Mtb specific T-cells of different shaped, but its effect in the tuberculosis pathogenesis is still indeterminate.Scavenger cell: scavenger cell plays an important role in the immune response of human body to Mtb.Mtb can survive in the non-activated scavenger cell.When scavenger cell is activated, can produce cytokines such as IFN-γ, induce the generation of NO, active nitrate medium (RNI) and NO synthetic enzyme, thus the effect of performance immunological effect.Another approach of scavenger cell performance antimicrobial acivity is the fusion of phagosome.But Mtb infects fusion and acidifying that scavenger cell can suppress phagosome, and Mtb is survived in the scavenger cell.The CD4+T cell: Mtb antigen through MHC II quasi-molecule submission to the CD4+T cell.The CD4+T cell can produce cytokine performance effect effects such as IFN-γ, also can induce the apoptosis that is subjected to transfect cell, also can assist the activation of B cell, CD8+T cell.But Mtb infects scavenger cell and can cause scavenger cell MHC II quasi-molecule to express decline, thereby suppresses the immunological effect of CD4+T cell.CD8+T cell: Mtb is engulfed by the phagosome of scavenger cell, and its antigen or its excretory protein enter in the phagosome, see through the phagosome film again and enter scavenger cell, and then by digestion process and by MHC I quasi-molecule submission to the CD8+T cell.The CD8+T cell can produce cytokine performance effect effects such as IFN-γ, also can pass through Fas/Fasl approach, granzyme B etc. and directly bring into play cytotoxic effect.Studies show that BCG can not fully stimulate the CD8+T cell, may be because BCG lose crucial molten after birth element, Mtb antigen can not enter infected cell cytosol and by submission.Existing research mainly is about the aversion response of CD4+T cell in tuberculosis, the dependency of specific C D4+T cell immune response and disease different steps, just under study for action.And the mechanism of action of CD8+T cell in the tuberculosis is not clear and definite as yet.The CD8+T cell is activated many factors that depend on by single antigen peptide, and what wherein gain public acceptance is that it is in the body or the key of external " initially " t cell activation that there is highdensity peptide/MHC mixture in the APC surface.Dendritic cell (DC) is expressed high-level MHC, plays an important role in activating " initially " T cell.About activating the report of the required peptide/MHC complex molecule number of " initially " T cell, have than big-difference, from 300 to 10000 in every cell.But generally, the 100-1000 of the required peptide of the reaction needed of " initially " T cell activation memory cell/MHC complex molecule number doubly.And there are some researches show that 90% by the peptide of CD8+T cell recognition, combine with its MHC I quasi-molecule high affinity.Mostly be 8-10 amino acid from the peptide of HLA I quasi-molecule wash-out.And with studies show that synthetic peptide carries out, length be the amino acid whose peptide of 15-20 also can be in conjunction with HLA I quasi-molecule activated T cell.The length of HLA II quasi-molecule bonded peptide then has suitable flexibility.

Present TB vaccine can be divided into 4 classes: subunit vaccine, dna vaccination, full bacterial vaccine and mixed vaccine.Subunit vaccine is based on one of hypothesis or a few antigen promptly is enough to induce protective immunological reaction.Existing research is tested to subunit vaccine on mouse model, comprises that nutrient solution filters albumen, single albumen, egg white mixture and fusion rotein.Though this class vaccine is very safe, the antigen presentation of this pattern can only stimulate the specific T-cells of limited quantity, and great majority are CD4+T cells, and generally, this class reaction is ofer short duration.Dna vaccination can stimulate CD4+ and CD8+T cell response and lasting, but its security is still worrying, comprises the risk of genome conformity.So far after tested, the most successful TB dna vaccination is Ag85A.Many researchs to full bacterial vaccine are also arranged.Remove individual gene such as virulence factor from Mtb, may reduce bacterial virulence but still the ability of maintenance stimulation CD4+, CD8+ and non-traditional T cell.Security for these mutons is still disputable.Mixed vaccine is to try hard to utilize above-mentioned dominance of strategies, can be as BCG through engineered, and the one or more immunogenicity/protective proteins of overexpression.As, BCG is through the engineered listerlysin that secretes, and this makes BCG albumen can enter the endochylema of infected cell, strengthens the ability that it stimulates the CD8+T cell.Equally, through engineered BCG overexpression Ag85, in the cavy experiment, be better than using separately BCG.

