CN106749674A - A kind of new asthma polypeptide vaccine and preparation method thereof - Google Patents
A kind of new asthma polypeptide vaccine and preparation method thereof Download PDFInfo
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- CN106749674A CN106749674A CN201611207485.7A CN201611207485A CN106749674A CN 106749674 A CN106749674 A CN 106749674A CN 201611207485 A CN201611207485 A CN 201611207485A CN 106749674 A CN106749674 A CN 106749674A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5437—IL-13
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Mycology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to biomedicine field, and in particular to a kind of novel polypeptide asthma vaccine and preparation method thereof.The technical problem to be solved in the present invention is for the treating asthma of this area provides a kind of new effective selection.The present invention solve technical problem technical scheme be E3 polypeptides nitrogen end connection DiTOX polypeptides or PADRE polypeptides obtain new fused polypeptide, the polypeptide can be prepared into asthma vaccine as main active.Polypeptide vaccine of the invention can effective inducing specific humoral immunity, can be tolerated with break immune, there is good application prospect in terms of as treating asthma polypeptide vaccine.
Description
Technical field
The invention belongs to biomedicine field, and in particular to a kind of new asthma polypeptide vaccine and preparation method thereof.
Background technology
Counted according to the World Health Organization, the current whole world there are the about 300,000,000 positive suffering from asthma of people.Asthma is most common in children
NCD.Asthma is not only the public health problem of high-income countries, and it occurs in All Countries, no matter developing water
Flat height.Most of dead generations relevant with asthma are in low income and middle and low income country.
Bronchial astehma (Bronchial asthma, abbreviation asthma) is a kind of main NCD, to breathe
Difficult and recurrent exerbation of panting is characterized, it be by various kinds of cell (such as eosinophil, mast cell, T lymphocytes, in
Property granulocyte, human airway epithelial cells etc.) and the chronic airway inflammation disease that participates in of cellular component.This chronic inflammation and air flue
High response is related, generally occurs within reversible airflow limitation changeable extensively, and causes the panting of repeated relapsing, out of breath, chest
The symptoms such as vexed or cough, often break out in night and (or) early morning, aggravation, and most of patients spontaneous remission or can be alleviated through treatment.Branch
San bronchial asthma such as diagnosis and treatment not in time, air flue irreversibility constriction and Airway Remodeling can be produced with the extension of the course of disease.At present, asthma
Related gene is not yet completely clear and definite, but related to airway hyperreactivity, IgE regulations and atopic reaction there are some researches show there are
Gene, these genes play an important role in the morbidity of asthma.Mainly include some excitation conditions, such as dirt in environmental factor
The various special and non-specific inhalation (inhalatio) such as mite, pollen, fungi, animal dander, sulfur dioxide, ammonia；Infection, such as bacterium, disease
Poison, protozoon, parasite etc.；Food, such as fish, shrimp, crab, eggs, milk；Medicine, such as Propranolol (inderal), aspirin
Deng；Climate change, motion, gestation etc. are all probably the excitation condition of asthma.
IL-13 is the multifunctional cytokine with regulation inflammation and immune response produced by Th2 cells, by activation
Eosinophil, reduces its apoptosis, promotes the generation of B cell and the secretion of cell factor IgE etc., and it is various that it participates in asthma etc.
The generation of allergic disease, also increase with various adhesion molecules and chemokine expression, the secretion of bronchus goblet cell it is glutinous
Liquid increases relevant with air flue subepithelial fibrosis.Therefore, the function of suppressing IL-13 has become the promising target for the treatment of asthma.
At present, the monoclonal antibody and solubility IL-13 acceptors for having had IL-13 enter clinical II, III phase, but do not go out also
Existing IL-13 polypeptide related vaccines.
The content of the invention
It is the asthma of this area it is an object of the invention to provide a kind of novel therapeutic asthma polypeptide vaccine and preparation method thereof
Treatment provides a kind of new effective selection.
In order to solve the above-mentioned technical problem, the present invention adopts the technical scheme that a kind of fused polypeptide of offer, and the fusion is more
Peptide is formed by connecting by the polypeptide fragment E3 and helper T lymphocyte antigenic polypeptide fragments in IL-13 sources.
Fused polypeptide of the present invention be E3 polypeptides nitrogen end connection DiTOX polypeptides or PADRE polypeptides form；Described E3
The amino acid sequence of polypeptide is LTLKELIEELSNITQ；The amino acid sequence of described DiTOX polypeptides is
AYNFVESIINLFQVVHNSY；The amino acid sequence of described PADRE polypeptides is aK-Cha-VAaWTLKAa.
