CN102775495A - Polypeptide mixture, T cell vaccine and application thereof - Google Patents
Polypeptide mixture, T cell vaccine and application thereof Download PDFInfo
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Abstract
The invention relates to the field of immunology, and particularly relates to a polypeptide mixture, a T cell activated by the polypeptide mixture and application of the polypeptide mixture in preparation of drugs preventing or treating Aspergillus fumigatus induced diseases. The polypeptide mixture contains three polypeptides, the amino acid sequences of which are respectively shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3. Preferably, the three polypeptides are in a molar ratio of 1:0.5-2:0.5-2. The invention also provides a method for preparation of a T cell vaccine from the polypeptide mixture.
Description
Technical field
The present invention relates to field of immunology, be specifically related to polypeptide mixture, its activated T cell and cause the application in the medicine of disease preparation prevention or treatment Aspergillus fumigatus.
Background technology
Aspergillus is one type of fungi that nature extensively exists, to the human disease have 40 kinds approximately, modal is Aspergillus fumigatus, black mold and flavus, secondly is terreus, excellent aspergillus, aspergillus versicolor, Aspergillus nidulans etc.The mankind are normally caused a disease through sucking airborne aspergillus spore.(Invasive Aspergillosis is to comprise the host deep aspergillus infection that causes by pathogenic aspergillus IA) to the aggressive aspergillosis, and the state of an illness is dangerous, and lethality rate is high.The aspergillar intracellular toxin can cause tissue necrosis, and focus can be the granular diffuse lesion of infiltration, consolidation, cavity, peribronchitis or grain.
Cellullar immunologic response is the important channel of immunity of organism defence and immunosurveillance, is mainly accomplished by the T cell, therefore identifies t cell epitope, and development T cell vaccine induces body to produce cellullar immunologic response, is the important channel of preventing pathogenic infections such as virus.
Cytotoxic T cell CTL is the cell that has killing activity in the T lymphocyte populations, in the body cell immunity, plays an important role, and effect is particularly outstanding in interior bacterium of particularly antiviral, anti-cell and the antitumor cell.CTL divides CD8
+CTLs.CD4
+The several subgroups of CTLs and CD4-CD8-CTLs, it derives from the T cell bank of lymph appearance hemopoietic stem cell, in thymus gland, develops into heat, and activation in the Lymphoid tissue around.Effect CTL carries out the approach that kills and wounds to target cell and mainly contains two: the molten cytosis of the apoptosis effect of Fast-Fas mediation and the mediation of granule exocytosis approach.Because CTL effect in antiviral is remarkable, thereby receives extensive attention.
CTL is from the T cell bank of lymph appearance hematopoiesis T cell, moved in the full thymus gland by marrow and accomplishes ripening process, becomes initial CTL (naibve CTL).Initial CTL gets into peripheral lymphoid tissue, and the antigen peptide of offering with antigen presenting cell: MHC I quasi-molecule is discerned mutually, become activatory CTL precursor cell (CTL precursor, CTLp).CTLp passes through cell surface molecule; Like CD28, CD2, VLA-4, LFA-1 etc. and costimulatory signal molecule at the antigen presenting cell surface expression; Like combinations such as B7-1, B7-2, LFA-3, ICAM-1, VACM-1; Part CTLp continues differentiation and becomes effect CTL, moves full target cell performance effect; Another part CTLp breeds, and becomes the CTLp cell with memory function, and subinfection is again carried out immunne response.
CTL removes virus infected cell, malignant conversioning cell and variant cell through granule exocytosis secretion pore-forming protein, granzyme, and action-reaction is rapid, tool Ca
2+Dependency and part half Guang L-Aspartase (caspase) dependency.
The immunotherapy of existing aspergillus infection is in the experimental study stage, and is complicated because of existing antigenic component, is mostly the crude protein extract, and complicated component is chaotic, and antigenicity and immunogenicity are poor, and the T cell clone that is difficult to prepare stability and high efficiency is applied to clinical treatment.Cause substituting or supplementary means of disease as pharmacological agent aspergillus, immunotherapy has potential application foreground.
Summary of the invention
The objective of the invention is defective to antigenic component complicacy in the aspergillus infection immunotherapy of prior art and antigenicity and immunogenicity difference; A kind of polypeptide mixture is provided; Contain three peptide species; Its aminoacid sequence is respectively ALWCSAPSL, HLLGQLWLL, YTAAALAAV, respectively shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3.
As preferably, said three peptide species mol ratios are 1: 0.5-2: 0.5-2.
As preferably, said three peptide species mol ratios are 1: 1: 1.
Confirm through external and in vivo tests; Polypeptide mixture according to the invention can and make its secrete cytokines IFN-γ at external remarkable activating T cell, also can imitate in vivo and kill and wound Aspergillus fumigatus, and antigenic component is single; Purity is high, and security and validity significantly are superior to existing Aspergillus fumigatus antigen.
The present invention also provides said polypeptide mixture to be used for preventing or treating the application that aspergillus causes the medicine of disease in preparation, and as preferably, said disease is the aggressive infection by Aspergillus fumigatus.
The aggressive infection by Aspergillus fumigatus is the host deep aspergillus infection that is caused by Aspergillus fumigatus, and the state of an illness is dangerous, and lethality rate is high.The intracellular toxin of Aspergillus fumigatus makes tissue necrosis, and focus can be the granular diffuse lesion of infiltration, consolidation, cavity, peribronchitis or grain.Can invade segmental bronchus, lung, gi tract, neural system or bone, severe patient causes septicemia.
Immunogenicity is meant the ability that can stimulate body to form specific antibody or primed lymphocyte, refers to that promptly antigen can stimulate specific immunocyte, makes activated immune cell, propagation, differentiation, finally produces the characteristic of immunologic effector substance antibody and primed lymphocyte.
The antigen of specificity active immunity must have two kinds of effects: the B cell in the immune response stimulating system, and then produce antibody; Can activate CD4
+With CD8
+T cell, and then inducing producing specificity cell immune response.Polypeptide according to the invention can be combined into mixture with HLAI type and HLAII type molecule, and then activates CD8
+With CD4
+The T cell produces specific cell immunoreaction.
Prepare the antigen peptide specific T-cells with said polypeptide mixture in the embodiments of the invention and be divided into two stages; The one, polypeptide is to the subcutaneous active immunity stage of mouse; The 2nd, external antigen presenting cell with the antigen peptide load stimulates lymphoglandula T cell stage; The cellular immunization therapeutic process of anthropomorphic dummy's aggressive aspergillosis in the mouse body verifies that its T cell vaccine has the passive immunization effect, thereby for polypeptide mixture according to the invention is used as active immunity treatment theoretical basis is provided.
The present invention also provides a kind of T cell vaccine, comprises by aminoacid sequence polypeptide activated T cell shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3.