When Mtb and some other intracellular pathogen are grown in the substratum, also discharge protein (substratum filtration albumen) and enter in the extracellular matrix.There is the scholar to cultivate filtered solution, identified the different protein site of kind more than 200 with two-phase polyacrylamide agarose electrophoretic analysis Mtb.With produce in the born of the same parents with substratum in the Mtb that grows produce albumen and compare analysis with two-phase polyacrylamide agarose electrophoresis, can find significant difference.Purifying such as Horwitz has also been identified at Mtb and has been cultivated 6 kinds of the abundantest protein in the filtered solution, comprised antigen 85A, antigen 85B, superoxide-dismutase, hsp71, Mpt51, Mpt63.Can make it to obtain immunizing power with these protein purification immune guinea pigs to Mtb.

Although many antigens have potential cytotoxic T lymphocyte (cytotoxic Tlymphocyte, CTL) epi-position, but have only minority can produce ctl response in first set reaction, main epi-position is called the immunodominance determinant, and other epi-position is called less important determinant or structural determinant.Processed and the submission of less important determinant energy, but when the immunodominance determinant existed, less important determinant can not excite ctl response.Structural epi-position can not may be had problem by submission in the antigen treating processes, comprise specificity and because the flanking sequence of the rich epi-position of bag that the albumen corpusculum is sheared.The factor that influences immunodominance comprises: it is invalid that the antigen of (1) APC is handled; (2) lack suitable TCR with identification epi-position/HLA complex molecule; (3) competition of CD8+T cell antigen submission; (4) the inhibition that produces by immunodominance CD8+T cell to other determinant reaction.For the TB vaccine, especially should consider these influence factors, because almost everyone can be exposed to the bacterium in the environment, BCG and/or Mtb.Though vaccine research concentrates on the immunodominance epi-position, immune response may be concentrated on the epi-position that to escape immunity identification to the research of less important determinant.Report is arranged,, can induce the protective immunological reaction of significant anti-TB, and can not produce such aversion response with the immunodominance epi-position that has with the less important determinant immune mouse of ESAT-6.Therefore, the epitope of the protective immunological reaction of research exciting human anti-tubercle bacillus comprises immunodominance determinant and less important determinant, and is very necessary and urgent.

Summary of the invention:

The objective of the invention is to filter out the molecular mimic peptide of the epitope of protective immunological reaction that can the exciting human anti-tubercle bacillus; study protective immunological reaction mechanism lungy; further develop novel multi-joint poly epi-position Vaccinum Calmette-Guerini, more effectively the generation and the development of prevention and controlling tuberculosis.

Integrated application genome sequence, information biology, functional genomics and immunology research; the invention provides a kind of can exciting human anti-tubercle the molecular mimic peptide of epitope of protective immunological reaction of bacillus, described peptide contains and is selected from following aminoacid sequence:

RLLALLCAAV(SEQ ID NO：2)，

KLILTQPFDV(SEQ ID NO：5)，

RLSQSADQYL(SEQ ID NO：10)，

FLTREMPAWL(SEQ ID NO：12)，

KLIANNTRV(SEQ ID NO：14)，

GLPVEYLQV(SEQ ID NO：15)。

The present invention also provide a kind of can exciting human anti-tubercle the screening method of molecular mimic peptide of epitope of protective immunological reaction of bacillus, it is characterized in that it may further comprise the steps:

Prediction of I epi-position and molecular mimic peptide are synthetic

Application data base SYFPEITHI carries out the prediction of HLA-A*0201 restricted CTL epitope to tubercule bacillus Rv3840c, Rv0309, Rv0129c, Rv0173, synthesizes the molecular mimic peptide that above-mentioned prediction obtains with the F-moc chemical method;

The above-mentioned molecular mimic peptide of II detects and specific antibody detects through avidity analysis, stability analysis, cell proliferation experiment, cytoactive, filters out that avidity is strong, good stability, can produce strong proliferative response, has than the molecular mimic peptide that can produce specific antibody after cytotoxic activity and the immunity by force.

