CN103724405A - Type A foot-and-mouth disease CTL (cytotoxic T lymphocyte) epitope peptide and screening method thereof - Google Patents
Type A foot-and-mouth disease CTL (cytotoxic T lymphocyte) epitope peptide and screening method thereof Download PDFInfo
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Abstract
The invention discloses type A foot-and-mouth disease CTL (cytotoxic T lymphocyte) epitope peptide and a screening method thereof. The CTL epitope peptide consists of nine amino acid residues, and has an amino acid sequence of Ala-Met-Leu-Arg-Ala-Ala-Thr-Tyr-Tyr. The epitope peptide has stronger combining capability with SLA (swine leukocyte antigen)-?? proteins from pigs with different strains and causees cell toxicity immune response, and is applicable to the preparation of prevention and treatment vaccines for foot-and-mouth disease virus of pigs with various strains and wide in application range. According to the type A foot-and-mouth disease CTL epitope peptide and the screening method thereof, constructed SLA-I single-stranded molecules of pigs with six strains are utilized for in vitro combination with CTL simulated epitope peptide of foot-and-mouth disease virus, polypeptide which can be combined with complex is determined and screened by using a mass spectrum, and simulated epitope peptide which can induce the production of T cell immune response capability is determined through ELISPOT (enzyme linked immunospot assay) detection. The invention provides a method for screening and identifying a large number of foot-and-mouth disease virus CTL epitopes in the future, and lays a foundation for the development of multi-epitope vaccines for the foot-and-mouth disease of pigs.
Description
Technical field
the invention belongs to molecular immunology field, be specifically related to SLA-I in conjunction with and can cause the A type foot and mouth disease CTL epitope peptide that cytotoxic immune is replied and utilize the method for CTL epitope peptide described in SLA-I complex body screening and identification.
Background technology
Pig major histocompatibility complex (major histocompatibility complex, MHC), that is the human leucocyte antigen of pig (swine leukocyte antigen, SLA) are the important immunne response molecules of pig.SLA is divided into three classes, is respectively SLA-I, SLA-II, SLA-III.Wherein SLA-I quasi-molecule comprises heavy chain and light chain, and heavy chain has polymorphism, and functional gene is mainly SLA-1 ,-2 ,-3.And light chain is by Beta 2 microglobulins (Beta 2 microglobulin, β 2m) genes encoding.At endocytoplasmic reticulum, SLA-I heavy chain, antigenic peptide are combined with non covalent bond with light chain, form SLA-I-peptides mixture, after golgi body processing, submission, to cell surface, is combined with CD8+ t lymphocyte receptor, causes cytotoxic T lymphocyte (cytotoxic T lymphocyte, CTL) immunne response, the CTL release cells factor is also killed by the target cell of virus infection.Can be combined with SLA-I quasi-molecule and can cause that the polypeptide that cytotoxic immune is replied is CTL epi-position.
Foot and mouth disease virus (foot-and-mouth disease virus, FMDV) be harm artiodactyls a kind of acute, hot, height contagious disease cause of disease, serotype is divided into O, A, C, SAT1, SAT2, SAT3 and Asia1 type, various between without Immunogenicity power.Because the epidemic strain of foot and mouth disease virus constantly makes a variation, traditional vaccine can not adapt to the requirement of foot and mouth disease control.The epiposition vaccine that contains various serotype and different epidemic strains can play the requirement that prevents and treats different serotypes foot and mouth disease virus.The key of development foot and mouth disease virus epiposition vaccine is to determine epi-position, and cell epitope comprises B cell epitope, helper T cell (T helper, Th) epi-position and CTL epi-position.At present, multiple B cell epitopes and Th epi-position are reported.Foot and mouth disease belongs to strict cytozoon, and humoral immunization is no doubt important, but cellular immunization is essential.Recent research shows, in the control of FMDV acute attack stage, is mainly to be played a role by neutralizing antibody, at the immunology of FMDV persistent infection phase, mainly by CTL, is played a role.Recently, some scholars CTL epi-position of foot and mouth disease virus that begins one's study.2006, Barfoed(Antiviral Res, 2006,72 (3): 178-89) etc. confirmed can in Mice Body, cause ctl response by a restrictive CTL epi-position KYKEAKEWL who derives from foot and mouth disease virus Nonstructural protein 2C of mouse H-2Kd.In addition, (the J Gen Virol such as Guzman, 2008,89 (Pt 3): 667-75) also confirmed recently the restrictive foot and mouth disease virus CTL of an ox BoLA N*02201 epi-position, this epi-position is positioned at 795-803 amino-acid residues of O type foot and mouth disease UKG/2001 strain structural protein, and this epi-position can cause target cell lethal effect.But up to the present, the foot and mouth disease virus CTL epi-position of all reports is verified by animals such as mouse, oxen, does not also report so far the epi-position through the foot and mouth disease virus CTL of the direct submission of pig SLA-I quasi-molecule.