The T cell vaccine is according to the special polypeptide binding motif synthetic polypeptide of MHC-I quasi-molecule, at the antigen-specific CTL of external evoked generation, is used to treat virus disease.Said polypeptide activated T cell be have the polypeptide epitope specific t-cell receptor, can be in external and/or body by this polypeptide antigen activated T cells.
The T cell of said T cell vaccine from self T cell of this vaccine therapy patient for use or from the allosome T cell of the akin donor of this vaccine therapy patient for use or HLA coupling or part coupling.
As preferably, said T cell vaccine comprises by aminoacid sequence polypeptide activated T cell, aminoacid sequence polypeptide activated T cell and aminoacid sequence polypeptide activated T cell shown in SEQ ID No.3 shown in SEQ ID No.2 shown in SEQ ID No.1.
The present invention also provides said T cell vaccine to be used for preventing or treating the application that Aspergillus fumigatus causes the medicine of disease in preparation.
As preferably, said disease is the aggressive infection by Aspergillus fumigatus.
The application contriver utilizes bioinformatics technique; Prediction and synthetic Aspergillus fumigatus specific antigens peptide sequence; And utilize said polypeptide mixture as antigen prepd T cell vaccine, the T cell vaccine is fed back the cellular immunization therapeutic process of anthropomorphic dummy's aggressive aspergillosis in the mouse body to invasive pulmonary aspergillosis model transgenic mice; Verify that its T cell vaccine has the passive immunization effect, for the clinical cellular immunization of Aspergillus fumigatus is treated the new scheme that provides.
The present invention also provides a kind of preparation method of T cell vaccine, comprises following steps:
Step 1: the PMBC crowd is provided, and it comprises the T cell that obtains with the immunity of three peptide species, and the aminoacid sequence of said three peptide species is respectively shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3;
Step 2: adding the antigen presenting cell of offering said polypeptide stimulates; Repeat this step one or many;
Step 3: sub-elect CD8
+The T cell.
Antigen presenting cell be can be through the MHC molecule with endogenous or exogenous antigen peptide submission in the cell of cell surface.Like monokaryon-scavenger cell, BMDC, bone-marrow-derived lymphocyte.Non-full-time property antigen presenting cell such as endotheliocyte, inoblast, various epithelium and mesothelial cell etc. also have certain angtigen presentation function.Antigen presenting cell according to the invention comprises macronucleus-scavenger cell, BMDC, bone-marrow-derived lymphocyte, endotheliocyte, inoblast etc.
As preferably, said antigen presenting cell is a BMDC.
More preferably, said T cell be this vaccine therapy patient for use self T cell or from the allosome T cell of the akin donor of this vaccine therapy patient for use or HLA coupling or part coupling.
T cell vaccine according to the invention can be used other biology or immunological method preparation; As further utilize limiting dilution technique separately with three kinds of antigen peptide specific T-cells, carry out respectively that TXi Baoshouti TCR protein sequencing, corresponding gene are synthetic, vector construction, transfection from means such as body T cells at these three kinds of antigen peptide specific T-cells of external artificial preparation.
T cell vaccine to the method for the invention preparation carries out phenotypic evaluation, wherein to ALWCSAPSL (P1), HLLGQLWLL (P2), the specific CD8 of YTAAALAAV (P3)
+The T cell accounts for all CD8
+The T cells ratio is 10.66%, 7.29%, 5.03%, and summation is 22.98%.
It is external by Aspergillus fumigatus antigen activatory ability that the enzyme linked immunological spotting method detects T cell vaccine according to the invention, and T cell vaccine according to the invention is a large amount of secretion of gamma-IFN of Aspergillus fumigatus peptide specific T cell.The spore fragmentation test shows; Activity of Aspergillus fumigatus through T cell vaccine culture supernatant liquid according to the invention effect reduces, and under the effect of optical dye FUN-1, presents yellow-green fluorescence, and is approaching with the high-temperature inactivation spore color; And the good spore of active condition is sent out fluorescent orange; Explain that sporocyst culture supernatant liquid the inside composition kills and wounds, spore is dead, and perhaps metabolic function weakens.
T cell vaccine according to the invention or irrelevant antigen peptide (control peptide) Control Peptide (CP) specific T-cells and Aspergillus fumigatus mycelia are directly acted on; The difference that compares both sterilizing abilities; T cell vaccine mycelia kill rate according to the invention is 81.48 ± 5.51%; Irrelevant antigen peptide CP specific T-cells mycelia kill rate is 25.90 ± 12.54%, and both have statistical difference (p＜0.05), explain that the mycelia killing-efficiency is that epitope is dependent.
Electron microscope observation spore and mycelia are by the morphological change after killing and wounding; Investigate the fragmentation effect of CTL to aspergillus fumigatus spores and mycelia; The result show T cell vaccine according to the invention or irrelevant antigen peptide CP specific T-cells and Aspergillus fumigatus mycelia and swelling spore directly acted on after; Negative control group is similar with irrelevant antigen peptide specific T-cells control group result, and spore and mycelia configuration of surface are normal; And breakage appears through spore and mycelia surface that T cell vaccine according to the invention was handled, uneven, mycelia narrows down, shrinkage, and can see the direct effect of T cell and spore.
The in vivo tests result shows: to be Aspergillus fumigatus antigen peptide specific CTL have sure curative effect to the adoptive immunotherapy of invasive pulmonary aspergillosis mouse to T cell vaccine according to the invention, and reduction and the pathological change of lung tissue that shows as prolongation, the lung tissue fungi carrying capacity of infecting mouse survival time for example germinateed, and mycelia quantity reduces, the lung tissue destructiveness alleviates.
Immunosuppressed mice intranasal inoculation Aspergillus fumigatus; Import T cell vaccine according to the invention or PBS by the tail vein after 2 hours, observe and record is respectively organized mouse and counted constantly by kind of a bacterium, to thorough dead time of being experienced; The result shows; T cell vaccine test group according to the invention is compared with CP control group and blank group, obviously prolongs the mouse survival time, and difference has significance statistical significance (p＜0.05).
Mouse lung organizes fungi carrying capacity result to show, T cell vaccine according to the invention is that Aspergillus fumigatus antigen peptide specific CTL treatment group bacterium carrying capacity is (14.56 ± 6.53) * 10
4CFU is with CP specific T-cells treatment group (22.66 ± 16.23 * 10
4CFU) and non-treatment group (23.83 ± 12.46 * 10
4CFU) compare, all have statistical difference (p＜0.05).
Behind the mouse infection the 24th hour, get the mouse lung tissue, 10% formalin fixed; Paraffin embedding, section, with GSM (hexamine silver) dyeing relatively behind the adoptive immunotherapy mouse lung organize mycelia invasion and attack situation; Different grouping mouse lung tissue pathological slice observations shows: compare with non-treatment group with CP specific T-cells input group; T cell vaccine according to the invention be Aspergillus fumigatus peptide specific CTL treatment group mycelia quantity still less, it is slight to invade lung tissue, only has the focal mycelia of small pieces to distribute.