Wherein, the amino acid sequence of above-mentioned polypeptide is：
AYNFVESIINLFQVVHNSYNLTLKELIEELSNITQ；
Or be aK-Cha-VAaWTLKAaLTLKELIEELSNITQ.A therein represents D-alanine；- Cha- represents L- rings
Hexyl alanine.
Wherein, the N-terminal amino quilt of above-mentioned fused polypeptide or acetylation.
Wherein, the C-terminal carboxyl of above-mentioned fused polypeptide is amidated.
Further, the amino acid sequence of above-mentioned fused polypeptide is：
Ac-AYNFVESIINLFQVVHNSYNLTLKELIEELSNITQ-NH2；
Or be Ac-aK-Cha-VAaWTLKAaLTLKELIEELSNITQ-NH2.
Wherein Ac- represents that the amino of connected amino acid is acetylation；-NH2The carboxyl that expression connects amino acid is amidated；
A represents D-alanine；- Cha- represents L- Cyclohexylalanines；
Present invention also offers purposes of the above-mentioned polypeptide in asthma vaccine is prepared.
Present invention provides a kind of asthma vaccine.The asthma vaccine is prepared by main active of above-mentioned polypeptide
Form.
Wherein, above-mentioned asthma vaccine also contains adjuvant.
Wherein, the adjuvant described in above-mentioned asthma vaccine is aluminum hydroxide adjuvant, cationic-liposome adjuvant, GM-CSF
At least one in (granulocyte-macrophage colony stimutaing factor), gamma interferon.In addition present invention also offers in preparation
The method of the fused polypeptide stated and the method for preparing the above-mentioned asthma vaccine of right.
The beneficial effects of the present invention are：Fused polypeptide molecular weight of the present invention is small, and synthesis is convenient, in that context it may be convenient to use
Fmoc solid phase polypeptide synthesis synthesize, and can also add adjuvant, such as aluminium hydroxide, cationic-liposome, GM-CSF, γ-interference
Element etc. is made various clinical applicable vaccine dosages；And more importantly polypeptide vaccine of the invention can be induced effectively specifically
The humoral immunity of property, can be tolerated with break immune, to there is very outstanding curative effect in animal model in asthma, prepare treating asthma
Property polypeptide vaccine aspect have good application prospect.
Brief description of the drawings
Fig. 1, three kinds of IL-13 antigen small peptide related fusion polypeptides carry out the mice serum antibody titer trend after animal immune
Figure.
Mice serum antibody titer tendency chart after Fig. 2, the animal immune of four kinds of fused polypeptides treatment asthma.
Fig. 3, the animal immune of four kinds of fused polypeptide treatment asthma test bronchoalveolar lavage fluid (BALF) cell counts.
The relevant cell factor testing result figure that Fig. 4, the animal immune of four kinds of fused polypeptide treatment asthma are tested.
The testing result figure of Fig. 5, four kinds of animal immune experiment OVA for treating asthma polypeptide special antibody titer.
Fig. 6：Four kinds of animal immune experimental pathology analysis result figures for treating asthma polypeptide.
Specific embodiment
Fused polypeptide of the present invention is the polypeptide fragment E3 and the connection of helper T lymphocyte antigenic polypeptide fragments originated by IL-13
Form.Specifically, the present invention is to have obtained a kind of new in the nitrogen end connection DiTOX polypeptides or PADRE polypeptides of E3 polypeptides
Fused polypeptide.
The amino acid sequence of described E3 polypeptides is LTLKELIEELSNITQ；The amino acid sequence of described DiTOX polypeptides
It is AYNFVESIINLFQVVHNSY；The amino acid sequence of described PADRE polypeptides is aK-Cha-VAaWTLKAa.
Wherein, the amino quilt of above-mentioned fused polypeptide or acetylation.Carboxyl is acetylation as that can increase polypeptide stability.
Wherein, the C-terminal carboxyl of above-mentioned fused polypeptide is amidated.Carboxyl is amidated as that can increase polypeptide stability.
Further, the amino acid sequence of above-mentioned fused polypeptide is：
Ac-AYNFVESIINLFQVVHNSYNLTLKELIEELSNITQ-NH2；
Or be Ac-aK-Cha-VAaWTLKAaLTLKELIEELSNITQ-NH2.
Wherein Ac- represents that the amino of connected amino acid is acetylation；-NH2The carboxyl that expression connects amino acid is amidated；
A represents D-alanine；- Cha- represents L- Cyclohexylalanines；
Present invention also offers purposes of the above-mentioned polypeptide in asthma vaccine is prepared.