The molecular mimic peptide of epitope that the present invention also provides above-mentioned protective immunological reaction that can the exciting human anti-tubercle bacillus is preparation immunogen, immune animal or produce application on the immunoprotection.

The present invention also provides the purposes of molecular mimic peptide on the multi-joint poly epi-position Vaccinum Calmette-Guerini of preparation of the epitope of above-mentioned protective immunological reaction that can the exciting human anti-tubercle bacillus.

This multi-joint poly epi-position Vaccinum Calmette-Guerini, it contains above-mentioned molecular mimic peptide SEQ ID NO:2, one or more peptide sections that are formed by connecting directly or indirectly in 5,10,12,14,15.

The present invention goes far towards prevention lungy and treatment, especially to the control of many endurance strains, also helps studying molecule, cytosis mechanism in the tuberculosis, promotes the solution of many resistances difficult problem that puzzlement is clinical.

Description of drawings

Fig. 1 predicts avidity and the stability analysis result of antigen peptide and HLA-A*0201

The proliferative response that Fig. 2 negre antigen peptide stimulates PBMCs to produce

Fig. 3 negre antigen inducing peptide PBMC produces specific CTL killing activity

The humoral immune reaction of Fig. 4 tuberculosis antigen inducing peptide

Embodiment:

Below in conjunction with drawings and Examples the present invention is further described.

Embodiment 1:

1 materials and methods

1.1 research object

Pulmonary tuberculosis patient diagnosed's 200 examples, positive 100 examples of HLA-A*0201 wherein, negative 100 examples of HLA-A*0201 are all cultivated check through radiology and clinical phlegm.100 routine HLA-A*0201 are positive, and other chronic lung diseases are the disease control group, and 100 routine HLA-A*0201 are positive, and the healthy volunteer is the normal control group.

1.2 reagent and test kit:

Lymphocyte separation medium Lymphoprep ^TMAvailable from Norway AXIS-SHIELD company;

LPS, people's recombinant il-2, DMSO, BFA, β ₂-mg is all available from Sigma company;

People's leukine, people recombinate IL-4 available from R﹠amp; D company;

RPMI1640 substratum and foetal calf serum are available from Gibco company; (Sigma);

Anti--CD3 of FITC mark, anti--CD8, anti--HLA.A2 streaming monoclonal antibody and detect cell intrinsic factor FITC traget antibody (IFN-γ, TNF-α, IL-10) and be Caltag company product;

RNA extraction agent box is available from Qiagen company;

Time resolved fluorescence DELFIA cell proliferation (AD0200) and cytotoxicity (AD0016) detection kit are all available from Wallac Oy company;

Polypeptide is given birth to worker's biotechnology company limited by Shanghai and is synthesized;

The T2 cell strain is available from U.S. ATCC cell bank.

1.3 key instrument

Flow cytometer (Coulter EPICS XL)

Time resolved fluorescence detector (DELFIA 1235, Perkin Elmer)

1.4 epi-position prediction and polypeptide are synthetic

Application data base SYFPEITHI carries out the prediction of HLA-A*0201 restricted CTL epitope to tubercule bacillus Rv3840c, Rv0309, Rv0129c, Rv0173, whether according to the amino acid on the polypeptide is that anchor residues, auxiliary residue or advantage residue are kept the score, and it might be t cell epitope that score value is higher than 21 fens persons.Give birth to the polypeptide that worker's biotechnology company limited obtains with the synthetic above-mentioned prediction of F-moc chemical method by Shanghai, the synthetic antigen peptide is all through high-efficient liquid phase chromatogram purification (purity＞95%), and identifies through mass spectrum.Lyophilized powder is stored in 4 ℃, with preceding with sterile distilled water recovery polypeptide (2 μ g/ul).