Above result shows; Polypeptide mixture according to the invention and to its specific T cell vaccine the treatment Aspergillus fumigatus cause that disease has using value; Can be used as the pharmacological agent Aspergillus fumigatus and cause substituting or supplementary means of disease, show that the disease that T cell vaccine immunotherapy Aspergillus fumigatus causes has a good application prospect.
Embodiment:
The invention discloses a kind of polypeptide mixture, its activated T cell and be used for preventing or treating the application that aspergillus causes the medicine of disease in preparation, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Product of the present invention, preparation method and application are described through preferred embodiment; The related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
Below in conjunction with embodiment, further set forth the present invention:
Embodiment 1: bioinformatic analysis and polypeptide are synthetic
1.HLA-A*0201 epi-position prediction
Utilize bibliographical information and online information biology resource to carry out the proteic HLA-A*0201 epi-position prediction of Aspergillus fumigatus antigen A sp f16; Its network address is not for being: National Center for Biotechnology Information (http://bimas.dcrt.nih.gov/molbio/hla_bind/); The University of Oklahoma Health Sciences Center, (http://syfpeithi.bmi-heidelberg.com).The proteic aminoacid sequence of Asp f16 is shown in SEQ ID No.4.
After the proteic total length ORF sequence of Asp f16 pressed software requirement input; Prediction ability and HLA-A*0201 (Chinese are modal) bonded antigen peptide; Filter out on several websites prediction mark antigens with higher epi-position with Peptide synthesizer synthetic after, contrived experiment is verified.
2.Asp f16HLA-A*0201 and MHC bonded structural simulation
The candidate polypeptide that will have better MHC avidity carries out the computer organization simulation, utilizes Autodock 2.0software to calculate epitope and HLA-0201 molecule bonded degree of agreement on space structure.
3. according to the computer software The selection result, confirm 3 restricted 9 peptides of HLA-A*0201: P1:ALWCSAPSL is shown in SEQ ID No.1; P2:HLLGQLWLL is shown in SEQ ID No.2; P3:YTAAALAAV is shown in SEQ ID No.3; Control group CP also is 9 peptides, and its aminoacid sequence is CLAVEEVSL, is leukemia cell antigen, and is irrelevant with Aspergillus fumigatus.
4.P1, P2, P3, four peptide sequences of CP give Shanghai shine by force bio tech ltd (ChinaPeptides Co., Ltd.) synthetic, 10 every kind, every 1mg, purity＞80%.Embodiment 2: the foundation of invasive pulmonary aspergillosis mouse model
1. bacterial strain: Aspergillus fumigatus BMU1200 is the bacterial strain of clinical separation from patient's cerebral tissue focus of infection.
2. the preparation of bacteria suspension
(1) the bacterial classification Aspergillus fumigatus BMU1200 commentaries on classics of-70 ℃ of preservations is planted in PDA substratum test tube slant, select single bacterium colony, the PDA substratum was cultivated 5 days for 37 ℃.
(2) contain the normal saline flushing test tube slant of 0.1%Tween20 with 2ml.
(3) collect spore liquid, filter to remove mycelia and substratum composition with 8 layers of sterile gauze.
(4) count with blood counting chamber at microscopically, adjustment spore liquid concentration is 3 * 10
8CFU/ml (AF1).
3. the foundation of infection by Aspergillus fumigatus animal model
Laboratory animal: C57BL/6 transgenic mice (HLA-A*0201), age in 6-8 week, body weight 22～25g, SPF level.Putting into laminar-flow rack raises.Feed is the aseptic feed that Co60 shone, and adds tsiklomitsin (500 μ g/ml) prevention of bacterial in the drinking-water and infects.
Set up the immunosuppressive condition mouse model: at inoculating spores suspension mouse peritoneal injection in preceding 4 days endoxan 300mg/kg (Hengrui Medicine Co., Ltd., Jiangsu Prov.); Preceding 1 day intraperitoneal injection of cyclophosphamide (100mg/kg) of inoculating spores suspension; 3 days intraperitoneal injection of cyclophosphamide (100mg/kg) behind the inoculating spores suspension; Behind the inoculating spores suspension 6 days (100mg/kg), dead until the laboratory animal morbidity.
Anesthesia: infect the same day, the aseptic absorbent cotton of a slice is put into transparent 1L volumetrical glass jar and poured an amount of ether into, mouse is put into cylinder, cover lid immediately sees that mouse changes inhibition over to by originally state of excitation, after whole body loosens, takes out mouse immediately.
Inoculation: left hand is grabbed mouse and is made it to be vertical, and middle finger compresses oral area, makes it to use nasal respiration, and the nameless light belly of pressing makes on the diaphram and moves.Draw 30-50 μ l spore suspension (concentration 3 * 10 with the application of sample rifle
8), splash into by the nostril, relax the abdomen, and keep the mouse erectility about 1 minute.
4. whether checking invasive pulmonary aspergillosis model is successful
Behind inoculating spores suspension 24-36 hour, get the lung tissue of 5 mouse, 10% formalin fixed, paraffin embedding, section, with HE dyeing, PAS dyeing and silver dye, and observe the phenomenon that whether has spore and mycelia to invade lung tissue under the light microscopic and take place.
The HE dyeing process is following:
(1) section was gone into YLENE I 10 minutes;
(2) YLENE II is 5 minutes;
(3) absolute ethyl alcohol I is 1 minute;
(4) absolute ethyl alcohol II is 1 minute;
(5) 95% alcohol I 1 minute;
(6) 95% alcohol II 1 minute;
(7) 90% alcohol 1 minute;
(8) 80% alcohol 1 minute;
(9) wash 1 minute from the beginning;
(10) haematoxylin dyeing is 15 minutes;
(11) wash 1 minute from the beginning;
(12) 1% hydrochloride alcohols differentiation 20 seconds;
(13) wash 5 minutes from the beginning;
Anti-blue 30 seconds of (14) 1% weak ammonias;
(15) wash 1 minute from the beginning;
(16) Yihong dyeing is 5 minutes;
(17) wash 30 seconds from the beginning;
(18) 85% alcohol 20 seconds;
(19) 90% alcohol 30 seconds;
(20) 95% alcohol I 1 minute;
(21) 95% alcohol II 1 minute;
(22) absolute ethyl alcohol I is 2 minutes;
(23) absolute ethyl alcohol II is 2 minutes;
(24) YLENE I, II, III, each is 2 minutes;
(25) neutral gum mounting.
Periodic acid-Xue Fu (PAS) dyeing process is following: (the sharp PAS staining kit of Beijing benefit)
(1) section dewaxes to water;
(2) distillation washing;
(3) 0.5% periodic acids were handled 10 minutes;
(4) the distillation washing is 2 * 1 minutes;
(5) drip Schiff liquid 2ml dyeing 10-15 minute;
(6) flowing water was washed 3 minutes;
(7) dehydration is transparent;
(8) optical resin gum mounting.