Present invention provides a kind of asthma vaccine.The asthma vaccine is prepared by main active of above-mentioned polypeptide
Form.
Wherein, above-mentioned asthma vaccine also contains adjuvant.
Wherein, the adjuvant described in above-mentioned asthma vaccine is aluminum hydroxide adjuvant, cationic-liposome adjuvant, GM-CSF
At least one in (granulocyte-macrophage colony stimutaing factor), gamma interferon.
The use approach of above-mentioned polypeptide vaccine includes but is not limited to intramuscular injection, hypodermic injection, intracutaneous injection, vein note
Penetrate with schneiderian membrane instil etc..
I.e. the formulation of aforementioned polypeptides vaccine can be injection, instillation or spray.Further, described injection is
Any one in intramuscular dose, subcutaneous injection agent, intracutaneous injection agent or intravenous injection.Certainly, described injection can be with
It is freeze-dried or parenteral solution.
In addition present invention also offers the method and the above-mentioned asthma vaccine of preparation right for preparing above-mentioned fused polypeptide
Method.
Polypeptide in the present invention method such as can manually synthesize and be obtained.The method that one of which can be selected is solid for Fmoc
Phase polypeptide synthesis,
The method comprises the following steps：
1) using chloromethyl polystyrene resin as insoluble solid phase carrier, an amino is closed group first
The amino acid of Fmoc- protections, adds appropriate condensing agent, catalyst and alkali, and amino acid is covalently attached on solid phase carrier, then
The blocking groupses of amino are sloughed with 10~50% piperidine solution, such first amino acid is attached on solid phase carrier.
2) another amino is closed the amino acid of group Fmoc- protections, adds appropriate condensing agent, catalyst and alkali,
Amino acid is covalently attached on the solid phase carrier for being connected to amino acid, then amino is sloughed with 10~50% piperidine solution
Blocking groupses, a dipeptides is just connected to so on solid phase carrier.
3) above-mentioned peptide bond reaction of formation is repeated, makes polypeptide to be synthesized peptide chain is grown from C-terminal to N-terminal in order, directly
Required peptide chain length is reached, the ester bond between hydrolysis peptide chain and solid phase carrier can be obtained with high-efficient liquid phase chromatogram purification
To synthetic polypeptide.
Wherein, the condensing agent in the above method is DCC (dicyclohexylcarbodiimide), EDCI (1- (3- dimethylaminos third
Base) -3- ethyl-carbodiimide hydrochlorides), PyBOP (1H- BTA -1- base oxygen tripyrrole alkyl hexafluorophosphate), HBTU
(BTA-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester), TBTU (2- (1H- benzo trisazo- -1- bases) -1,1,3,
3- tetramethylurea tetrafluoro boric acids ester) at least one；The catalyst is DMAP (N, N- lutidines), HOBT (1- hydroxyls
Base BTA) at least one；The alkali is in triethylamine, DIEA (diisopropyl ethyl amine), N-methylmorpholine
It is at least one.
Wherein, the catalyst in methods described is DMAP (N, N- lutidines), HOBT (1- hydroxy benzo triazoles)
In at least one.
Alkali wherein in the above method is at least in triethylamine, DIEA (diisopropyl ethyl amine), N-methylmorpholine
Kind.
More specifically, tool synthesis step is as follows：
1. the Fmoc- amino acid starting materials with Side chain protective group may be referred to use following raw material
2. synthesis in solid state
Using HBTU/HOBT method activated amino acids, it is connected on amino resins according to sequence：
By taking 10mmol scales as an example, 20 grams of synthesis in solid state resin (resin charging ratio is 0.5mmol/g) is weighed, pour into polypeptide
In synthesizer reactor, the corresponding band protection group ammonia of 40mmol is weighed from C- ends to N- ends according to the amino acid sequence of target polypeptides
Base acid, and be arranged in synthesizer.At ambient temperature, completion synthetic reaction is carried out automatically respectively according to computer program.Synthesis
After end, the polypeptide resin with Side chain protective group is obtained.Polypeptide resin is taken out, is put into after drying 2 hours in vacuum desiccator and is claimed
Weight.
3. Deprotection and precipitation：
Target polypeptides resin with protection group is inserted in the conical flask with plug, lytic reagent such as following table is added：
Constant temperature under the conditions of 25 DEG C, stirring reaction 2 hours；Filtering, collection filtrate, resin are washed with a small amount of trifluoroacetic acid,
Filtering merges collects filtrate.Under agitation, 3000mL ice ether (- 10 DEG C) is added dropwise, white precipitate is obtained, filters, use a small amount of ice
Ether washs crude product, and crude product is put into vacuum desiccator is dried overnight.
