CN103965288A - Dengue virus specific HLA-A*2402 restricted epitope peptides and application thereof - Google Patents
Dengue virus specific HLA-A*2402 restricted epitope peptides and application thereof Download PDFInfo
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Abstract
The invention discloses a group of dengue virus specific HLA-A*2402 restricted epitope peptides having relatively high immunogenicity for the first time. The epitope peptides are originated from dengue virus, and can induce a high-level cytotoxic T lymphocyte reaction.
Description
Technical field
The invention belongs to biotechnology and field of medicaments; More specifically, the present invention relates to dengue virus specificity HLA-A*2402 restricted epitope peptide and application.
Background technology
Dengue virus (Dengue Virus, DV) be Flavivirus single strand plus RNA virus, comprise four kinds of serotypes (D1V, D2V, D3V and D4V), 3 kinds of structural protein of genome encoding (C, PrM/M and E) and 7 kinds of Nonstructural Proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5).DV can be bitten propagation by mosquito (Aedes aegypti and Aedes albopictus etc.), cause slight singapore hemorrhagic fever (Dengue fever, or severe dengue hemorrhagic fever (Dengue hemorrhagic fever DF), DHF)/Dengue shock syndromes (Dengueshock syndrome, DSS), and DHF/DSS mortality up to 0.5-3.5%.Add up according to WHO: the whole world approximately has 5,000 ten thousand-100,000,000 DF patients (comprising approximately 250,000-500,000 DHF/DSS case) every year.
Large quantity research shows: DV specific antibody can pass through ADE (reinforcing effect that antibody relies on) effect and promote the infection of DV, thereby do not have immunoprotective effec.Early stage research prompting IFN-γ plays a role in control DV infects.Research subsequently shows: DV specificity cell toxicity T lymphocyte (CTL, the i.e. CD8 of activation
+t cell) can limit DV infection by the target cell of secrete cytokines (IFN-γ etc.) or direct killing virus infection, body is had to provide protection.Recent a large amount of experimentation on animalies further confirm: DV specific CTL (CD8 that especially can secretion of gamma-IFN
+t cell) can remove DV, reduce DV titre, it is absolutely necessary in the anti-DV infection immunity of host process.Because DV specific CTL plays a significant role in control DV copies and infects, for fear of ADE effect, the vaccine based on CTL epi-position is a kind of new generation vaccine getting a good chance of.
Due to current dengue virus serious harm human health, therefore in the urgent need to developing vaccine, the especially security and the high CTL epitope peptide vaccine of specificity that can be used for prevention or treatment dengue virus infection disease.
Summary of the invention
The object of the present invention is to provide dengue virus specificity HLA-A*2402 restricted epitope peptide and application.
In a first aspect of the present invention, a kind of isolated polypeptide is provided, described polypeptide is the polypeptide that is selected from aminoacid sequence as shown in SEQ IDNO:1, SEQ ID NO:2 or SEQ ID NO:3.
In a preference, described polypeptide derives from dengue virus.
In another aspect of this invention,, there is for the preparation of induction the pharmaceutical composition that cytotoxic T lymphocyte (CTL) reacts in the purposes of the polypeptide described in providing.
In a preference, the cell of described cytotoxic T lymphocyte secretion of gamma-IFN.
In another preference, described cytotoxic T lymphocyte is the restrictive cytotoxic T lymphocyte of HLA-A*2402.
In another aspect of this invention, the purposes of the polypeptide described in providing, for the preparation of the pharmaceutical composition of prevention or treatment dengue virus infection disease.
In a preference, described dengue virus infection disease includes, but is not limited to: singapore hemorrhagic fever, dengue hemorrhagic fever and Dengue shock syndromes.
In another aspect of this invention, provide a kind of pharmaceutical composition, described pharmaceutical composition contains:
One or more described polypeptide of significant quantity; With
Pharmaceutically acceptable carrier.
In a preference, described pharmaceutical composition is vaccine.
In another aspect of this invention,, there is for the preparation of induction the medicine box that cytotoxic T lymphocyte (CTL) reacts in the purposes of the pharmaceutical composition described in providing.