1.5 antigen peptide avidity is analyzed

Adopt the T2 cell strain to carry out the analysis of antigen peptide avidity, its principle mainly is to utilize the characteristics of T2 clone.The HLA-A2 developed by molecule of T2 cell surface zero load is extremely unstable, very fast degraded behind the submission, antigen peptide with it in conjunction with after stablized HLA-A2 expression, the bonding force of antigen peptide and HLA-A2 is strong more, the expression amount of T2 cell surface HLA-A2 molecule is just high more.And owing to antigenic peptide translocator (TAP) essential in the endogenous antigen submission approach of T2 cell lacks, so the increase of the expression amount of T2 cell surface HLA-A2 molecule has reflected the bonding force of exotic antigen peptide and HLA-A2 intuitively.

Concrete grammar is: the T2 cell makes the MHCI class mixture sex change of cell surface with Trisodium Citrate-phosphate buffered saline buffer pre-treatment 1min of pH3.2, uses excessive RPMI1640 (Gibco BRL) nutrient solution to wash immediately 1 time, regulates cell concn to 3 * 10 ⁵, with 1ml/ hole kind in 24 porocyte culture plates.Stimulate with antigen peptide (20ug/ml), and additional Brefeldin (BFA, 10ug/ml) with β 2-MG (1ug/ml), 37 ℃ of 5%CO ₂Saturated humidity is hatched 4h, and is the blank hole not add antigen peptide.Collecting cell, wash 1 time with FACS (PBS that contains 0.2%FCS and 0.1% sodium azide), mouse anti human HLA-A*0201 monoclonal antibody dyeing with the FITC mark, normal temperature dark place reaction 30min, FACS liquid is washed 2 times, suspend with sheath fluid, flow cytometer (Coulter EPICS XL) detects average fluorescent strength.Each experiment repeats 3 times, and the antigen peptide that average fluorescent strength is higher than blank hole average fluorescent strength+3SD has the HLA-A*0201 high-affinity.

1.6 antigen peptide stability analysis

The same pre-treatment of T2 cell is increased to 80 μ g/ml with antigen peptide concentration, 37 ℃ of 5%CO ₂Saturated humidity is hatched 18h, replenish BFA to final concentration be 10ug/ml, continue to hatch 3h.Collecting cell, the same equal fluorescence intensity of lining.Average fluorescent strength is higher than the antigen peptide of blank hole average fluorescent strength+3SD and carries out cell proliferative response and cytotoxicity experiment.

1.7 antigen peptide is induced the CTL preparation

Density gradient centrifugation separates peripheral blood mononuclear cell (the peripheral blood mononuclear cells in the venous blood of EDTA anti-freezing, PBMCs), separate the PBMCs kind obtain in 6 porocyte culture plates, in containing the RPMI1640 substratum of 10%FCS, 37 ℃ of 5%CO ₂Hatch 1.5h.Collect suspension cell (being mainly lymphocyte), it is standby to be stored in liquid nitrogen with 10% methyl-sulphoxide, 90% calf serum.Attached cell is antigen presenting cell-dendritic cell (DC), replenishes to contain GM-CSF (1000U/ml, R﹠amp; D), IL-4 (1000U/ml, R﹠amp; D) and contain the RPMI1640 nutrient solution of 10%FCS, 37 ℃ of 5%CO2 cultivate 7d, replenish fresh medium when needing.Behind the 5d, (1 μ g/ml, Sigma) behind the cultivation 2d, gamma-radiation makes it lose proliferation activity to add LPS.Will through the irradiation DC and antigen peptide (10 μ mol/L) in nutrient solution, 37 ℃ of 5%CO ₂Incubate altogether and spend the night.With 1 * 10 ⁵Load has the DC and 1 * 10 of antigen peptide ⁶24 porocyte culture plates from body PBMCs kind in the RPMI1640 complete culture solution that contains 10%FCS, 37 ℃ of 5%CO ₂Behind the mixed culture 3d, (20U/ml Sigma) continues to cultivate 10d, carries out cell proliferation experiment and cytoactive and detects to add rIL-2.