PAS stained positive judgement criteria is that various hypha,hyphaes and spore are painted, is scarlet.
Urotropin silver (GMS) dyeing process is following:
At first prepare hexamine silver dye liquor: 3% hexamethylenetetramine aqueous solution 5ml and 2.5% silver nitrate aqueous solution 0.5ml mix, and rock, and become clear, add 2.5% sodium tetraborate 2ml adding distil water again to 40ml.Subsequent use.
(1) tissue dewaxes to water;
(2) distillation washing;
(3) 0.5% periodic acids were handled 10 minutes, and flowing water flushing 3min uses distilled water flushing 2 times again;
(4) go into hexamine silver solution, be positioned over 60 ℃ of constant water bath box interior 1 hour;
(5) distillation washing;
(6) drip gold trichloride and handle, flowing water flushing 5min;
(7) dehydration is transparent;
(8) optical resin gum mounting.
GMS stained positive judgement criteria is that mycelia and the spore of various fungies is painted, is tangible chocolate or brownish black.
The plantation spore suspension is after 24 hours, and mouse lung is organized HE dyeing, and PAS dyeing is gone up visible lung tissue structure with silver-colored dyeing and destroyed, a large amount of aspergillus fumigatus silks formation, and the phenomenon of invading blood vessel is arranged.
Behind inoculating spores suspension 24-36 hour, get mouse, open the thoracic cavity after disconnected neck is put to death, cut off postcava, inject PBS lavation systemic pulmonary circulation from right ventricle with the 5ml syringe, white until lung tissue by red stain.Cut whole lung tissues, insert sterile petri dish, surperficial blood stains are removed in the rinsing of PBS liquid, and aseptic filter paper is drawn excessive moisture.Get and respectively organize the same area lung tissue and place the aseptic plastic pipe, weigh, add 1ml saline water; Grind with tissue homogenizer, the tissue homogenate that dilutes 1,10,100,1000 times is coated on the PDA substratum, count colony-forming unit (colony forming unit behind 37 ℃ of cultivation 36h; CFU); Aspergillus fumigatus carrying capacity in the Units of Account lung tissue, result show that the plantation spore suspension is after 36 hours; The mouse lung tissue through homogenization, doubling dilution, be coated with ware and after 36 hours, count colony-forming unit, the checking mouse becomes mould rate and lung tissue fungi charge capacity situation.Empirical tests totally becomes the mould rate 97%.
Embodiment 3: antigen peptide and lymphocytic the obtaining of control peptide specific C D8+T
1. antigen presenting cell (Antigen presenting cell, preparation APC)
1.1 BMDC (dendritic cells, cultivation DC)
Get 20 C57BL/6 transgenic mices (HLA-A*0201), put to death, immerse immediately in 75% alcohol through taking off cervical vertebra.After 5-10 minute, aseptic free shin bone in super clean bench is removed muscle and reticular tissue totally as far as possible, puts into aseptic PBS then.Cut the bone two ends, wash medullary space repeatedly, turn white until the backbone with containing RPM I1640 complete culture solution (Hyclone) asepsis injector.Stem cell suspension inserted in the RPM I1640 complete culture solution that contains 10% foetal calf serum (Gibco) that (contain penicillium mould 100U/ml, Streptomycin sulphate 100 μ g/ml Gibco), place 37 ℃, 5%CO2 incubator (Thermo) to cultivate.The soft not adherent cell that goes to suspend of inhaling behind the 4h is to wash three times in the RPM I1640 complete culture solution, to remove not attached cell gently.What more renew contains 10% foetal calf serum and antibiotic nutrient solution, adds GM-CSF (R&D, final concentration 20ng/ml) and IL-4 (R&D, final concentration 10ng/ml) simultaneously.Full dose was changed 1 nutrient solution in the 4th day, and composition is the same.The 5th day to culture system adding TNF-α (R&D, final concentration 10ng/ml).The interior cell of results culture system was sophisticated BMDC in the 7th day.
When attached cell is cultivated 3 days at mGM-CSF and mIL-4, DC quantity showed increased, and the part cell suspension is arranged, suspension cell has little projection, a large amount of half adherent half colonies that suspend occur, and the cell on colony surface has little projection.In the time of the 4th day, the cell increase is more, and long dendritic processes appears in the surface.Added TNF-α on the 5th day and impel its maturation, the most of cell to the 7th day nutrient solution is mature on the whole, and becomes bigger spheroidal, and there is the thorn-like projection on the surface.
1.2 the phenotypic evaluation of sophisticated BMDC
Collect the DC that cultivated the 7th day, transfer to 1 * 10
7/ ml is hatched with following fluorescence antibody, is all U.S. Biolegend Company products: FITC-Anti Mouse I-Ab (AF6-120.1), PE-Anti MouseCD80 (16-10A1); FITC-Anti Mouse CD1lc (N418), PE-Anti Mouse CD86 (GL-1).Control antibodies is respectively: FITC-Mouse Ig2a κ Isotype Ctrl (MOPC-173); PE-Armenian Hamster IgG IsotypeCtrl (HTK-888); FITC-Armenian Hamster IgG Isotype Ctrl (HTK-888), PE-Rat IgG2a κ Isotype Ctrl (RTK2758).100 μ l cells and fluorescence monoclonal antibody are reacted 30min at 4 ℃, usefulness after PBS washes 3 times, (BD LSR) detects phenotype to flow cytometer.
The result shows: cultivate 7 days DC; After the mark through four kinds of fluorescence antibodies; Show that expressing the corresponding surface molecular cell quantity per-cent of various traget antibodies is: MHC II-32.80%, CD80-52.39%, CDllc-55.38%; CD86-43.15% shows that the dendritic cell of being cultivated have reached the phenotype maturation of certain degree.
1.3 three antigen 9 peptides (synthetic) of loaded dendritic cell and dissolving and the packing of control peptide CP: the polypeptide of lmg is dissolved among the DMSO (sigma) of 20 μ l, and wherein P1, P2, P3 use polypeptide with 1: 1: 1 mixed as experimental group.Every EP manages 5 μ l packing ,-30 ℃ of freezing preservations.
1.4 sophisticated BMDC antigen peptide load
Ripe DC is divided into two groups, adds three antigens of DMSO dissolved, 9 peptide experimental group and CP control group polypeptide respectively, makes its concentration be all 50 μ g/ml, 37 ℃, 5%CO
2Condition was cultivated 1.5-2 hour, and after the RMPI 1640 training base washings of serum-free 3 times, microscopically is counted, and is with 25Gy rad equivalent irradiation cell, subsequent use as the APC of stimulated in vitro T cell.