In another aspect of this invention, the purposes of the pharmaceutical composition described in providing, for the preparation of the medicine box of prevention or treatment dengue virus infection disease.
In another aspect of this invention, provide a kind of medicine box, in described medicine box, contain: the polypeptide described in one or more; Or described pharmaceutical composition.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Brief description of the drawings
Fig. 1, polypeptide NS3
548-556mass spectrum.
Fig. 2, polypeptide NS3
22-30mass spectrum.
Fig. 3, peptide C
49-57mass spectrum.
Fig. 4, NS3
548-556bonding force with HLA-A*2402 molecule.The polypeptide NS3 of different concns (redness is that 1 μ g/ml, green are that 10 μ g/ml, blueness are 100 μ g/ml)
548-556educate altogether with positive epitope peptide and the positive B cell strain of HLA-A*2402 of FITC mark.Streaming instrument is measured cell average fluorescent strength (MFI).
Fig. 5, NS3
22-30bonding force with HLA-A*2402 molecule.The polypeptide NS3 of different concns (redness is that 1 μ g/ml, green are that 10 μ g/ml, blueness are 100 μ g/ml)
22-30educate altogether with positive epi-position and the positive B cell strain of HLA-A*2402 of FITC mark.Streaming instrument is measured cell average fluorescent strength (MFI).
Fig. 6, C
49-57bonding force with HLA-A*2402 molecule.The peptide C of different concns (redness is that 1 μ g/ml, green are that 10 μ g/ml, blueness are 100 μ g/ml)
49-57educate altogether with positive epitope peptide and the positive B cell strain of HLA-A*2402 of FITC mark.Streaming instrument is measured cell average fluorescent strength (MFI).
In the HLA-A*2402 positive Peripheral Blood mononuclearcell (PBMC) of Fig. 7, epitope peptide stimulated in vitro, the ELISPOT of peptide specific CTL detects.Upper figure is part ELISPOT result figure, and figure below is the statistical graph of the ELISPOT result of the PBMC that stimulates of the positive healthy volunteers' of 5 HLA-A*2402 epitope peptide, and * represents peptide stimulating group and without relatively there being significant difference between peptide stimulating group.
The CTL killing experiments of the HLA-A*2402 positive Peripheral Blood mononuclearcell (PBMC) of Fig. 8, epitope peptide stimulated in vitro.The positive healthy volunteer's of HLA-A*2402 of epitope peptide stimulated in vitro PBMC (effector cell) kills and wounds the effect (target cell) (every group of 5 people) of the positive B cell strain of HLA-A*2402 of the immortalization of the identical epitope peptide of load, effector cell/target cell (E/T)=1 or 5.
Embodiment
The inventor, through research deeply and widely, discloses one group of dengue virus specificity HLA-A*2402 restricted epitope peptide with high immunogenicity first.Described epitope peptide is dengue virus source, the restrictive CTL epitope peptide of HLA-A*2402, can induce high-caliber cytotoxic T lymphocyte (cytotoxic lymphocyte, CTL) reaction.
The invention discloses three dengue virus specificity HLA-A*2402 restricted epitope peptide (NS3
548-556, NS3
22-30and C
49-57).Epitope peptide forms by 9 natural amino acid residues, and its sequence is respectively: SYKVASEGF, IYRILQRGL and AFMAFLRFL.The invention has the advantages that: epitope peptide of the present invention is dengue virus source, the restrictive CD8 of HLA-A*2402
+t cell (CTL) epi-position, can induce high-caliber CD8
+t cell.
CTL is also TC cell (cytotoxic T cell), is leukocytic sub-portion, is the special T cell of one, secretes specially various cytokines and participates in immunization.Known in this field, CTL has lethal effect to antigenic substances such as virus, tumour cells, can form with natural killer cell the important defence line of body disease-resistant poison, antineoplastic immune.