4.HPLC is purified
Target polypeptides sterling trifluoroacetic acid salting liquid (purity ＞ 95%) is prepared by the anti-phase purifying of HPLC.
1. chromatographic column：
Can select 10 μm of the Kromasil RP-18 of 50mm*250mmPreparative chromatography post
2. mobile phase：
A：0.1% trifluoroacetic acid aqueous solution
B：0.1% trifluoroacetic acid acetonitrile solution
3. load solution：
It is 8.0mg/mL that polypeptide crude product (purity about 40%~50%) is made into concentration with 0.1% trifluoroacetic acid aqueous solution
The solution of (being calculated by crude product), and by 0.22 μm of membrane filtration.
4. elution requirement：
Using linear gradient elution, flow velocity is 100mL/min, ultraviolet 254nm detections, gradient such as table 1：
Table 1
| Time (min) | Mobile phase B |
| 0-0.5 | 6% |
| 0.5-5 | 6%-20% |
| 5-15 | 20%-40% |
| 15-20 | 40% |
5. sample collection：
During 5-20 minutes, main elution peak is collected according to 50mL/ bottles of distribution, and inspection is analyzed to each sample liquid
Survey.Merge sample liquid of all purity more than 98%.
(4) salt is changed
The sample liquid that will have been purified prepares polypeptide sterling acetate solution (purity ＞ by the anti-phase salt that changes of HPLC
98%).1. chromatographic column：
50mm*250mm Kromasil RP-18 10μmPreparative chromatography post
2. mobile phase：
A：0.5% acetic acid aqueous solution
B：0.5% acetic acid acetonitrile solution
3. load solution：
Isometric ultra-pure water is added to the pure solution of polypeptide
4. elution requirement：
Using linear gradient elution, flow velocity is 100mL/min, ultraviolet 254nm detections, gradient such as table 2：
Table 2
5. sample collection：
Collect all main elution peak solution
6. acetonitrile is removed：
Polypeptide solution is poured into round-bottomed flask, 25 DEG C, rotary evaporation under the conditions of -0.1MPa removes all acetonitriles, raffinate
Body remains freeze-drying by 0.22 μm of membrane filtration.
5. freeze-drying：
Target polypeptides aqueous acetic acid is poured into vacuum freeze drier sample disc, is freezed according to computer program.
Gained polypeptide is both needed to verify correct by mass spectrum.
Obvious those skilled in the art can also synthesize the polypeptide being related in the present invention with other method.
Further description is carried out to present disclosure below by way of specific embodiment combination accompanying drawing.
Screening and determination of the present invention of embodiment 1 to asthma IL-13 small peptides
The compatibility size that IL-13 antigens small peptide is combined with MHC, and IL-13 have been filtered out by consulting library, detection
Interaction between antigen small peptide and IL-13 acceptors, establishes IL-13 antigen small peptides optimal in new asthma vaccine.
Specific method：From Allele Frequency Net Database (AFND, http://
Www.allelefrequencies.net/ the related coverage rate of asthma from 13.5% (DRB1*09) is screened in database:01) arrive
1% (DRB1*14:05) human leucocyte DR epitopes, the Chinese population of total covering 95.9%, obtain 20 epitopes.As
The candidate antigen polypeptide of vaccine, it needs have high-affinity with MHC, so 20 China's HLA-DRB1 epitopes are passed through
Immune Epitope Database(IEDB,http://www.iedb.org) in six kinds of conventional Forecasting Methodologies sieved
Choosing.In this 20 epitopes, the epitope that can be combined with MHC molecules is screened out first；Secondly, HLA-DRB1 tables are being considered
Position compatibility in, by Accelrys Discovery Studio softwares establish ranking before 95 and 5% or less than this hundred
The epitope of fraction can be rested against on IL-13 acceptors and and acceptor interaction.
Then, molecular dynamics can will be passed through with the HLA-DR epitopes of IL-13 acceptor interactions with high-affinity
Simulate to determine the stability of its molecular conformation in IL-13 albumen and polypeptide vaccine.However, it is difficult to be tested on atomic level
Simulate the interaction between the IL-13 polypeptide vaccines and IL-13 acceptors of candidate.Therefore also need to further be worked.