2. the preparation of the mouse lymph nodal cell suspension of antigen peptide preimmunization
2.1 initial immunity
Under the aseptic condition lmg experimental group polypeptide antigen (P1, P2, P3 with 1: 1: 1 mixed) and contrast CP are dissolved in respectively among the 10 μ l DMSO, are dissolved in again among the 0.5ml PBS, with 0.5ml Freund's complete adjuvant (CFA; Sigma, St.Louis, MO) complete mixing.The mixing method: the syringe that polypeptide solution and freund's adjuvant are housed respectively with two 2ml links to each other with segment transfusion device sebific duct, pushes away left and right sides mixing mutually 2000 times, and it leaves standstill the down not stratified sign of mixing that is spending the night.
Get 10 C57BL/6 transgenic mices (HLA-A*0201), with its root of the tail portion of 75% cotton ball soaked in alcohol sterilization, subcutaneous antigen peptide and the about 50 μ l of freund's adjuvant homomixture of injecting respectively in its root of the tail portion left and right sides.
2.2 booster immunization: under the aseptic condition lmg experimental group polypeptide antigen (P1, P2, P3 with 1: 1: 1 mixed) and contrast CP are dissolved in respectively among the 10 μ l DMSO, are dissolved in again among the 0.5mlPBS, with 0.5ml Freund's incomplete adjuvant (IFA; Sigma, St.Louis, MO)) complete mixing.The mixing method: the syringe that polypeptide solution and freund's adjuvant are housed respectively with two 2ml links to each other with segment transfusion device sebific duct, pushes away left and right sides mixing mutually 2000 times, and it leaves standstill the down not stratified sign of mixing that is spending the night.
Behind the initial immunity 10-14 days, implement immunity once more.With its root of the tail portion of 75% cotton ball soaked in alcohol sterilization, subcutaneous antigen peptide and the about 50 μ l of freund's adjuvant homomixture of injecting respectively in its root of the tail portion left and right sides.
2.3 the preparation of LNC suspension
Put to death mouse after 7-10 days the immunity back through taking off cervical vertebra once more, immerses immediately in 75% alcohol.After 5-10 minute, in super clean bench, carefully take out the inguinal lymph nodes (about each) of enlargement, it is subsequent use to put into the RPMI RPMI-1640.After collecting neat all mouse inguinal lymph nodes (20 every group), every group places respectively on 150 eye mesh screens, grinds with the 5ml syringe piston, washes screen cloth with nutrient solution frequently in the process of lapping, obtains single cell suspension.With RPMI-1640,400g washed twice in 10 minutes, and transferring cell concn is 1 * 10
6/ ml is subsequent use.
3. antigen peptide specific C D8
+The lymphocytic preparation of T
3.1 with the DC behind the irradiation and the LNC suspension after adherent 4 hours not attached cell with DC: the mixed of T Cell=1: 10-20 is even, and nutrient solution is for containing 10% foetal calf serum and contain antibiotic RPMI 1640, beginning external activation of the first round.Change liquid to cell after 5 days, at this moment begin to add in addition IL-2 (R&D, final concentration 10ng/ml), add the DC behind the freshly prepd irradiation after 5-7 days once more, the stimulation that beginning second is taken turns, two kinds of cells are without the hungry stage of IL-2.By that analogy, altogether through the stimulated in vitro of 3-4 wheel, obtain the higher T lymphocyte populations of specific antigens peptide purity (note,, reduce by half one by one) from the second antigen peptide concentration of taking turns in order to loaded dendritic cell along with the increase that stimulates the wheel number.
3.2 erythrocyte splitting
10 * erythrocyte cracked liquid stoste (BD) with 10 times of aseptic double-distilled water dilutions, is obtained the erythrocyte cracked liquid working fluid.With the T cell suspension with 500g, 5 minutes centrifugal after, careful supernatant discarded; Mix with 1ml erythrocyte cracked liquid working fluid, the piping and druming mixing was hatched under the room temperature condition 5-10 minute; Then 20ml serum-free RPMI RPMI-1640 is added in the cracking system, mix 500g; 5 minutes centrifugal, gives a baby a bath on the third day after its birth repeatedly time.
3.3 dead cell is removed
In the process that cell cultures and specificity improve constantly, can produce a large amount of dead cells and cell debris, use dead cell and remove test kit (Dead Cell Removal Kit, Miltenyi Biotec Germany) is removed.
Preparation 1 * damping fluid:, promptly get 1 * damping fluid, i.e. working fluid with 20 * damping fluid and aseptic double-distilled water mixed with 1: 20.
Light microscopic is counting experimental group and the various TCSs of control group culture system down.
With experimental group and control group lymphocyte suspension with 300g, 10 minutes centrifugal after, the careful suction abandoned supernatant.
Marked by magnetic bead: according to per 10
7Individual cell adds the ratio of 100 μ l immunomagnetic beadses, and cell precipitation and magnetic bead suspension are mixed room temperature held 15 minutes, lucifuge.
Sorting cells: with MS or LS sorting post (Miltenyi Biotec, Germany) place the magnetic bead sorting device (Miltenyi Biotec, Germany) on; Behind working fluid profit post; The marked by magnetic bead cell of 0.5-3ml working fluid dilution is added to cylinder, notes not staying bubble, treat its slowly flow out cylinder collection tube down interior after; Add the working fluid flushing cylinder of 3 times of column volumes one by one, effluent is all collected.
Cell in the collection tube is subsequent use after the washing of centrifugal, RPMI RPMI-1640.
3.4 immunomagnetic beads negativity sorting CD8
+The T lymphocyte
The preparation of damping fluid: in the phosphate buffered saline buffer (PBS) of pH 7.2, add foetal calf serum and YD 30 (EDTA) and make its concentration be respectively 0.5% and 2mM, be damping fluid.Used kit is the CD8a+T Cell Isolation Kit that German Miltenyi Biotec company produces.
With experimental group and control group lymphocyte suspension with 300g, 10 minutes centrifugal after, the careful suction abandoned supernatant.According to per 10
7The ratio of individual cell 40 μ l adds damping fluid, per 10
7The ratio of individual cell 10 μ l adds Biotin-Antibody Cocktail, the piping and druming mixing, and 4-8 ℃ of lucifuge hatched 10 minutes.
According to per 10
7The ratio of individual cell 30 μ l adds damping fluid, per 10
7The ratio of individual cell 20 μ l adds Biotin-Antibody Cocktail, the piping and druming mixing, and 4-8 ℃ of lucifuge hatched 15 minutes.
With the damping fluid washed cell of 10-20 times of volume, 300g, 10 minutes centrifugal after, the careful suction abandoned supernatant.
Sorting cells: LS sorting post is placed on the magnetic bead sorting device; Behind 3ml damping fluid profit post; The marked by magnetic bead cell of 1ml working fluid dilution is added to cylinder, notes not staying bubble, treat its slowly flow out cylinder collection tube down interior after; Add the working fluid flushing cylinder of 3 times of column volumes one by one, effluent is all collected.