The inventor is first by adopting t cell epitope forecasting software SYFPEITHI prediction to dengue virus (D1V), many the antigen peptide that may be combined and induce body to produce ctl response with HLA-A*2402 molecule have been synthesized in selection, in conjunction with experiment, filter out the epitope peptide NS3 with HLA-A*2402 molecule with high-bond by competitive peptide
548-556, NS3
22-30and C
49-57, and its immunogenicity is assessed, find that these peptides can induce the restrictive CTL of HLA-A*2402 of peptide specific in the positive healthy volunteer's peripheral blood mononuclear cell of HLA-A*2402 (PBMC).
As used herein, described " the restrictive CTL epitope peptide of HLA-A*2402 " refers to polypeptide (as table 1) or its derived peptide with aminoacid sequence shown in SEQID NO:1 or SEQ ID NO:2 or SEQ ID NO:3, it is that dengue virus is specific, has the function of induction ctl response.Described " the restrictive CTL epitope peptide of HLA-A*2402 " is in the text also referred to as " epitope peptide of the present invention " or " described epitope peptide "." CTL epitope peptide " as herein described refers to the albumen of the aminoacid sequence that contains CTL epi-position; This albumen or the composition that contains this albumen can cause mammalian immune and reply.In the present invention, term " polypeptide ", " albumen " are used interchangeably.
Table 1
| Polypeptide | Zero position | Sequence |
| NS3 548-556 | 548-556 | SYKVASEGF(SEQ ID NO:1) |
| NS3 22-30 | 22-30 | IYRILQRGL(SEQ ID NO:2) |
| C 49-57 | 49-57 | AFMAFLRFL(SEQ ID NO:3) |
As used herein, " separation " refers to that material separates (if natural substance, primal environment is natural surroundings) from its primal environment.For example, polynucleotide and polypeptide under the native state in active somatic cell do not have separation and purification, if but other materials that same polynucleotide exists together with native state with polypeptide separate, for separation and purification.
As used herein, " immunocompetence " or " immunogenicity " refers to by the specificity humoral in natural, restructuring or synthetic vaccine-induced mammalian body and/or the ability of cellullar immunologic response.
As used herein, " immunne response " comprises cellularity and/or body fluid immunne response, and they are enough to suppress or prevent dengue virus infection; Or prevent or suppress dengue virus infection disease.
As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or Mammals and without excessive bad side reaction (as toxicity, stimulation and transformation reactions), has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various vehicle or thinner.
As used herein, " significant quantity " or " immune significant quantity " refers to that giving individual amount with single dose or a continuous agent part is effective to treatment or prevention.This consumption according to the preparation of the ability of treated individual healthy state and physiological situation, the individual classification for the treatment of (as non-human primates etc.), individual immunity system synthesis antibody, required degree of protection, vaccine, treatment doctor the assessment to medical conditions and other correlative factor determine.Estimate that this consumption, by relatively wide scope, can determine by normal experiment.
Epitope peptide of the present invention and encoding gene thereof
Epitope peptide of the present invention can be recombinant polypeptide, synthetic polypeptide.Epitope peptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology for example, to produce from protokaryon or eucaryon host (, bacterium, yeast and mammalian cell).
The present invention also comprises fragment, derivative and the analogue of described epitope peptide.As used herein, term " fragment ", " derivative " refer to and substantially keep biological function or the active polypeptide that epitope peptide of the present invention is identical with " analogue ".Epitope peptide fragment of the present invention, derivative and analogue can be: (1) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferably conservative amino acid residue), or (2) have the polypeptide of substituted radical in one or more amino-acid residues, or (3) mature polypeptide and another compound are (such as extending the compound of polypeptide transformation period, for example polyoxyethylene glycol) merge the polypeptide that forms, or (4) additional aminoacid sequence is blended in this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for peptide sequence or the proteinogen sequence of purifying, or with the fusion rotein of the formation of antigen I gG fragment).These fragments, derivative and analogue belong to the known scope of those skilled in the art.
The purposes of epitope peptide of the present invention is: directly as chemoprophylaxis or treatment dengue virus infection disease; Or there is the pharmaceutical composition of cytotoxic T cell (CTL) reaction in preparation induction.