1.8 cell proliferation experiment

Employing time resolved fluorescence DELFIA cell proliferation detecting kit (AD0200, Wallaroy), will be through radiating 5 * 10 ³The DC100ul of load antigen peptide and 5 * 10 ⁴The antigen peptide inductive from body CTL100ul kind in 96 porocyte culture plates, and with the DC of not load antigen peptide as negative control.Add the 20ulBrdU marking fluid and hatch 10h, centrifugal 1 time, the anti-BrdU monoclonal antibody of adding Eu mark, incubated at room 2h, washing, fixing.Time resolved fluorescence detector (DELFIA 1235) fluorescence intensity, each sample are established 3 multiple holes, and averaging is detected result.Growth coefficient (SI)=contain the average fluorescent strength of the average fluorescent strength/negative control of antigen peptide, the antigen peptide of SI＞2 is considered as can induction of lymphocyte propagation.

1.9 cytotoxicity analysis

Employing time resolved fluorescence DELFIA EUTDA cell toxicity test (AD0116, Wallaroy), it is effector cell (E) that antigen peptide is induced CTL, the T2 cell of load antigen peptide is target cell (T), detects killing activity.Operate with reference to specification sheets, the ratio of effector cell and target cell is 40: 1, cultivates 4h.Time resolved fluorescence detector (DELFIA 1235) fluorescence intensity, each sample are established 3 multiple holes, and averaging is detected result.Cell killing activity=(treating gaging hole fluorescence intensity-natural release aperture fluorescence intensity)/(maximum release aperture fluorescence intensity-natural release aperture fluorescence intensity) * 100%.

1.10 experimentation on animals

1.10.1 animal and grouping

20 of C57BL/6 mouse, 2 monthly ages, body weight (18 ± 2) g, male and female are not limit, and are divided into 4 groups at random, 5 every group.

1.10.2 immunization method

The tuberculosis antigen peptide that filters out with experiment in vitro is an immunogen, injects altogether 3 times, and each 2 weeks at interval are adjuvant first with CFA, and booster immunization is adjuvant with IFA.Mouse reach first the booster immunization amount be 50 μ g/ only, after the complete emulsification of 0.3ml adjuvant, at armpit, inguinal region, armpit is subcutaneous and the abdominal cavity multi-point injection.

1.10.3 the collection of sample with separate

Back 21 days of first immunisation, second immunisation and three immunity, respectively 5/group mouse is taken a blood sample at eye socket, separation of serum, and frozen standby in-20 ℃ of refrigerators.

1.10.4 detection specificity antibody

Employing standard indirect elisa method is measured antibody titers in the serum, measures every treated animal specific antibody titre respectively.By 96 hole elisa plates, antigen concentration is 20 μ g/ml by 100 holes bag, and 4 ℃ are spent the night; With 37 ℃ of sealings of 5%BSA-PBS 120min, wash 3min/ time 3 times; Add the tested animal serum of multiple proportions dilution, 90min is hatched for 37 ℃ in 100 holes.After washing 3 times, add the HRP ELIAS secondary antibody, 37 ℃ of 30min; After washing 3 times, add the DAB substrate, 37 ℃ of 10min, termination reaction.Enzyme connection instrument is measured the absorbance A value in 490nm, makes negative control with normal mouse serum.The result is with two multiple equal value representations of hole A value.With sample A value to be measured/negative control A value＞2 positive standards, with high dilution that positive reaction occurs as antibody titers in this sample.

1.10.5 the malicious protectiveness of attacking of immune mouse is tested

8 weeks attacked poison at interval to after every group of remaining 5 C57BL/6 mouse immunity three times, attacking malicious injection site is mouse tail vein, and injected dose is 1 * 10 ⁶CFU, attacking strain is H37Rv.Attack the poison back and 8 weeks killed mouse, lungs, spleen homogenate dilution are inoculated on the solid Russell medium, all around the back live bacterial count.

2 results

2.1 the synthetic result of epi-position prediction and polypeptide

Through the information biology prediction, select following peptide sequence to synthesize.The synthetic polypeptide identifies that through mass spectrum purity is analyzed all greater than 95% through high pressure liquid chromatography (HPLC).