Cell in the collection tube is subsequent use after the washing of centrifugal, RPMI RPMI-1640, obtains the CD8 of purity more than 90%
+The T lymphocyte.
4. antigen peptide specific C D8
+The lymphocytic phenotypic evaluation of T
The design of P1-P3 epitope specificity PE-Tetramer is with synthetic: designed and synthesized by Dutch Sanquin company.
Get the CD8 after 6 components are selected
+The T lymphocyte, every group of 2ml, cell concn are 1 * 106/ml.Get wherein 3 groups as experimental group, 300g, 10 minutes centrifugal after; The careful suction abandoned supernatant; Add 40 μ l and contain the PBS of 0.5% foetal calf serum, mix with P1-PE-Tetramer, P2-PE-Tetramer, the P3-PE-Tetramer of 10 μ l respectively, under 18-25 ℃ of condition, hatched 20 minutes; (clone 53-6.7, Biolegend) 2 μ l mix, and hatch 30 minutes under 4 ℃ of conditions with APC anti-mouse CD8a respectively again.The dyeing antibody of 3 control groups is APC-Rat IgG2a, and κ Isotype Ctrl (clone RTK2758, Biolegend)) 2 μ l mix, and hatch 30 minutes under 4 ℃ of conditions.After the PBS washing three times, 300g, 10 minutes are centrifugal, and each group is all transferred concentration 1 * 10
6/ ml, 4 ℃ of preservations, (BD LSR) detects phenotype to sample presentation through flow cytometer immediately.
The result shows; The T cell in the draining lymph node source that subcutaneous in advance immunity obtains; Stimulation through APC (BMDC of load antigen peptide) stimulated in vitro 2-4 wheel: preceding 5 days of the first round did not add cytokine IL-2; Purpose is to guarantee non-specific T necrocytosis, to improve the purity of T cells with antigenic specificity; Take turns since second, the cell fast breeding every other day will go down to posterity; To 3-4 week specific T-cells reach certain purity, red through dissolving, remove dead, CD8
+The moon selects after a series of processing, dyes jointly with the CD8a fluorescence antibody of P1, P2, the P3 specificity tetramer and the APC mark of PE mark.The result shows that CD8 molecule positive rate is more than 90%, and the positive expression rate of P1, P2, P3 is that the shared ratio of various 9 peptide specific T cells is respectively 10.66%, 7.29%, 5.03%, and summation is 22.98%.2-20％
Embodiment 4: the external test that kills and wounds the Aspergillus fumigatus ability
1. (Enzyme-linked immunosorbent assay ELISPOT) detects CD8 to the enzyme linked immunological spotting method
+The T lymphocyte is to the reaction of specific anti primary stimuli, and agents useful for same is Mouse IFN-γ Precoated ELISPOT kit (reaching section is Bioisystech Co., Ltd).
Experiment is divided into groups:
1. PHA (Positive Control, sigma)+Antigen specific T cells; The phytohaemagglutinin positive controls
2. Irradiated antigenloaded-DC alone; The BMDC control group
3. Antigen specific T cells alone; T cell control group
4. Antigen specific DC+Antigen specific T cells; Aspergillus fumigatus source peptide specific T cell experiment group
5. CP specific DC+CP specific T cells; CP peptide specific T cell experiment group
6. Blank DC+Antigen specific T cells alone; Non-specific BMDC stimulates Aspergillus fumigatus source peptide specific T cell control group
7. the non-specific BMDC stimulation of CP of Blank DC+CP specific T cells peptide specific T cell control group.
Concrete experimentation is following:
First day, add cell and antigenic stimulation thing (needing aseptic technique)
Encapsulate the activation of plate in advance: every hole 200 μ l add the RPMI-1640 serum-free medium, and room temperature left standstill after 5-10 minute deducts it.
Add cell suspension, every hole 100 μ l: positive control wells, the T cell count is every hole 1 * 10
4Individual; Test holes, T cell count are every hole 5 * 10
4Individual; Background is born control wells, adds RPMI-1640 and contains the serum nutrient solution.Each sample is done 3 multiple holes.
Add stimulator, every hole 10 μ l: positive control wells adds PHA (Sigma-Aldrich), final concentration 2 μ g/ml; Test holes according to the difference of dividing into groups, adds the irradiation DC or the PBS of different peptide loads respectively, and cell count is 5 * 10
3Individual; Background is born control wells, adds RPMI-1640 and contains the serum nutrient solution.Each sample is done 3 multiple holes.
After all samples and stimulator add, build cover plate, put into 37 ℃, 5%CO
2Incubator 16-36 hour.
Second day, cultivate the back operation
Lysing cell: topple over hole inner cell and substratum.Add ice-cold deionized water, 200 μ L/well, 4 ℃ of refrigerators are placed 10 minutes hypotonic lysing cell.
Wash plate: topple over liquid in the hole, 1 * Washingbuffer, 200 μ L/well wash 5-7 time.The each stop 30-60 second.For the last time, on thieving paper, buckle and do.
Detect antibody incubation: will dilute good biotin labeled IFN-gamma antibodies working fluid and add each experimental port, 100 μ L/well.37 hatched 1 hour.
Wash plate: topple over liquid in the hole, 1 * Washingbuffer, 200 μ L/well wash 5 times.The each stop 30-60 second.For the last time, on thieving paper, buckle and do.
Enzyme joins avidin and hatches: will dilute good enzyme mark avidin working fluid and add each experimental port, 100 μ L/well.37 hatched 1 hour.
Wash plate: topple over liquid in the hole, 1 * Washingbuffer, 200 μ L/well wash 5 times.The each stop 30-60 second.For the last time, on thieving paper, buckle and do.
Colour developing: the AEC colour developing liquid working fluid that will at present join adds each experimental port, 100 μ L/well.The room temperature lucifuge leaves standstill 15-45 minute (at 20-25 ℃, develop the color 5-25 minute more suitable).If room temperature is lower than 20 ℃, suggestion is done colour developing at 37 ℃ of incubators, and inspection in every separated 5-10 minute once.
Color development stopping: topple over liquid in the hole, open the plate base, with deionized water/tap water washing pros and cons and base 3-5 time, color development stopping.Plate is placed on the shady and cool place of room temperature, treats the base that closes after it dries naturally.ELISPOT plate spot counting, and the various parameters of record spot are done statistical study, detect the situation of CD8+T lymphocytic emiocytosis IFN-γ.