Downloaded from PDB databases and obtain IL-13/IL-13R composite structures, and the IL-13 antigen small peptide structures of candidate by
Accelrys Discovery Studio softwares and the simulation of Amber 12package assembly modules are obtained.In molecular dynamics mould
Before plan, each analogue system requires energy minimization to avoid complicated and the conflicting between solvent in space.In balance mould
When plan, use PME (Particle Mesh Ewald) methods mutual for electrostatic long-range between treatment space structure
Effect, and this electrostatic breaking distance and van der Waals interaction are established as 10 angstroms units.In network terminal system, this
Simulation system is heated to 300K from 0 gradually with the speed of 100ps.Finally, molecular dynamics analog product is placed at when this long
500ns is detected in NPT environment under periodic boundary condition.Meanwhile, the tolerance of all covalent bonds is controlled with SHAKE methods
10-5Angstrom unit value.The coupling constant and pressure value of temperature are arranged on 1.0ps.For sampling parameter, the coordinate of total complex
Holding time is at intervals of 0.1ps.
As a result：Based on the above method, go out three kinds with molecular dynamics simulation and run being answered with IL-13 acceptors for 500ns
The IL-13 antigen short peptide stretch of conjunction, has as the prospect of vaccine.
The 3 kinds of fragments of antigen small peptide for obtaining are E1 (DTKIEVAHF), E2 (NSYTKQLFRHGPF), E1
(LTLKELIEELSNITQ)。
In order to improve immunogenicity, it is contemplated that the nitrogen end coupling helper T lymphocyte antigen in above-mentioned antigen small peptide is more
PEPD itox (AYNFVESIINLFQVVHNSYN), the RMSD values with IL-13R compounds are respectively 0.17,0.19 and 0.23nm.
By artificial chemistry route of synthesis, using Fmoc solid phase polypeptide synthesis, and amino in nitrogen end carries out the carboxylic of acetylation, carbon teminal
Base carries out amidatioon, has obtained polypeptide Ditox-E1 (V1), Ditox-E2 (V2) and Ditox-E3 (V3), the amino of V1, V2, V3
Acid sequence and the modification for carrying out are referring to table 3.
Following scheme is pressed respectively and prepares polypeptide vaccine, by every total μ l system of dosage 200 of mouse, adding less than 200 μ l
Enter physiological saline to mend to 200 μ l, due to being coupled identical T cell auxiliary epitope, then every identical molecular weight is exempted from
Epidemic disease, every group of dosage of 5 mouse is prepared, and every mouse dosage is as follows：
1) NS groups：200μl PBS
2) DiTOX-E1 (V1) group：200μg DiTOX-E1+1000μg Al(OH)3；
3) DiTOX-E2 (V2) group：225μg DiTOX-E2+1125μg Al(OH)3；
4) DiTOX-E3 (V3) group：244μg DiTOX-E3+1220μg Al(OH)3；
2. immunization protocol
From 6~8 week old, the female Balb/c mouse of 18-20g or so are randomly divided into above-mentioned five groups, and multiple spot is subcutaneous to be given
Medicine.It was administered once respectively at the 0th, 2,4,6,8,10 weeks, eye socket takes blood within 1,3,5,7,9,11 weeks.Serum is separated, detects that it is induced
Humoral immune reaction.
Testing index and method：By the production of the antibody titer in ELISA method detection each group mice serum.
ELISA detection method is：By polypeptide LTLKELIEELSNITQ-NH2 coating buffer solution (0.05M carbonate buffers
Liquid, pH9.6) 1 μ g/ml are diluted to, 100 μ l are added per hole in 96 orifice plates, 4 DEG C are overnight.Next day, discard solution in hole, PBST
Washing 3 times.Closed 1 hour for 37 DEG C with 5% skimmed milk power, PBST is washed 3 times.Blood serum sample dilutes (1 with 5% skimmed milk power:
100-1:12,800) blood serum sample for, adding 100 μ l to dilute per hole, 37 DEG C are incubated 1 hour.PBST is washed 5 times.Thereafter, add per hole
Enter 100 μ l HRP-Pr.A (1:5000 dilutions), 37 DEG C are incubated 1 hour.PBST is washed 5 times.Extemporaneous are added in each reacting hole
The μ l of tmb substrate solution 100, color development at room temperature is after 20 minutes, adds 1N H2SO4100 μ l terminating reactions, 450nm wavelength readings.
Result is shown in Fig. 2, and compared to physiological saline immune group and two other experimental group, DiTOX-E3 (V3) is this new to be controlled
Energy inducing mouse produces the immune response result for persistently increasing after treating the vaccine injection of asthma.It is thus determined that E3 is optimal IL-
13 antigen small peptides, as the basis of follow-on test.