Rv0309：

VLAPVSLAV(SEQ ID NO：1)

RLLALLCAAV(SEQ ID NO：2)

SLAVVNPWFA(SEQ ID NO：3)

VLAPVSLAVV(SEQ ID NO：4)

Rv0173：

KLILTQPFDV(SEQ ID NO：5)

FLGKLDTFT(SEQ ID NO：6)

VLLVLALLL(SEQ ID NO：7)

RVMVADVWV(SEQ ID NO：8)

YLVGALKLI(SEQ ID NO：9)

RLSQSADQYL(SEQ ID NO：10)

Rv0129c：

GLPVEYLQV(SEQ ID NO：11)

FLTREMPAWL(SEQ ID NO：12)

Rv3804c：

AIYGPQQFV(SEQ ID NO：13)

KLIANNTRV(SEQ ID NO：14)

GLPVEYLQV(SEQ ID NO：15)

2.2 antigen peptide avidity and stability analysis result

The 99% T2 cell of cultivating is all expressed the HLA-A*0201 molecule, but average fluorescent strength a little less than.Above-mentioned synthetic antigen peptide combines activity with HLA-A*0201 and sees Table 1.Raising appears in the cell average fluorescent strength after antigen peptide 1-15 handles, and wherein raises the most obvious with antigen peptide 2 and 14.The average fluorescent strength of average fluorescent strength/the no antigen peptide after T2 cell average fluorescent strength growth multiple=antigen peptide after antigen peptide is handled is handled, with antigen peptide 14 handle the highest, antigen peptide 15 next.Antigen peptide and HLA-A*0201 molecule bonded stability analysis result show, antigen peptide is with after HLA-A*0201 molecule on the T2 cell combines, and antigen peptide 2,3,5,7,8,10,12,14,15 can make the stable (see figure 1) that raises of T2 cell HLA-A*0201 developed by molecule.According to bonding force and stability result, select antigen peptide 2,3,5,7,8,10,12,14,15 to continue the downstream experiment.

The post-stimulatory T2 cell of table 1 antigen peptide HLA-A*0201 average fluorescent strength

Antigen peptide SEQ ID NO	Aminoacid sequence	Simulation source: amino acid position	Average fluorescent strength
				1	VLAPVSLAV	Rv0309：aa.20～28	10.69
2	RLLALLCAAV	Rv0309：aa.3～12	10.86
				3	SLAVVNPWFA	Rv0309：aa.25～34	10.73
4	VLAPVSLAVV	Rv0309：aa.20～29	9.18
				5	KLILTQPFDV	Rv0173：aa.302～311	11.54

6	FLGKLDTFT	Rv0173：aa.191～199	8.08
				7	VLLVLALLL	Rv0173：aa.20～28	11.32
8	RVMVADVWV	Rv0173：aa.69～77	12.46
				9	YLVGALKLI	Rv0173：aa.296～304	8.12
10	RLSQSADQYL	Rv0173：aa.260～269	12.37
				11	GLPVEYLQV	Rv0129c：aa.51～59	10.65
12	FLTREMPAWL	Rv0129c：aa.144～153	11.53
				13	AIYGPQQFV	Rv3804c：aa.178～186	8.29

6	FLGKLDTFT	Rv0173：aa.191～199	8.08
				14	KLIANNTRV	Rv3804c：aa.242～250	13.51
15	GLPVEYLQV	Rv3804c：aa.48～56	12.84
				Negative control	-	-	4.21

2.3 antigen peptide stimulates cellular proliferation

With the ability of cell proliferative response evaluation antigen peptide 2,3,5,7,8,10,12,14 and 15 inducing antigen-specific CTL in tuberculosis patient PBMC, identify further whether it is the specific cell epi-position.Wherein, positive tuberculosis patient 100 examples of HLA-A*0201, negative tuberculosis patient 100 examples of HLA-A*0201,100 routine HLA-A*0201 are positive, and other chronic lung diseases are the disease control group, 100 routine HLA-A*0201 are positive, and the healthy volunteer is the normal control group.If to stimulate coefficient (SI)＞3 is the proliferative response positive.The result shows that tuberculosis patient (the HLA-A*0201 positive and negative patient) PBMC all produces stronger positive proliferative response to antigen peptide 2,5,10,12,14 and 15, and significant proliferative response (see figure 2) do not occur at disease control group and normal healthy controls group.