The result shows, antigen presenting cell and CD8
+The T lymphocyte produces the different amts of the T cell of cytokine IFN-γ through surplus 20 hours effect in the different grouping hole: 1. positive controls under the stimulation of lower concentration mitogen PHA, a part of Aspergillus fumigatus peptide specific T emiocytosis IFN-γ; Not secretion of gamma-IFN of antigen presenting cell (DC behind the irradiation) is 2. only arranged; When 3. not having the stimulation of APC, Aspergillus fumigatus peptide specific T cell is secretion of gamma-IFN hardly, and cell count is merely 4.0 ± 3.6spots/well; 4. under the stimulation of specificity APC, the Aspergillus fumigatus peptide specific T emiocytosis IFN-γ that quantity is a lot of is up to 266.0 ± 39.2spots/well; 5. under CP load DC stimulates, the CP peptide specific T emiocytosis IFN-γ that quantity is a lot; 6. non-antigen load DC stimulates down, seldom the Aspergillus fumigatus peptide specific T emiocytosis IFN-γ of number; 7. non-antigen load DC stimulates down, seldom the CP peptide specific T emiocytosis IFN-γ of number.
2. spore fragmentation test
Experiment is divided into groups: the spore of 1. living is as negative control; 2. 85 ℃ of 30 minutes deactivation spores are as positive control; 3. the experimental group spore that to be P1-P3 antigen peptide specific CTL stimulate the culture supernatant liquid after the 24h to hatch altogether through the corresponding antigens DC of irradiation (after the load P 1-P3 antigen peptide through) once again.
Optical dye FUN-1 (Molecule probes, Invitrogen) mark is respectively organized spore in advance: FUN-1 concentration is 25 μ M, hatches 45 minutes under the room temperature lucifuge condition, the centre is softly shaken for several times, finishes the back and washes twice with RPMI, adjustment spore concentration 10
6/ ml, 4 ℃ of preservations are subsequent use.
The supernatant of experimental group and control group and ordinary culture medium mix with the preliminary making spore suspension at 1: 1, spend the night.
Respectively organize spore suspension 500g after experiment accomplished, 5 minutes of short duration centrifugal, abandons supernatant; With spore composition smear; The try one's best lucifuge operation of whole process, under fluorescent microscope, observe and show: the activity of Aspergillus fumigatus reduction through the effect of overactivation CTL culture supernatant liquid presents yellow-green fluorescence under the effect of optical dye FUN-1; With the high-temperature inactivation spore color near (green fluorescence); And the good spore of active condition is sent out fluorescent orange, and the illustrative experiment group is dead by the spore that culture supernatant liquid the inside composition kills and wounds, and perhaps metabolic function weakens.
3. mycelia fragmentation test
Experiment is divided into groups: 1. experimental group T cell+mycelia; 2. CP control group T cell+mycelia; 3. acellular (having only mycelia) group; 4. acellular and mycelia is through 10% Superlysoform deactivation group.
Mycelia generates: blood counting chamber counting 1.5 * 10
6Individual aspergillus fumigatus spores joins in the EP pipe that 1mlRPMI 1640 is housed, 45 ℃ of water-baths 16 hours.
In the different holes of 24 well culture plates (Corning), T cells with antigenic specificity and irrelevant antigen peptide CP specific T-cells number mix with 3: 1 ratio and mycelia, and 37 ℃, 5%CO
2Hatch 2h, middle constantly mixing cell and mycelia.Each sample is done 3 multiple holes.
Every hole is used for termination reaction with the ice-cold aseptic double-distilled water of 1ml.Violent piping and druming mixing is to realize the fragmentation dissolving of cell.
Centrifugal 10 minutes of 4 ℃, 3000g, every hole add the XTT that contains 40 μ g/mlCoenzyme Q (Sigma) of prepared fresh, and (37 ℃, 5%CO2 was hatched 1 hour for 0.5mg/ml, Sigma) 400 μ l, middlely blew and beat mixing once with sample injector (Eppendorf).
Every pipe is got 100 μ l supernatants and is joined flat 96 orifice plates (Corning), and (ELISA Reader Bio-Rad) surveys wavelength 450nm and 650nm absorbance respectively, (the 650nm absorbance is used for the non-specific absorption of Quality Control) at ELIASA.
Fungal cell's damage calculation formula:
[A450 of 1-(mycelia and cell be the A450 of the A450-T cells with antigenic specificity pipe of incubation tube altogether)/viable bacteria fiber tube] * 100%
Aspergillus fumigatus antigen peptide specific T-cells nothing to do with antigen peptide CP specific T-cells and Aspergillus fumigatus mycelia are directly acted on; The difference that compares both sterilizing abilities; Change (being directly proportional) through XTT staining analysis absorbancy with the metabolic activity of viable bacteria silk; Calculating the mycelia kill rate is: Aspergillus fumigatus antigen peptide specific T-cells 81.48 ± 5.51%; Irrelevant antigen peptide CP specific T-cells 25.90 ± 12.54%, both have statistical difference (p＜0.05), explain that the mycelia killing-efficiency is that epitope is dependent.
4. electron microscope observation spore and mycelia are investigated the fragmentation effect of CTL to aspergillus fumigatus spores and mycelia by the morphological change after killing and wounding
Spore that preparation experiment is required and mycelia: with the fresh spore blood counting chamber counting 1.5 * 10 of Aspergillus fumigatus
6Individual aspergillus fumigatus spores joins in the EP pipe that 1ml RPMI 1640 is housed, and 45 ℃ of water-baths 2 hours obtain the spore of swelling state; Continued to hatch the short mycelia that has been germinateed 10 hours.
With spore, mycelia, T cell with 1: 1: 2 mixed in 24 orifice plate plate holes, 37 ℃, 5%CO2 was hatched 2 hours, and is middle with sample injector piping and druming mixing 1-2 time.
Each hole suspension is collected centrifuge tube, 500g, 10 minutes centrifugal after, abandoning supernatant gained deposition is scanning electron microscope example.Scanning electron microscope example fixing 20h in 3% (v/v) LUTARALDEHYDE phosphoric acid buffer (100mmol/L, pH 6.8), 1% osmic acid is fixed, through serial acetone dehydration, isoamyl acetate displacement, stagnation point CO
2After dry, sticking platform, the spraying; Under the JEOL-JSM5600LV ESEM, observe and take pictures; The result show Aspergillus fumigatus antigen peptide specific T-cells nothing to do with antigen peptide CP specific T-cells and Aspergillus fumigatus mycelia and swelling spore directly acted on after; Acellular control group is similar with irrelevant antigen peptide specific T-cells control group result, and spore and mycelia configuration of surface are normal; And breakage appears in spore that experimental group CTL handled and mycelia surface, and uneven, mycelia narrows down, shrinkage, and can see the direct effect of T cell and spore.
Embodiment 5: experiment in the body
1. laboratory animal is divided into groups:
1. the aspergillus fumigatus spores group is not inoculated in immunosuppression;
2. invasive pulmonary aspergillosis is not done cell treatment group;
3. the invasive pulmonary aspergillosis antigen peptide specific CTL treatment group of adopting;
4. the invasive pulmonary aspergillosis CP specific T-cells treatment experimental group of adopting.