The animal immune experiment and the assessment of antibody titer of four kinds of polypeptides that embodiment 3 is built based on E3
In E3 to be defined as three kinds of candidate's IL-13 antigen small peptides, after epitope optimal, with treatment asthma function,
The present invention considers the different helper T cell epitope polypeptide of coupling to strengthen the immunogenicity of IL-13 antigen small peptides, so as to obtain energy
The best candidate polypeptide for the treatment of asthma disease is reached, is vaccine to develop.It is even due to it is determined that during IL-13 antigen short peptide epitopes
Helper cell DiTOX epitope peptides (AYNFVESIINLFQVVHNSYN) for having joined.But, not affirming that the epitope has increases
The optimal effect of IL-13 antigen small peptide immunogenicities, so, we screen other three kinds of epitope peptides：PADRE；TT8；TT9；Point
Coupling acquisition TT8-E3 (V4) is not carried out with E3, ((V6) carries out phase to TT9-E3 (V5) and PADRE together with DiTOX-E3 (V3)
The therapeutic effect assessment of pass.Synthesis mode is identical with DiTOX-E3 synthesis steps, also carries out acetylation, carbon in the amino of nitrogen end
The carboxyl at end carries out amidatioon.
Fused polypeptide V1~V6 in the present invention of table 3
Experimental design and packet：
Following scheme is pressed respectively and prepares polypeptide vaccine, by every total μ l system of dosage 200 of mouse, adding less than 200 μ l
Enter physiological saline to mend to 200 μ l, epitope is aided in due to being coupled different T cells, then every identical molecular weight is exempted from
Epidemic disease, every group of dosage of 5 mouse is prepared, and every mouse dosage is as follows：
1) NS groups：200μl PBS；
2) DiTOX-E3 (V3) group：244μg DiTOX-E3+1220μg Al(OH)3；
3) TT8-E3 (V4) group：205μg DiTOX-E2+1025μg Al(OH)3；
4) TT9-E3 (V5) group：250μg DiTOX-E3+1250μg Al(OH)3；
6) PADRE-E3 (V6) group：178μg DiTOX-E3+890μg Al(OH)3；
Immunization protocol：From 6~8 week old, the female Balb/c mouse of 18~20g or so are randomly divided into above-mentioned five groups, many
Point subcutaneous administration.It was administered once respectively at the 0th, 2,4,6,8,10 weeks, eye socket takes blood within 1,3,5,7,9,11 weeks.Then carry out asthma
The structure of model.
Asthmatic model builds：At the 0th, 1,2 weeks, finite concentration OVA (ovalbumin) abdominal cavity is carried out within continuous three weeks to mouse
Sensitization, every mouse peritoneal priming dose is 20 μ g OVA+2mg Al (OH) 3, at the 3rd week every time with the concentration of 1%OVA to every
Group mouse carries out atomization modeling, once a day, continuous three days.Mouse is put to death after 36-72, bronchoalveolar lavage fluid is collected, separated laggard
Row cell count and relevant cell factor are detected；Pluck eyeball and take blood, separate serum, detection relevant cell factor and OVA specificity
Antibody.
Testing index and method：By the production of the antibody titer in ELISA method detection each group mice serum
ELISA detection method is：By polypeptide LTLKELIEELSNITQ-NH2 coating buffer solution (0.05M carbonate buffers
Liquid, pH9.6) 1 μ g/ml are diluted to, 100 μ l are added per hole in 96 orifice plates, 4 DEG C are overnight.Next day, discard solution in hole, PBST
Washing 3 times.Closed 1 hour for 37 DEG C with 5% skimmed milk power, PBST is washed 3 times.Blood serum sample dilutes (1 with 5% skimmed milk power:
100-1:12,800) blood serum sample for, adding 100 μ l to dilute per hole, 37 DEG C are incubated 1 hour.PBST is washed 5 times.Thereafter, add per hole
Enter 100 μ l HRP-Pr.A (1:5000 dilutions), 37 DEG C are incubated 1 hour.PBST is washed 5 times.Extemporaneous are added in each reacting hole
The μ l of tmb substrate solution 100, color development at room temperature is after 20 minutes, adds 1N H2SO4100 μ l terminating reactions, 450nm wavelength readings.