The polynucleotide of epitope peptide of the present invention of encoding can be DNA form or rna form.DNA can be strand or double-stranded.Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise the epitope peptide of the present invention of encoding, and can be also the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or the fragment of polypeptide, analogue and derivative with epitope peptide of the present invention.
Polynucleotide of the present invention can obtain by the method for pcr amplification method, recombination method or synthetic conventionally.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.Described recombination method is normally cloned into carrier by described polynucleotide, then proceeds to cell, is then separated and obtains relevant sequence from the host cell propagation by ordinary method.Can synthesize relevant sequence by the method for synthetic in addition.
At present, can be completely obtain the DNA sequence dna of code book invention epitope peptide (or its fragment, or derivatives thereof) by chemosynthesis.Then this DNA sequence dna can be introduced in various existing DNA moleculars as known in the art (or as carrier) and cell.In addition, also can will suddenly change and introduce in protein sequence of the present invention by chemosynthesis.
The present invention also relates to the carrier that comprises polynucleotide of the present invention, and the host cell that produces through genetically engineered of carrier of the present invention or epitope peptide encoding sequence, and the method for producing epitope peptide of the present invention through recombinant technology.
By conventional recombinant DNA technology (Science, 1984,224:1431), can utilize polynucleotide sequence of the present invention to express or the epitope peptide of the present invention of Restruction.In general there are following steps: (1) with the polynucleotide (or varient) of coding epitope peptide of the present invention, or transform or the suitable host cell of transduceing with the recombinant expression vector that contains these polynucleotide; (2) in suitable substratum, cultivate host cell; (3) separation, protein purification from substratum or cell.
In the present invention, the polynucleotide of the described epitope peptide of encoding can be inserted in recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.In a word, as long as can copy in host and stablize, any plasmid and carrier can be used.A key character of expression vector is conventionally to contain replication orgin, promotor, marker gene and translation controlling elements.
Method well-known to those having ordinary skill in the art can be used for building containing described polynucleotide sequence and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, to instruct mRNA synthetic.In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of the host cell transforming, as eukaryotic cell is cultivated Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of use, or for colibacillary tsiklomitsin or amicillin resistance.
The carrier of the polynucleotide that comprise the epitope peptide described in above-mentioned coding and suitably promotor or control sequence, can be for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, yeast etc.For different host cells, also can carry out described polynucleotide sequence codon optimizedly, to obtain improved expression effect, codon optimized technology is well known in the art.
Persons skilled in the art are all known the suitable carrier of How to choose, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host is prokaryotic organism during as intestinal bacteria, the competent cell that can absorb DNA can, in exponential growth after date results, be used CaCl
2method processing, step used is well-known in this area.Another kind method is to use MgCl
2.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method is as microinjection, electroporation, liposome packaging etc.
The transformant obtaining can be cultivated by ordinary method, to express described epitope peptide.According to host cell used, substratum used in cultivation can be selected from various conventional mediums.Under the condition that is suitable for host cell growth, cultivate.
Described epitope peptide can or be secreted into extracellular at cell inner expression.Can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation processing, with the combination of protein precipitant processing (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Composition
The present invention also provides the composition that comprises epitope peptide of the present invention, particularly pharmaceutical composition, and described composition also comprises vaccine.Said composition can be used for induction cytotoxic T cell (CTL) reaction occurs, and also i.e. induction produces the cytotoxic T cell (CTL) of peptide specific.Said composition can be used for prevention or treatment dengue virus infection disease, and described disease includes, but is not limited to: singapore hemorrhagic fever, dengue hemorrhagic fever and Dengue shock syndromes.
The composition that comprises epitope peptide of the present invention can comprise the buffer reagent selected by the practical use of epitope peptide; Also can comprise other material that is applicable to intended purpose.Those skilled in the art are good at selecting suitable buffer reagent, and known in the art have numerous buffers to be applicable to intended purpose.In some example, said composition can contain pharmaceutically acceptable vehicle, known in the art have multiple and without discussing in detail at this.Pharmaceutically acceptable various vehicle is at the existing detailed description of multiple publication, comprise as " Remington ' sPharmaceutical Sciences " (" Lei Mingdun pharmaceutical science ", the 19th edition (1995) Mack PublishingCo.).