2. cell input
, polish off after 2 hours at via intranasal application plantation aspergillus fumigatus spores, do not have the fur skin with 70% warm alcohol wipe and expand to impel its blood vessel with the hair of fine sandpaper with mouse tail dorsal part to be injected.Extract cell suspension (concentration 1 * 10 with 1ml syringe (BD Ultra-Fine Insulin Syringe)
6Individual/ml, be suspended among the PBS 4 ℃ of preservations), every mouse is through tail vein injection 100 μ l, and the aspergillus fumigatus spores group is not inoculated in immunosuppression and invasive pulmonary aspergillosis is not done cell treatment group: injection PBS100 μ l.
3. the mouse survival time is counted
Immunosuppressed mice intranasal inoculation Aspergillus fumigatus by tail vein input T cell or PBS, was observed and record is respectively organized mouse and counted constantly by kind of a bacterium after 2 hours, to thorough dead time of being experienced, hour be unit calculating.Every group of experiment mice sample size is 30, and each is organized survival time and is 1. experimental group (Antigen Specific): 70.62 ± 10.12 hours; 2. CP control group (CPControl): 63.85 ± 8.39 hours; 3. blank group (Infected without Therapy) is 62.38 ± 6.75 hours.2. 3. there is not significant difference (p＞0.05) between the group, 1. 2., 1. significant difference (p＜0.05) is all arranged between 3..
4. mouse lung organizes the fungi carrying capacity to calculate
To the mouse infection the 36th hour, get and respectively organize mouse, after putting to death, disconnected neck opens the thoracic cavity, cut off postcava,, white by red stain with the 5ml syringe until lung tissue from right ventricle injection PBS lavation systemic pulmonary circulation.Cut whole lung tissues, insert sterile petri dish, surperficial blood stains are removed in the rinsing of PBS liquid, and aseptic filter paper is drawn excessive moisture.Get and respectively organize the same area lung tissue and place the aseptic plastic pipe; Weigh, add 1ml saline water, grind with tissue homogenizer; The tissue homogenate of 1,10,100,1000 times of dilution is coated on the PDA substratum; 37 ℃ cultivate count behind the 36h colony-forming unit (colony forming unit, CFU), Aspergillus fumigatus carrying capacity in the Units of Account lung tissue.
The result shows that Aspergillus fumigatus antigen peptide specific CTL treatment group bacterium carrying capacity is (14.56 ± 6.53) * 10
4CFU is with CP specific T-cells treatment group (22.66 ± 16.23 * 10
4CFU) and non-treatment group (23.83 ± 12.46 * 10
4CFU) compare, all have statistical difference (p＜0.05), after no difference of science of statistics (p＞0.05) between the two.
5. different grouping mouse lung tissue pathological slice is observed and is compared
To the mouse infection the 24th hour, get the mouse lung tissue, 10% formalin fixed; Paraffin embedding; Section, with GSM (hexamine silver) dyeing relatively behind the adoptive immunotherapy mouse lung organize mycelia invasion and attack situation, find more than Aspergillus fumigatus peptide specific CTL treatment group mycelia of CP specific T-cells input group and non-treatment group; The infringement lung tissue is even more serious, and the latter only has the focal mycelia of small pieces to distribute.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (12)
1. a polypeptide mixture contains three peptide species, and its aminoacid sequence is respectively shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3.
2. polypeptide mixture according to claim 1 is characterized in that, said three peptide species mol ratios are 1: 0.5-2: 0.5-2.
3. polypeptide mixture according to claim 1 is characterized in that, said three peptide species mol ratios are 1: 1: 1.
4. claim 1 or 2 or 3 said polypeptide mixture are used for preventing or treating the application that Aspergillus fumigatus causes the medicine of disease in preparation.
5. application according to claim 4 is characterized in that, said disease is the aggressive infection by Aspergillus fumigatus.
6.T cell vaccine comprises by aminoacid sequence polypeptide activated T cell, aminoacid sequence polypeptide activated T cell and aminoacid sequence polypeptide activated T cell shown in SEQ ID No.3 shown in SEQ ID No.2 shown in SEQ ID No.1.
7. T cell vaccine according to claim 6 is characterized in that, said T cell be this vaccine therapy patient for use self T cell or from the allosome T cell of the akin donor of this vaccine therapy patient for use or HLA coupling or part coupling.
8. each said T cell vaccine of claim 6-7 is used for preventing or treating the application that Aspergillus fumigatus causes the medicine of disease in preparation.
9. application according to claim 8 is characterized in that, said disease is the aggressive infection by Aspergillus fumigatus.
10. the preparation method of a T cell vaccine comprises following steps:
Step 1: the PMBC crowd is provided, and it comprises the T cell that obtains with the immunity of three peptide species, and said amino acid sequence of polypeptide is respectively shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3;
Step 2: adding the antigen presenting cell of offering said polypeptide stimulates; Repeat this step one or many;
Step 3: sub-elect CD8
+The T cell.
11. preparation method according to claim 10 is characterized in that, said antigen presenting cell is a BMDC.
12. preparation method according to claim 11 is characterized in that, said T cell be this vaccine therapy patient for use self T cell or from the allosome T cell of the akin donor of this vaccine therapy patient for use or HLA coupling or part coupling.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL2019156B1 (en) * | 2017-06-30 | 2019-01-10 | Academisch Ziekenhuis Leiden H O D N Leids Univ Medisch Centrum | Treatment of haematological malignancies |
| US11352389B2 (en) * | 2017-06-30 | 2022-06-07 | Academisch Ziekenhuis Leiden (H.O.D.N. Leids Universitair Medisch Centrum) | Treatment of haematological malignancies |
| CN114656572A (en) * | 2022-02-25 | 2022-06-24 | 北京大学第一医院 | BP180 antibody rapid detection kit and preparation method thereof |
-
2011
- 2011-05-12 CN CN2011101231705A patent/CN102775495A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 赵作涛等: "烟曲霉抗原Asp f16 HLA-A*0201限制性的CD8+ CTL抗原表位生物信息学预测与实验室鉴定", 《中国真菌学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL2019156B1 (en) * | 2017-06-30 | 2019-01-10 | Academisch Ziekenhuis Leiden H O D N Leids Univ Medisch Centrum | Treatment of haematological malignancies |
| US11352389B2 (en) * | 2017-06-30 | 2022-06-07 | Academisch Ziekenhuis Leiden (H.O.D.N. Leids Universitair Medisch Centrum) | Treatment of haematological malignancies |
| EP3645560B1 (en) * | 2017-06-30 | 2023-12-20 | Academisch Ziekenhuis Leiden (H.O.D.N. Leids Universitair Medisch Centrum) | Treatment of haematological malignancies |
| CN114656572A (en) * | 2022-02-25 | 2022-06-24 | 北京大学第一医院 | BP180 antibody rapid detection kit and preparation method thereof |
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