Research shows, utilizes the method for external structure MHC I heavy chain molecule, peptide and light chain complex body can screen in vitro virus epitopes.White etc. (J Immunol, 1999,162 (5): 2671-6) are connected the heavy chain of mouse MHC-I with a Linker with light chain, built MHC-I molecule, and proved it can with antigen peptide molecule specific combination.(the Eur J Immunol such as Denberg, 2000,30 (12): 3522-32) with a polypeptide, Linker, heavy chain and light chain β 2m, built people's HLA-A2 single chain molecule, and proved that the single chain molecule of the Binding peptide building has the function of identification cell receptor.In seminar's early-stage Study, built six strain pig SLA-I complex body albumen, and expressed at pMAL-p2X, expressing protein is soluble proteins after testing, and keeps good protein conformation.The present invention, on the basis of early-stage Study, utilizes the six strain pig SLA-I single chain molecules that build in vitro in conjunction with the CTL analogue epi-peptide of foot and mouth disease virus, and mass spectroscopy screens the polypeptide that can be combined with complex body.Combinative polypeptide is through the CTL activity of ELISPOT experiment detection polypeptide.The present invention provides method for screening in a large number from now on foot and mouth disease virus CTL epi-position, and lays the foundation for developing swine foot-and-mouth disease virus polyepitope vaccines.
Summary of the invention
The object of the present invention is to provide a kind of can combination with SLA-molecule also can cause the A type foot and mouth disease CTL epitope peptide that cytotoxic immune is replied.
To achieve these goals, the technical solution adopted in the present invention is a kind of A type foot and mouth disease CTL epitope peptide, described A type foot and mouth disease CTL epitope peptide called after QA4, it is comprised of nine amino-acid residues, its aminoacid sequence is: Ala-Met-Leu-Arg-Ala-Ala-Thr-Tyr-Tyr(SEQ ID No:1), i.e. L-Ala-methionine(Met)-leucine-arginine-Ala-Ala-Threonine-Tyr-Tyr.
Described A type foot and mouth disease CTL epitope peptide (QA4) derives from A type FMDV VP1 albumen, and it all has stronger binding ability with the SLA-albumen that derives from different lines pig, and can induce pig PBMC extremely significantly to discharge IFN-γ.
A type foot and mouth disease CTL epitope peptide of the present invention (QA4) is applied to the preparation of foot and mouth disease polypeptide vaccine.
The screening method of A type foot and mouth disease CTL epitope peptide as above (QA4), it comprises the steps:
(1) utilize biological information software netMHCpan2.4 (http://www.cbs.dtu.dk/services/ NetMHCpan), input FMDV VP1 protein sequence, select nonapeptide, under SLA gene prediction obtain can with SLA-protein binding, and offer the CTL analogue epi-peptide that cell surface causes that cytotoxic immune is replied, with artificial chemistry method, synthesize the CTL analogue epi-peptide that above-mentioned prediction obtains;
(2) CTL analogue epi-peptide step (1) being obtained and the external association reaction of SLA-albumen, reaction mixture obtains solid powdery SLA--polypeptide complex after C-18 post desalination, refining, lyophilize;
(3) SLA--polypeptide complex screens the CTL analogue epi-peptide that can be combined with SLA-through mass spectroscopy;
(4) take can be with the protein bound CTL analogue epi-peptide of SLA-as antigen, take the pig spleen lymphocyte that separates the infection of foot-and-mouth disease obtaining as cell to be measured, carry out ELISPOT detection, determine and can induce the analogue epi-peptide that produces T cellullar immunologic response ability.
In the screening and identification method of above-mentioned CTL epitope peptide, the aminoacid sequence of the described CTL analogue epi-peptide of step (1) is preferably SEQ ID No:1 ~ 4.
In the screening method of above-mentioned CTL epitope peptide, the described FMDV VP1 protein sequence of step (1) can derive from the foot and mouth disease virus of different serotypes, and its serotype can be O, A, C, SAT1, SAT2, SAT3 and Asia1 type etc.