Composition of the present invention can be prepared into various formulations, as injection, granula, tablet, pill, suppository, capsule, suspension, spraying, transdermal drug etc.Be applicable to pharmaceutical grade other organic or inorganic carrier and/or thinner of oral or local use, can be used for the various compositions that preparation comprises therapeutical active compound.Thinner known in the art comprises aqueous medium, vegetalitas and animality oil & fat.Also the salt of available stablizer, wetting agent and emulsifying agent, change osmotic pressure or the various buffer reagents and the skin penetration enhancer etc. that maintain suitable pH value are as complementary material.
When as vaccine, described vaccine can adopt the whole bag of tricks to prepare.Conventionally,, by the whole bag of tricks well known in the art, prepare vaccine of the present invention or medicine with suitable pharmaceutical carrier and/or vehicle (vehicle).Suitable carrier is Sterile Saline.Also can use other water-based and non-aqueous isotonic sterile injection liquid and water-based and non-aqueous sterile suspensions (known is all pharmaceutically acceptable carrier well-known to those skilled in the art) for this reason.
In addition, the preparation of vaccine of the present invention also can contain other composition, comprises as adjuvant, stablizer, pH adjusting agent, sanitas etc.These compositions are that vaccine those skilled in the art are known.Adjuvant class comprises (but being not restricted to) Alum adjuvant; Saponin adjuvant; Ribi adjuvant (Ribi ImmunoChem Research In., Hamilton, MT); Montanide ISA adjuvant (Seppic, Paris, France); Hunter ' s TiterMax adjuvant (CytRx Corp., Norcross, GA); Gerbu adjuvant (Gerbu Biotechnik GmbH, Gaiberg, Germany) etc.In addition, in preparation, also can comprise other composition (IL-12, CpG oligodeoxynucleotide (CpG-ODN) etc.) that regulates immunne response.
When as vaccine, epitope peptide of the present invention is applied to object by available known method.Conventionally adopt the route of administration identical with conventional vaccine to use these vaccines.While adopting the form of vaccine composition, also can comprise pharmaceutically acceptable carrier.In addition, this composition also can comprise adjuvant, correctives or stablizer etc.
The routine and the pharmaceutically acceptable approach that give the present composition comprise: in nose, interior, the intravenously of interior, subcutaneous, the intracutaneous of intramuscular, tracheae, lung, intranasal, oral administration or other administered parenterally approach.Can combination medicine-feeding approach if needed, or regulate by antigen peptide or disease situation.Vaccine can single dose or multiple doses give, and can comprise and give booster dose to cause and/or to maintain immunizing power.
Should give epitope peptide of the present invention with " significant quantity ", the amount of epitope peptide is enough to cause immunne response in selected administration path, can effectively impel protection host to resist dengue virus infection disease.
The amount of selected epitope peptide in each vaccine dose part is to determine without the amount of significantly side effect by causing protective immune response.Conventionally, give approximately 1 μ g-100mg epitope peptide/kg body weight, preferably 0.01mg-10mg epitope peptide/kg body weight, more preferably 0.1mg-10mg epitope peptide/kg body weight.Can be with comprising that the level of epitope peptide specific CTL in the object of observation and the research on standard method of other reaction determine the optimum amount of concrete vaccine, the immunity level that can provide by monitoring vaccine determines whether to strengthen dosage.Having assessed after the level of the epitope peptide specific CTL in blood, may need to select enhancing dosage immunization.Use adjuvant and/or immunostimulant and just can improve the immunne response to epitope peptide of the present invention.
Medicine box
The present invention also provides a kind of and has prevented and treated dengue virus infection or because infecting the medicine box of the complication that causes, the composition that wherein contains epitope peptide of the present invention or contain this epitope peptide.In addition, in order to facilitate administration, in described medicine box, also can contain the pin of injection, and/or pharmaceutically acceptable carrier, and/or working instructions.