2.4 antigen peptide induces CTL to kill and wound toxicity

T2 cell with the load antigen peptide is a target cell, and antigen peptide inductive CD8+CTL is the effector cell, and imitating target is 20: 1 than (E/T), analyzes the CTL cytotoxic activity of negre antigen inducing peptide through time resolved fluorescence DELFIA EUTDA cell toxicity test.The result shows, through antigen peptide 2,5,10,12,14 and the 15 CD8+CTL cytotoxic activity (see figure 3)s stronger for the effector cell has that stimulate.

2.5 the dynamic measurement of mouse antibodies titre

2 beginnings in week after the first immunisation, get 1 time/week of blood, separation of serum, with the tuberculosis antigen peptide is envelope antigen, and the ELISA indirect method is measured the specific antibody titre, and first immunisation can detect specific antibody after 3 weeks, antibody titers is in minute, antibody titers increases gradually, and in 8 weeks after the first immunisation, antibody titers reaches the highest 1: 64.Antibody titers raise with immune time and time, and negative control group does not detect the antibody (see figure 4).

2.6 vaccine is to the protection effect analysis of immunized mice

With the mouse of attacking after malicious 8 weeks is experiment material, has studied each vaccine-induced protection and has renderd a service.Experiment shows that the bacterial count of each immune group mouse lungs and spleen significantly reduces.Its protection efficient such as table 2.

The protective response ability that table 2 is vaccine-induced

* the method for calculation of protection ratio are: the logarithmic value that negative group bacterial count logarithmic value deducts the bacterial count of vaccine or BCG group obtains.The big more protection efficient of numerical value is high more.

3 conclusions

Integrated application genome sequence, information biology, functional genomics and immunology research, the result confirms, the molecular mimic peptide of following epitope, protective immunological reaction that can the exciting human anti-tubercle bacillus:

(1.RLLALLCAAV SEQ ID NO:2), Rv0309:aa.3～12, simulation source;

(2.KLILTQPFDV SEQ ID NO:5), Rv0173:aa.302～311, simulation source;

(3.RLSQSADQYL SEQ ID NO:10), Rv0173:aa.260～269, simulation source;

(4.FLTREMPAWL SEQ ID NO:12), Rv0129c:aa.144～153, simulation source;

(5.KLIANNTRV SEQ ID NO:14), Rv3804c:aa.242～250, simulation source;

(6.GLPVEYLQV SEQ ID NO:15), Rv3804c:aa.48～56, simulation source.

SEQUENCE LISTING

＜110〉Second Military Medical University, PLA

＜120〉epitope of the protective immunological reaction of exciting human anti-tubercle bacillus and uses thereof

＜130〉specification sheets, claims

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<170>PatentIn version 3.1

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＜213〉artificial sequence

<400>10

Arg Leu Ser Gln Ser Ala Asp Gln Tyr Leu

1 5 10

<210>11

<211>9

<212>PRT

＜213〉artificial sequence

<400>11

Gly Leu Pro Val Glu Tyr Leu Gln Val

1 5

<210>12

<211>10

<212>PRT

＜213〉artificial sequence

<400>12

Phe Leu Thr Arg Glu Met Pro Ala Trp Leu

1 5 10

<210>13

<211>9

<212>PRT

＜213〉artificial sequence

<400>13

Ala Ile Tyr Gly Pro Gln Gln Phe Val

1 5

<210>14

<211>9

<212>PRT

＜213〉artificial sequence

<400>14

Lys Leu Ile Ala Asn Asn Thr Arg Val

1 5

<210>15

<211>9

<212>PRT

＜213〉artificial sequence

<400>15

Gly Leu Pro Val Glu Tyr Leu Gln Val

1 5

Claims (3)

1. the molecular mimic peptide of the epitope of the protective immunological reaction of energy exciting human anti-tubercle bacillus is characterized in that the aminoacid sequence of described peptide is shown in SEQ ID NO:14.

2. the application of molecular mimic peptide on the preparation immunogen of the epitope of the protective immunological reaction of energy exciting human anti-tubercle bacillus as claimed in claim 1.

3. the purposes of molecular mimic peptide on the multi-joint poly epi-position Vaccinum Calmette-Guerini of preparation of the epitope of the protective immunological reaction of energy exciting human anti-tubercle bacillus as claimed in claim 1.

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