In the screening method of above-mentioned CTL epitope peptide, one or more in landrace, Yorkshire Pigs, EmilZatopek pig, Hebao pig, Yantai pig or Laiwu Black Pigs of described SLA-dietary protein origin.
Utilize the combination of mass spectrometric determination SLA-I single chain molecule and polypeptide, can point-devicely filter out the polypeptide that can be combined with SLA-I molecule, specificity is high.Applicant of the present invention has built bar horse miniature pig SLA-I single chain molecule by early-stage Study, and has carried out external combination screening foot-and-mouth disease virus polypeptide, thereby has proved the science of the method.But because SLA-I quasi-molecule exists restrictedly, the SLA-I that derives from different lines pig may be in conjunction with different polypeptide.In addition, because the restrictive polypeptide epitope of SLA-I quasi-molecule may have common anchor residues, different lines pig SLA-I also may be in conjunction with common polypeptide epitope.If energy Isolation and screening is adapted to the polypeptide of multiple strain pig SLA-I jointly, can contribute to development to there is the foot-and-mouth disease virus multi-epitope vaccine of prevention effect.The present invention goes out with the SLA-albumen that derives from Yorkshire Pigs, Hebao pig, Yantai pig or Laiwu Black Pigs all to have good binding ability by aforesaid method screening and identification, and can induce pig PBMC significantly to discharge the CTL epitope peptide (QA4) of IFN-γ.CTL epitope peptide of the present invention is applicable to the preparation of the control vaccine of multiple strain swine foot-and-mouth disease virus, applied range.
Beneficial effect of the present invention: 1. A type foot and mouth disease CTL epitope peptide of the present invention (QA4) all has stronger binding ability with the SLA-albumen that derives from different lines pig and can cause that cytotoxic immune replys, be applicable to the preparation of the control vaccine of multiple strain swine foot-and-mouth disease virus, applied range.2. the present invention provides method for a large amount of screening and identification foot and mouth disease virus CTL epi-positions from now on, and lays a good foundation for development Schweineseuche polyepitope vaccines.3. epitope polypeptide of the present invention also can be used for carrying out external Study on Crystallization etc. with SLA-I albumen from now on, is conducive to research and prevents and treats molecule, the cytosis mechanism in foot and mouth disease.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE electrophorogram of the fusion protein S LA-I-MBP that obtains through Amylose affinity chromatography, wherein: M, represent low molecular weight protein (LMWP) standard substance, 1-6, represents respectively the black and Laiwu Black Pigs SLA-I-MBP purifying protein of landrace, Yorkshire Pigs, EmilZatopek pig, Hebao pig, Yantai;
Fig. 2 is the SDS-PAGE electrophorogram of the SLA-I-MBP after Factor Xa cutting, wherein: M, represent low molecular weight protein (LMWP) standard substance, 1-6, represent respectively the albumen after the black and Laiwu Black Pigs SLA-I cutting of landrace, Yorkshire Pigs, EmilZatopek pig, Hebao pig, Yantai, wherein SLA-I albumen size is 41.6kDa, and the size of fusion rotein is 84.1 kDa;
Fig. 3 is the SDS-PAGE electrophorogram of the SLA-I after DEAE-dextrane gel anionresin column purification, wherein: M, represent low molecular weight protein (LMWP) standard substance, 1-6, represents respectively the black and Laiwu Black Pigs SLA-I purifying protein of landrace, Yorkshire Pigs, EmilZatopek pig, Hebao pig, Yantai;
Fig. 4 is the external combination mass spectrum measurement result of Q02 and six strain pig SLA-I;
Fig. 5 is the external combination mass spectrum measurement result of AS3 and six strain pig SLA-I;
Fig. 6 is QA4 and the external combination mass spectrum measurement result with six strain pig SLA-I;
In described Fig. 4～Fig. 6, described A, B, C, D, E and F represent respectively, corresponding polypeptide and the external combination mass spectrum of landrace SLA-I measurement result, the external combination mass spectrum of Yorkshire Pigs SLA-I measurement result, the external combination mass spectrum of EmilZatopek pig SLA-I measurement result, the black pig SLA-I external combination mass spectrum measurement result in Yantai and the external combination mass spectrum of Laiwu Black Pigs SLA-I measurement result;
Fig. 7 is that ELISPOT detects polypeptid induction CD8
+t lymphocyte produces IFN-γ ability, wherein: * * represents polypeptide to be measured extremely remarkable (the P < 0.01) of difference that compare with control peptide.