Major advantage of the present invention is:
Epitope peptide of the present invention is dengue virus source, the restrictive CTL epi-position of HLA-A*2402, can induce the high-caliber ctl response with lethal effect, it is the research to dengue virus infection disease incidence mechanism not only, and the research and development of dengue virus epiposition vaccine and dengue virus infection reagent for disease diagnosis are all had great importance.
HLA-A*2402 is a kind of MHC-I quasi-molecule in different ethnic populations with higher distribution, and positive rate is more than 40%, and therefore HLA-A*2402 restricted epitope peptide has high crowd's fraction of coverage.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, writes molecular cloning experiment guide, the third edition, Science Press, the condition described in 2002, or the condition of advising according to manufacturer conventionally as J. Pehanorm Brooker etc. according to normal condition.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Embodiment 1, competitive peptide are in conjunction with experiment
First dengue virus (D1V) is adopted to t cell epitope forecasting software SYFPEITHI prediction, synthesized many antigen peptide of being likely combined and inducing body generation ctl response with HLA-A*2402 molecule by conventional peptide synthetic method.Through primary dcreening operation, determine many peptides that carry out subsequent authentication, the sequence of partial peptide is in table 2.
The bonding force of table 2, dengue virus candidate epitope peptide and HLA-A*2402 molecule
Adopt the epitope peptide of competitive peptide in conjunction with experiment screening and HLA-A*2402 molecule high-bond.The positive B cell strain of the HLA-A*2402 surface expression HLA-A*2402 molecule of the immortalization that the inventor sets up, adopt citric acid treatment B cell strain can wash-out HLA-A*2402 molecular peptide engagement groove in the peptide of combination.
By restricted positive peptide (the Nakamoto Y of HLA-A*2402 of the open report of candidate's epitope peptide and FITC mark, et al.J Med Virol.2003,70:51-61.) jointly hatch with the positive B cell strain of HLA-A*2402 of citric acid treatment, if candidate's epitope peptide can be in conjunction with HLA-A*2402 molecule, the amount that the positive peptide of FITC mark is attached on HLA-A*2402 molecule reduces.Thereby, in the time that flow cytometer is tested, the average fluorescent strength (MFI) of cell reduces, so can utilize the percent inhibition (PI) of candidate's epitope peptide to positive epitope peptide to reflect the bonding force of candidate's epitope peptide and HLA-A*2402 molecule.
Concrete grammar is as follows:
Gather the positive healthy volunteer's peripheric venous blood of HLA-A*2402, adopt human lymphocyte parting liquid separating peripheral blood mononuclear cells (PBMC), adopt B95-8 cell (ATCC, the i.e. biological product of USS collecting center) culture supernatant to stimulate PBMC (simultaneously adding 1 μ g/ml cyclosporin A) 4 weeks to produce the positive B cell strain of HLA-A*2402 of immortalization.A large amount of above-mentioned B cell strain, collecting cells cultivated.Peptide with citric acid (0.131M/L citric acid and 0.061M/L Sodium phosphate dibasic, PH3.2) processing cell 90s with combination in wash-out cell surface HLA-A*2402 molecular peptide engagement groove.Washed cell, adds the restricted positive epitope peptide of HLA-A*2402 (FITC-FWAKHMWNF, 50 μ g/ml) of different concns (1 μ g/ml, 10 μ g/ml, 100 μ g/ml) candidate's epitope peptide and FITC mark in cell.Blank (not adding any peptide) and positive controls (only adding the positive epitope peptide of FITC mark) are set.By cell cultures 3h, adopt the average fluorescent strength (MFI) of cells were tested by flow cytometry cell.Adopt the percent inhibition (PI, Percentage Inhibition) of following formula calculated candidate epitope peptide to positive epitope peptide.In the time that peptide concentration is 100 μ g/ml, PI>50% represents that candidate's epitope peptide and HLA-A*2402 have high-bond.