Result is shown in Fig. 2, it can be found that by after four kinds of vaccine immunities, IgG antibody titre has been obtained necessarily in Mice Body
Degree is excited, and with the increase of immune time, antibody titer is presented obvious ascendant trend.After being immunized at the 4th time, drop
Though degree has certain rising, tend to be steady.V3 groups have reached 10000, V4 groups with V5 groups with the antibody titer of V6 groups
Data have also reached 1000.V3 groups are contrasted with V5 groups, significant significant difference (P ﹤ 0.05) is presented；It is right with V6 groups
Than its difference also has statistical significance.
Bronchoalveolar lavage fluid (BALF) cell of the animal immune experiment of four kinds of polypeptides that embodiment 4 is built based on E3
Count and relevant cell factor testing result：
Embodiment 3 is shown in grouping experiment treatment.
BALF count detection methods are：Alveolar wass is carried out during sacrifice with the physiological saline of precooling, about 1mL lungs are collected
Bubble irrigating solution；4 DEG C, 1200rpm centrifugations 10min collects supernatant, in -80 DEG C of preservations；Precipitation use >=300ul physiological saline is resuspended,
Send in the counting for carrying out GLP detection eosinophils.Supernatant is detected with related kit detection method.
IFN-γ：Detection kit is R＆D Systems Mouse IFN-gamma (Catalog Number:VAL607)
IL-4:Detection kit is R＆D Systems Mouse IL-4 (Catalog Number:VAL603)
IL-13:Detection kit is eBioscience Mouse IL-13 (Catalog Number:88-7137)
Plucking eyeball takes blood simultaneously, and supernatant is collected by centrifugation in -20 DEG C of preservations, prepares detection Total IgE.
IgE:Kit eBioscience Mouse IL-13 (Catalog Number:88-50460)
Result shows to be contrasted with Normal group mouse, total cell number (Tcc), acidophilia in bronchoalveolar lavage fluid after modeling
Granulocyte (Eos) and neutrophil cell (Neu) are above (Fig. 3) of non-modeling.Contrasted with asthma modeling NS groups, vaccine
Immunized mice cell number Tcc, Eos, Neu have a certain degree of downward, wherein, under these three cell counts of V3 groups
Drop is obvious, and its difference has statistical significance (P ﹤ 0.05), and the effect of V6 is also more significant.
Meanwhile, showing (Fig. 4) in the related cytokines measurement result of each asthma, four kinds of cell factors of immune group contain
Amount has obvious decline, wherein, compared to NS groups, V3 groups, two groups of down regulation trends of V6 are obvious.V3 groups IL-13,
IL-4, tetra- groups of testing results of IFN-γ and Total IgE are respectively, 1.86pg/ml, 15.34pg/ml, 9.44pg/ml and
5.650ng/ml.Every kind of cell factor of V3 groups is contrasted with other groups, is respectively provided with significant significant difference (P ﹤ 0.05),
With best effect, the effect of V6 is also more significant.
The detection of the OVA specific antibody titers of the animal immune experiment of four kinds of polypeptides that embodiment 5 is built based on E3
As a result
The production that the present embodiment passes through the OVA specific antibody titers in ELISA method detection each group mice serum.Packet
Experiment process is shown in embodiment 3.
ELISA detection method is：Ovalbumin is diluted with coating buffer solution (0.05M carbonate buffer solutions, pH9.6)
To 1 μ g/ml, 100 μ l are added per hole in 96 orifice plates, 4 DEG C overnight.Next day, solution in hole is discarded, PBST is washed 3 times.With 5%
37 DEG C of skimmed milk power is closed 1 hour, and PBST is washed 3 times.Blood serum sample dilutes (1 with 5% skimmed milk power:100-1:12,800),
The blood serum sample for adding 100 μ l to dilute per hole, 37 DEG C are incubated 1 hour.PBST is washed 5 times.Thereafter, 100 μ l HRP- are added per hole
Pr.A/HRP-IgE(1:5000 dilutions), 37 DEG C are incubated 1 hour.PBST is washed 5 times.Extemporaneous are added in each reacting hole
The μ l of tmb substrate solution 100, color development at room temperature adds 1N H after 20 minutes2SO4100 μ l terminating reactions, 450nm wavelength readings.
Result is shown in Fig. 5, and display is contrasted with the mouse of modeling group, the mouse OVA after new generation vaccine is immune special IgE
With IgG and parting has significant downward to act on.
The immunization experiment pathological analysis of the animal immune experiment of four kinds of polypeptides that embodiment 6 is built based on E3
Each experimental mice in embodiment 3 measures Mouse Weight for the 0th, 2,4,6,8,10 weeks in the immune cycle, and
Fixed during the heart, liver, spleen, lung, kidney are put in 4% paraformaldehyde preservation is collected when mouse is put to death.After tissue is embedded, cut into slices
Colouring method according to H＆E, PAS carries out staining pathologic section, analysis mouse inflammatory cell, cup after new asthmatic medicament is immunized
Whether the pathological phenomenons such as the infiltration of shape cell change.