embodiment
Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.
embodiment 1the preparation of six strain pig SAL-I albumen
The preparation of (1) six strain pig SLA-I/pMAL-p2X recombination bacillus coli
Six strain pig SLA-I/pMAL-p2X recombination bacillus colis, are University Of Dalian's Life Science and Technology institute molecular immunology laboratory and build preservation.That described six strain pigs comprise is long white, Yorkshire, EmilZatopek, pocket, Yantai and Laiwu Black Pigs.Described SLA-I/pMAL-p2X recombination bacillus coli is obtained by the method described in (Gene, 2012,502 (2): 147-153) such as high Fengshan.
(2) abduction delivering SLA-I recombination bacillus coli
The SLA-I/pMAL-p2X recombination bacillus coli bacterium liquid 5mL that gets respectively six strain pigs is seeded to 500mL LB substratum, and shaking culture in the 37 degree incubators of 170rpm, detects its OD every for some time
600, OD to be grown to
600in the time of between 0.6 to 0.8, add IPTG to 0.5mmol/L, 37 degree inductions 5 hours.
(3) fusion rotein MBP-SLA-I affinity purification
Will be with the IPTG inducing culture thalline of 5 hours in above-mentioned (2), 3000r/min, centrifugal 10min, abandon supernatant, add 50mL cross post buffer A [20 mmol/L Tris-HCl(pH 7.4), 200 mmol/L NaCl, 1mmol/L EDTA (pH8.0), 1 mmol/L sodium azide, 1 mmol/L DTT] resuspended thalline.Thalline is through ultrasonication 10min, 90W, and broken 5min stops 5min.The ultrasonic rear 4000r/min of thalline, centrifugal 10min, abandons precipitation, gets supernatant liquor.In supernatant liquor, contain the fusion protein S LA-I-MBP of solubility, utilize maltose binding protein (MBP) character that ligands specific is combined in pillar, carry out Amylose affinity chromatography, fusion protein S LA-I-MBP is adsorbed on Amylose pillar, remove not in conjunction with after foreign protein with the above-mentioned post buffer A wash-out of crossing of 12 times of column volumes, the character that recycling maltose can be combined with MBP advantage, elution buffer (crossing post buffer A+10mmol/L maltose) the wash-out target protein that contains 10 mmol/L maltose with 3 times of column volumes, liquid after wash-out concentrates, obtain the SLA-I-MBP protein concentrated solution of solubility.SLA-I-MBP protein concentrated solution SDS-PAGE electrophoresis, stripe size and the purity of check target protein.The results are shown in Figure 1.Fig. 1 result shows, six strain pig SLA-I-MBP purity approximately 85%, and size is 84.1kDa, meets expection SLA-I-MBP albumen size.
(4) fusion rotein excision MBP, separation and purification
Because the molecular weight of MBP own is larger, may interfere with the specific binding of target protein molecule and antigen peptide, therefore need to be by the MBP excision in fusion rotein.On pMAL-p2X carrier, between malE gene and multiple clone site, have an aminoacid sequence of being identified by Xa factor, Ile-Glu-Gly-Arg, can excise MBP.
Concrete cutting method is:
Factor Xa cutting for the MBP-SLA-I fusion rotein that step (3) is obtained, cutting condition is: 1mol/L NaCl 100 μ L, 100 mmoL/L CaCl
220 μ L, 1mol/L Tris-HCl 20 μ L, the SLA-I-MBP protein concentrated solution 400 μ L(approximately 1 mg SLA-I-MBP albumen that step (3) obtains), Factor Xa (1 U enzyme cuts 50 mg) 20 U, add sterilizing deionized water to 1 mL.21 ℃ of cutting 20 h, carry out SDS-PAGE and detect fusion rotein cutting situation after cutting, the results are shown in Figure 2.The demonstration of Fig. 2 result, at 43kDa place, six strain pig SLA-I-MBP all cut out a treaty 41kDa band, and 41.6kDa is consistent with target protein SLA-I size.