Peptide NS3
548-556, NS3
22-30and C
49-57mass spectrum see Fig. 1,2,3.As shown in table 2 and Fig. 4,5,6, peptide NS3
548-556, NS3
22-30and C
49-57have high-bond (PI value increases with the increase of peptide concentration, and when peptide concentration is 100 μ g/ml, its PI approaches 100%) with HLA-A*2402 molecule, these peptides are HLA-A*2402 restricted epitope peptide.And other candidate's epitope peptides and HLA-A*2402 bonding force are low.
The positive PBMC of embodiment 2, dengue virus candidate epitope peptide stimulated in vitro HLA-A*2402
Recruit the positive healthy volunteers of 5 HLA-A*2402 (not infecting dengue virus), gather EDTA anti-freezing peripheric venous blood, adopt human lymphocyte parting liquid separating peripheral blood mononuclear cells (PBMC).Adopt above-mentioned epitope peptide to stimulate in vitro PBMC tri-weeks (within every 3 days, half amount is changed liquid, and supplements the human IL-2 of 50 μ g/ml peptides and 10U/ml restructuring), stimulate first after surrounding, collect the PBMC (effector cell) that peptide stimulates.Afterwards, adopt ELISPOT and CTL killing experiments to detect the cell levels of epitope specificity secretion of gamma-IFN in effector cell.
In the positive PBMC of HLA-A*2402 that embodiment 3, epitope peptide stimulate, peptide specific CTL detects
Adopt people's IFN-γ enzyme linked immunological Spot Jest (ELISPOT) test kit (pre-coated pvdf membrane 96 orifice plates, Dutch U-CyTech company), carry out with reference to specification sheets.Process is as follows:
The 96 every holes of orifice plate add above-mentioned 5 × 10
4the PBMC (effector cell) (being suspended in 100 μ lRPMI1640 nutrient solutions) that peptide stimulates.Negative control hole (add cell and do not add epitope peptide) and epitope peptide are set and stimulate hole (add cell simultaneously add epitope peptide to make its final concentration be 10 μ g/ml).Plate is put in to 37 DEG C, 5%CO
2in incubator, cultivate 24h.Abandon every hole liquid, wash plate, every hole adds the detection antibody of the anti-IFN-γ of biotinylation that the fresh dilution of 100 μ l is good, cultivates 1h in 37 DEG C.Abandon liquid in hole, wash plate, every hole adds the streptavidin of horseradish peroxidase (HRP) mark of the fresh dilution of 100 μ l, cultivates 1h (lucifuge) in 37 DEG C.Abandon liquid in hole, wash plate, the ACE nitrite ion that every hole adds 100 μ l now to join, cultivates 10-30min (lucifuge) in 37 DEG C.Abandon every hole liquid, wash plate.Every hole spot is counted with ELISPOT spot analysis instrument.A spot represents a spot formation cell (Spots Forming Cells, SFC).Spot number is expressed as SFCs/5 × 10
4the PBMC that peptide stimulates.Epitope peptide stimulates the statistics between hole and negative control hole relatively to adopt t inspection.
As shown in Figure 7, in the positive PBMC of the HLA-A*2402 of epitope peptide stimulated in vitro, there is NS3 in result
548-556, NS3
22-30and C
49-57specific CTL (cell of secretion of gamma-IFN).
Embodiment 4, CTL killing experiments
The positive B cell strain of HLA-A*2402 of cultivating above-mentioned immortalization, adopts the ametycin of 1 μ g/ml to process cell 30min in 37 DEG C.Cell is divided into control tube (1 × 10
5cell) and experiment tube (1 × 10
5cell).Control tube adds the CFSE of final concentration 0.5 μ M, and (this is for CFSE
lowtarget cell) and experiment tube adds the CFSE of final concentration 5 μ M, and (this is for CFSE
hightarget cell), in 37 DEG C of dyeing 15min, adopt 40% foetal calf serum to end staining reaction.In experiment tube, add the epitope peptide of final concentration 50 μ g/ml, hatch 3h in 37 DEG C.By after control tube cell and experiment tube cytomixis with the positive PBMC (1 × 10 of the HLA-A*2402 of above-mentioned epitope peptide stimulated in vitro
5or 5 × 10
5, effector cell) and mix [effector cell/target cell (E/T)=1 or 5], hatch 24h in 37 DEG C.Adopt flow cytometer to detect different fluorescence intensity cells peak, calculate the ratio at hyperfluorescenceZeng Yongminggaoyingguang peak and hypofluorescence peak.CTL is [1-(CFSE to the percentage that kills and wounds of target cell
high/ CFSE
low)] × 100%.