Result is shown in Fig. 6, is displayed in the sxemiquantitative number by mouse lung HE and the PAS coloring pathological section after vaccine immunity
According to analysis, the inflammatory cell infiltration of its lung (Fig. 6 is left, tracheae week and alveolar week inflammation cul-de-sac scoring number) and goblet cell
Propagation (Fig. 6 is right, is alveolar goblet cell percentage number) carries out contrast with the mouse lung tissue of modeling group a certain degree of
Reduce, the effect of V6 is more significant, and the effect of V3 groups is the most notable.
Claims (10)
1. fused polypeptide, it is characterised in that：Formed in the nitrogen end connection DiTOX polypeptides or PADRE polypeptides of E3 polypeptides；
The amino acid sequence of described E3 polypeptides is LTLKELIEELSNITQ；The amino acid sequence of described DiTOX polypeptides is
AYNFVESIINLFQVVHNSY；The amino acid sequence of described PADRE polypeptides is aK-Cha-VAaWTLKAa.
2. fused polypeptide according to claim 1, it is characterised in that：The amino acid sequence of described polypeptide is：
AYNFVESIINLFQVVHNSYNLTLKELIEELSNITQ；
Or be aK-Cha-VAaWTLKAaLTLKELIEELSNITQ.
3. fused polypeptide according to claim 1 and 2, it is characterised in that：The N-terminal amino of described polypeptide is acetylation.
4. fused polypeptide according to claim 1 and 2, it is characterised in that：The C-terminal carboxyl of described polypeptide is amidated.
5. the fused polypeptide according to any one of Claims 1 to 4, it is characterised in that：Described polypeptide is：
Ac-AYNFVESIINLFQVVHNSYNLTLKELIEELSNITQ-NH2；
Or be Ac-aK-Cha-VAaWTLKAaLTLKELIEELSNITQ-NH2.
6. the fused polypeptide described in any one of Claims 1 to 5 prepare asthma vaccine in purposes.
7. asthma vaccine, it is characterised in that：Fused polypeptide described in any one of claim 1~6 is as main active system
It is standby to form.
8. asthma vaccine according to claim 7, it is characterised in that：Also contain adjuvant.
9. asthma vaccine according to claim 12, it is characterised in that：Described adjuvant is aluminum hydroxide adjuvant, cation
At least one in Liposome Adjuvant, GM-CSF, gamma interferon.
10. the fused polypeptide described in any one of Claims 1 to 4 or the asthma vaccine described in any one of claim 5~8 are prepared
Method.
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| CN201611207485.7A CN106749674A (en) | 2016-12-23 | 2016-12-23 | A kind of new asthma polypeptide vaccine and preparation method thereof |
| PCT/CN2016/113125 WO2018113023A1 (en) | 2016-12-23 | 2016-12-29 | Novel asthma polypeptide vaccine and preparation method therefor |
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| CN201611207485.7A CN106749674A (en) | 2016-12-23 | 2016-12-23 | A kind of new asthma polypeptide vaccine and preparation method thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101090729A (en) * | 2003-05-30 | 2007-12-19 | 艾更斯司股份有限公司 | Antibodies and related molecules that bind to psca proteins |
| TW201138815A (en) * | 2010-01-21 | 2011-11-16 | Daiichi Sankyo Co Ltd | Peptide vaccine |
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|---|---|---|---|---|
| GB0521509D0 (en) * | 2005-10-21 | 2005-11-30 | Novartis Ag | Organic molecules |
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- 2016-12-29 WO PCT/CN2016/113125 patent/WO2018113023A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101090729A (en) * | 2003-05-30 | 2007-12-19 | 艾更斯司股份有限公司 | Antibodies and related molecules that bind to psca proteins |
| TW201138815A (en) * | 2010-01-21 | 2011-11-16 | Daiichi Sankyo Co Ltd | Peptide vaccine |
Non-Patent Citations (2)
| Title |
|---|
| MA, YANBING等: "《A potential immunotherapy approach: Mucosal immunization with an IL-13 peptide-based virus-like particle vaccine in a mouse asthma model》", 《 VACCINE》 * |
| 刘杰等: "《两条肿瘤靶向抑制肽的修饰及作用靶点的初步研究》", 《第三军医大学学报》 * |
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