First egg white mixture after cutting passes through Amylose column purification, collects the protein sample of post buffer A wash-out.The protein sample that collection obtains passes through DEAE Ceramic HyperD F anionresin column separating purification albumen again, post damping fluid (the 20mmoL/L Tris-HCl(pH 8.0 of 0.15 moL/L NaCl) excessively with 10 column volumes containing 0.15 mol/L NaCl) wash-out, according to spectrophotometer, detect, collect elution peak, after concentrated, SDS-PAGE testing goal Protein S LA-I, the results are shown in Figure 3.Fig. 3 result shows, finally collects the albumen obtaining and presents single band, and size is 41.6 kDa, and purity has reached more than 95%, meets the requirement of carrying out protein function research.
The design of embodiment 2 foot and mouth disease virus analogue epi-peptides
Utilize biological information software netMHCpan2.4 (http://www.cbs.dtu.dk/services/ NetMHCpan), input O type FMDV VP1 aminoacid sequence (AJ539138), A type FMDV VP1 aminoacid sequence (GQ406249) and Asia1 type FMDV VP1 aminoacid sequence (EF149009), select nonapeptide, under SLA gene prediction obtain can with SLA-protein binding, and offer the CTL analogue epi-peptide that cell surface causes that cytotoxic immune is replied, 39 peptides that derive from FMDV VP1 albumen have been designed altogether, called after Q02 respectively, AS3 and QA4, its aminoacid sequence and other essential informations are in Table 1.Wherein Q02 derives from the outer epidemic isolates Tibet/CHA/99 of Present Domestic of O type foot and mouth disease; AS3 derives from Asia1 type foot and mouth disease strain epidemic strain Asia 1/Jiangsu/China/2005; QA4 derives from A type foot and mouth disease international popular strain A/VN/03/2009.In addition, design and synthesized irrelevant control peptide Co.Each epitope peptide is stored in-80 ℃ with lyophilised state, standby.
Combination and the screening of embodiment 3 SLA-I albumen and foot and mouth disease virus analogue epi-peptide
The each 1mL(protein content of the six strain pig SLA-I albumen 1mg/mL that embodiment 1 purifying is obtained) mix with 5mL PBS solution respectively, the centrifugal 20min of 4500r/min, remove filtrate, add PBS and be about 5mL to cumulative volume, repeated centrifugation 3 times, the PBS solution (containing SLA-I) of getting upper strata mixes with polypeptide solution (Q02, AS3, QA4 or Co) respectively, and the mol ratio of peptide and target protein is 10:1, and 37 ℃ are spent the night.Mixture is added to 30K super filter tube, the centrifugal 45min of 4500r/min.Add PBS, 37 ℃ of water-bath 2min, 4500r/min is centrifugal, about being 100uL to final volume.5mL citric acid-phosphate buffered saline buffer is joined in mixed solution, and room temperature is placed 2min.Proceed to 5K super filter tube, the centrifugal 5min of 4500r/min, collects the about 5mL of filtrate.Filtrate is respectively through C-18 post desalination, and method is as follows: Sep-Pak C18 is connected into 5ml syringe, washes post with 2 ~ 3ml acetonitrile (acetonitrile); Wash post with 2～3mL bi-distilled water (ddH2O); Add 1mL citric acid-Na
2hPO
4the polypeptide mixture that damping fluid washes out, allows it naturally rely under run by gravity; Wash post with 5ml bi-distilled water (ddH2O); Finally use 0.5ml 60% acetonitrile/40% ddH2O wash-out target protein (polypeptide), finally collect sample in 1mL eppendorf pipe, then be put in 0.5mL 60% acetonitrile/40% distilled water, put into freeze drier, lyophilize 12 hours, obtains solid powdery SLA-I-polypeptide complex peptide.Get 0.5 g sample spot to sample target, be covered with 8mg/mL CHCA matrix (50% acetonitrile, 0.1% TFA).After to be dried, sample target is put into 4800 MALDI-TOFTOF(Foster City, Applied Biosystems) mass spectrograph analyzes.One-level mass spectrum laser intensity 3700, mass range 800-4000, shots value 750, acceleration voltage 20KV.
The external combination mass spectrum measurement result of Q02 and six strain pig SLA-I is shown in Fig. 4.
The external combination mass spectrum measurement result of AS3 and six strain pig SLA-I is shown in Fig. 5.
The external combination mass spectrum measurement result of QA4 and six strain pig SLA-I is shown in Fig. 6.