Result as shown in Figure 8, NS3
548-556, NS3
22-30and C
49-57the positive PBMC of HLA-A*2402 of stimulated in vitro can kill and wound the positive B cell strain of HLA-A*2402 of the identical epitope peptide of load, kills and wounds per-cent and is directly proportional to effect target ratio.
Embodiment 5, pharmaceutical composition
Compounding pharmaceutical composition is as follows: the above-mentioned epitope peptide SEQ of synthetic ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, with freund's adjuvant emulsification, make pharmaceutical composition by epitope peptide.
Described pharmaceutical composition is placed in to container, is positioned in packaging, thereby make medicine box.
All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. an isolated polypeptide, is characterized in that, described polypeptide is the polypeptide that is selected from aminoacid sequence as shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
2. polypeptide as claimed in claim 1, is characterized in that, described polypeptide derives from dengue virus.
3. the purposes of polypeptide claimed in claim 1, is characterized in that, the pharmaceutical composition of cytotoxic T lymphocyte reaction occurs for the preparation of induction.
4. purposes as claimed in claim 3, is characterized in that, the cell of described cytotoxic T lymphocyte secretion of gamma-IFN.
5. purposes as claimed in claim 3, is characterized in that, described cytotoxic T lymphocyte is the restrictive cytotoxic T lymphocyte of HLA-A*2402.
6. the purposes of polypeptide claimed in claim 1, is characterized in that, for the preparation of the pharmaceutical composition of prevention or treatment dengue virus infection disease.
7. a pharmaceutical composition, is characterized in that, described pharmaceutical composition contains:
One or more polypeptide claimed in claim 1 of significant quantity; With
Pharmaceutically acceptable carrier.
8. the purposes of pharmaceutical composition claimed in claim 7, is characterized in that, the medicine box of cytotoxic T lymphocyte reaction occurs for the preparation of induction.
9. the purposes of pharmaceutical composition claimed in claim 7, is characterized in that, for the preparation of the medicine box of prevention or treatment dengue virus infection disease.
10. a medicine box, is characterized in that, in described medicine box, contains: one or more polypeptide claimed in claim 1; Or pharmaceutical composition claimed in claim 7.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008044567A1 (en) * | 2006-10-12 | 2008-04-17 | Nec Corporation | Hla-binding peptide, precursor thereof, dna fragment encoding the same and recombinant vector |
| CN101921310A (en) * | 2009-06-15 | 2010-12-22 | 温州医学院 | Dengue virus (DENV) specific HLA-A2 (Human Leukocyte Antigen-A2) restrictive epitope peptide and application thereof |
-
2013
- 2013-02-06 CN CN201310047583.9A patent/CN103965288B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008044567A1 (en) * | 2006-10-12 | 2008-04-17 | Nec Corporation | Hla-binding peptide, precursor thereof, dna fragment encoding the same and recombinant vector |
| CN101921310A (en) * | 2009-06-15 | 2010-12-22 | 温州医学院 | Dengue virus (DENV) specific HLA-A2 (Human Leukocyte Antigen-A2) restrictive epitope peptide and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| LUND O ET AL: "Human Leukocyte Antigen(HLA) class I restricted epitope discovery in yellow fewer and dengue viruses:importance of HLA binding strength", 《PLOS ONE》 * |
| TIAN J ET AL: "Identification and immunogenicity of two new HLA-A*0201-restricted CD8(+) T-cell epitopes", 《INTERNATIONAL IMMUNOLOGY》 * |
| 段志良等: "登革病毒抗原肽免疫小鼠诱导特异性CD4+T细胞反应", 《中国免疫学杂志》 * |
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