In Fig. 4～Fig. 6, A, B, C, D, E and F represent respectively, corresponding polypeptide and the external combination mass spectrum of landrace SLA-I measurement result, the external combination mass spectrum of Yorkshire Pigs SLA-I measurement result, the external combination mass spectrum of EmilZatopek pig SLA-I measurement result, the black pig SLA-I external combination mass spectrum measurement result in Yantai and the external combination mass spectrum of Laiwu Black Pigs SLA-I measurement result.
Fig. 4 result shows, Q02 presents very weak target peak with long white and Yorkshire Pigs SLA-I albumen, with the target peak that other strain pigs can not detect, illustrates that Q02 and all SLA-I albumen substantially can not be in conjunction with.
Fig. 5 result shows, AS3 presents very weak target peak with long white, Yorkshire, EmilZatopek and Laiwu Black Pigs SLA-I albumen, and present the target peak of medium tenacity with the black pig SLA-I of Hebao pig and Yantai albumen, illustrate AS3 can with the black pig SLA-I of Hebao pig and Yantai protein binding, but very weak with the combination degree of other strain pig SLA-I albumen.
The demonstration of Fig. 6 result, QA4 is white with length, EmilZatopek pig SLA-I albumen presents weak peak, presents the combination peak of medium tenacity, and present stronger combination peak with Yantai and Laiwu Black Pigs SLA-I albumen with Yorkshire, Hebao pig SLA-I albumen.Presentation of results QA4 can with Yorkshire, pocket, Yantai and Laiwu Black Pigs SLA-I protein binding, but very weak with long bletilla EmilZatopek pig SLA-I protein binding degree.
Six strain pig SLA-I are combined in vitro with control peptide Co, the combinable peptide of mass spectroscopy, does not detect sample peptide signal, illustrates that control peptide Co is not combined with described strain pig SLA-I molecule, the epitope peptide of method design of the present invention has specificity, and screening method has science.
Utilize the combination of mass spectrometric determination SLA-I single chain molecule and polypeptide, can point-devicely filter out the polypeptide that can be combined with SLA-I molecule, specificity is high.Applicant of the present invention has built bar horse miniature pig SLA-I single chain molecule by early-stage Study, and has carried out external combination screening foot-and-mouth disease virus polypeptide, thereby has proved the science of the method.But because SLA-I quasi-molecule exists restrictedly, the SLA-I that derives from different lines pig may be in conjunction with different polypeptide.In addition, because the restrictive polypeptide epitope of SLA-I quasi-molecule may have common anchor residues, different lines pig SLA-I also may be in conjunction with common polypeptide epitope.If energy Isolation and screening is adapted to the polypeptide of multiple strain pig SLA-I jointly, can contribute to development to there is the foot-and-mouth disease virus multi-epitope vaccine of prevention effect.Fig. 6 presentation of results QA4 has the protein bound ability of pig SLA-I stronger and multiple types (Yorkshire, pocket, Yantai and Laiwu Black Pigs), and QA4 is applicable to the preparation of the control vaccine of multiple strain swine foot-and-mouth disease virus, applied range.
embodiment 4eLISPOT detects the activity of epitope peptide
Because the activated CTL epitope peptide of tool can stimulate specific C D8
+emiocytosis gamma-interferon, utilizes this characteristic, to screen the CTL epitope peptide obtaining as antigen, take the pig spleen lymphocyte that separates the infection of foot-and-mouth disease obtaining as cell to be measured, carries out ELISPOT detection.
Concrete detection method is as follows:
Choose 4 of the purebred landraces (boar) at a monthly age, antibody test is determined without FMDV antibody.Stay 2 for control group, inoculate respectively Asia1 and A type foot and mouth disease virus for all the other 2 every, as experimental group.Pig is attacked poison and takes a blood sample and separate peripheral blood lymphocyte (PBMC) afterwards for 10 days, carries out after cell counting, utilizes ELISPOT test kit (MABTECH AB, Sweden) to measure the ability of polypeptid induction PBMC release IFN-γ to be measured.By the pig PBMC of control group and experimental group with 2 × 10
5the cell concn of cells/well is inoculated in ELISPOT test kit 96 orifice plates, adds each group of given the test agent by certain concentration simultaneously.Not add any sample as blank group (Blank), to add negative control PEPC o(final concentration as 10 μ g/ml) negative control group is set as stimulator, with sample peptide (Q02, AS3 and QA4, final concentration is 10 μ g/ml) for stimulator is as testing sample group, every group of sample established 3 parallel holes.According to reagent and described method, measure the ability of polypeptid induction PBMC release IFN-γ to be measured.The measurement result of experimental group and control group is shown in Fig. 7.
The demonstration of Fig. 7 result, in experimental group, QA4 stimulates the average spot number producing maximum, is then AS3, Q02, negative control group and blank group.Experimental group Q01 compared with control peptide Co, difference extremely significantly (
p< 0.01), illustrate that, compared with experimental group control peptide Co, QA4 can induce PBMC extremely significantly to discharge IFN-γ, and Q02 and AS3 can not discharge IFN-γ by obvious stimulation PBMC.In addition, to stimulate the average spot producing to count difference extremely remarkable for experimental group QA4 and control group QA4
p< 0.01), therefore QA4 is foot and mouth disease virus CTL epitope polypeptide.The significant difference although the spot number that other polypeptide Q02 and AS3 produce with the stimulation of control group corresponding polypeptide is compared (
p< 0.05), but because the difference of comparing with control peptide is not remarkable, therefore Q02 and AS3 are not effective CTL epitope peptides.Experimental result at least repeats 3 times.Read on plate instrument, to read spot, statistical study.
ELISPOT technology is called again enzyme linked immunological spot, and it is the extension of elisa technique, and difference is, ELISPOT reflects the cell of the secretion specific cells factor with the form of spot.Specific CD8
+t lymphocyte can be secreted gamma-interferon under the stimulation of active epitope peptide, i.e. IFN-γ.Each spot that ELISPOT produces represents the cell of a secretion of gamma-IFN, and spot number has reflected that polypeptide stimulates CD8
+t lymphocyte produces the ability of IFN-γ, thus indirect proof the CTL activity of polypeptide.The result showing according to Fig. 7, in experimental group, compared with control peptide Co, QA4 can induce PBMC extremely significantly to discharge IFN-γ, and Q02 and AS3 can not discharge IFN-γ by obvious stimulation PBMC.Presentation of results QA4 is Dominant Epitopes peptide, other two foot and mouth disease epitope peptide QO2 and AS3 irritation cell immunne response ability a little less than, be not Dominant Epitopes peptide.
Control peptide Co mensuration group and blank group also have small part spot produce, this be due in lymphocyte except CD8
+beyond T lymphocyte, also have natural killer cell (NK), scavenger cell etc. without antigenic stimulation in the situation that, to still have the ability of certain secretion of gamma-IFN.
Claims (2)
1. an A type foot and mouth disease CTL epitope peptide, is characterized in that: the aminoacid sequence of described epitope peptide is: Ala-Met-Leu-Arg-Ala-Ala-Thr-Tyr-Tyr.
2. CTL epitope peptide as claimed in claim 1 is prevented and treated in foot and mouth disease polypeptide vaccine and is applied in preparation.
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| CN105884869A (en) * | 2016-01-25 | 2016-08-24 | 大连大学 | Method for preparing tetramer from Asia I foot and mouth disease virus polypeptide |
| CN107247132A (en) * | 2017-05-22 | 2017-10-13 | 东莞市儿童医院 | A kind of screening appraisal procedure of polypeptide vaccine |
| CN111548389A (en) * | 2018-01-03 | 2020-08-18 | 中牧实业股份有限公司 | Immunopotentiator and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105504045A (en) * | 2015-12-18 | 2016-04-20 | 大连大学 | Method for refolding and crystallizing between Asian type-I foot and mouth disease virus polypeptide and heavy chain SLA-2 and light chain beta2m |
| CN105884869A (en) * | 2016-01-25 | 2016-08-24 | 大连大学 | Method for preparing tetramer from Asia I foot and mouth disease virus polypeptide |
| CN105884869B (en) * | 2016-01-25 | 2022-05-27 | 大连大学 | Method for preparing tetramer from Asia I type foot-and-mouth disease virus polypeptide |
| CN107247132A (en) * | 2017-05-22 | 2017-10-13 | 东莞市儿童医院 | A kind of screening appraisal procedure of polypeptide vaccine |
| CN107247132B (en) * | 2017-05-22 | 2019-06-11 | 东莞市儿童医院 | A kind of screening appraisal procedure of polypeptide vaccine |
| CN111548389A (en) * | 2018-01-03 | 2020-08-18 | 中牧实业股份有限公司 | Immunopotentiator and application thereof |
| CN111548389B (en) * | 2018-01-03 | 2021-11-26 | 中牧实业股份有限公司 | Immunopotentiator and application thereof |
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