CN103864905B - A kind of O type foot and mouth disease CTL epitope peptide and screening method - Google Patents

A kind of O type foot and mouth disease CTL epitope peptide and screening method Download PDF

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CN103864905B
CN103864905B CN201310742940.3A CN201310742940A CN103864905B CN 103864905 B CN103864905 B CN 103864905B CN 201310742940 A CN201310742940 A CN 201310742940A CN 103864905 B CN103864905 B CN 103864905B
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CN103864905A (en
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高凤山
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Dalian University
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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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention discloses a kind of O type foot and mouth disease CTL epitope peptide and screening method thereof and application.Described CTL epitope peptide is made up of nine amino-acid residues, does is its aminoacid sequence: Ala-Thr-Arg-Val-Thr-Glu-? Leu-Leu-Tyr.This epitope peptide all can have stronger binding ability and cytotoxic immune can be caused to reply with SLA-I albumen deriving from different lines pig, be applicable to the preparation of the control vaccine of multiple strain swine foot-and-mouth disease virus, applied range.The CTL analogue epi-peptide of the six strain pig SLA-I single chain molecules that the present invention utilizes structure combined mouth aphtovirus in vitro, mass spectroscopy screens the polypeptide that can be combined with complex body, and detect through ELISPOT, determine to induce the analogue epi-peptide producing T cell immunne response ability.The present invention is that a large amount of screening and identification foot and mouth disease virus CTL epi-position will provide method from now on, and lays a good foundation for developing Schweineseuche polyepitope vaccines.

Description

A kind of O type foot and mouth disease CTL epitope peptide and screening method
Technical field
The invention belongs to molecular immunology field, be specifically related to SLA-I and combine and the O type foot and mouth disease CTL epitope peptide that cytotoxic immune can be caused to reply and the method utilizing CTL epitope peptide described in SLA-I complex body screening and identification.
Background technology
Pig major histocompatibility complex (majorhistocompatibilitycomplex, MHC), that is the human leucocyte antigen (swineleukocyteantigen, SLA) of pig 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, and-2 ,-3.And light chain is by Beta2 microglobulin (Beta2microglobulin, 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 (cytotoxicTlymphocyte, CTL) immunne response, the CTL release cells factor is also killed by the target cell of virus infection.The polypeptide that also cytotoxic immune can be caused to reply can be combined with SLA-I quasi-molecule and be CTL epi-position.
Foot and mouth disease virus (foot-and-mouthdiseasevirus, FMDV) be the cause of disease of a kind of acute, hot, high degree in contact sexually transmitted disease of harm artiodactyls, serotype is divided into O, A, C, SAT1, SAT2, SAT3 and Asia1 type, without Immunogenicity power between various.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.Epiposition vaccine containing various serotype and different epidemic strain can play the requirement preventing and treating different serotypes foot and mouth disease virus.The key of development foot and mouth disease virus epiposition vaccine determines epi-position, and cell epitope comprises B cell epi-position, helper T cell (Thelper, Th) epi-position and CTL epi-position.At present, multiple B cell epi-position 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, is mainly played a role by neutralizing antibody in the control of FMDV acute attack stage, then plays a role primarily of CTL at the immunology of FMDV persistent infection phase.Recently, some scholars begin one's study the CTL epi-position of foot and mouth disease virus.2006, Barfoed(AntiviralRes, 2006,72 (3): 178-89) etc. confirm a restrictive CTL epi-position KYKEAKEWL deriving from foot and mouth disease virus Nonstructural protein 2C of mouse H-2Kd, ctl response can be caused in Mice Body.In addition, (the JGenVirol such as Guzman, 2008,89 (Pt3): 667-75) also demonstrate that an ox BoLAN*02201 restrictive foot and mouth disease virus CTL epi-position recently, this epi-position is positioned at the 795-803 amino-acid residue 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 the animal such as mouse, ox, does not also report the foot and mouth disease virus CTL epi-position through the direct submission of pig SLA-I quasi-molecule so far.
Research shows, utilizes the method for external structure MHCI heavy chain molecule, peptide and light chain complex body can screen virus epitopes in vitro.The heavy chain of mouse MHC-I is connected with light chain with a Linker by White etc. (JImmunol, 1999,162 (5): 2671-6), constructs MHC-I molecule, and demonstrate it can with antigen peptide molecule specific combination.(the EurJImmunol such as Denberg, 2000,30 (12): 3522-32) construct the HLA-A2 single chain molecule of people with a polypeptide, Linker, heavy chain and light chain β 2m, and prove that the single chain molecule of the Binding peptide built has the function identifying cell receptor.In seminar's early-stage Study, constructed six strain pig SLA-I complex protein, and expressed at pMAL-p2X, expressing protein is soluble proteins after testing, and keeps good protein conformation.The present invention is on the basis of early-stage Study, and utilize the CTL analogue epi-peptide of the six strain pig SLA-I single chain molecules combined mouth aphtovirus in vitro built, mass spectroscopy screens the polypeptide that can be combined with complex body.Combinative polypeptide detects the CTL activity of polypeptide through ELISPOT experiment.The present invention provides method for screening foot and mouth disease virus CTL epi-position from now in a large number, 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 and a kind ofly can merge the O type foot and mouth disease CTL epitope peptide that cytotoxic immune can be caused to reply with SLA-I molecular juction.
To achieve these goals, the technical solution adopted in the present invention is a kind of O type foot and mouth disease CTL epitope peptide, described O type foot and mouth disease CTL epitope peptide called after Q01, it is made up of nine amino-acid residues, its aminoacid sequence is: Ala-Thr-Arg-Val-Thr-Glu-Leu-Leu-Tyr(SEQIDNo:1), i.e. L-Ala-Thr-Arg-α-amino-isovaleric acid-TE-Leu-Leu-tyrosine.
Described O type foot and mouth disease CTL epitope peptide (Q01) derives from O type FMDV VP1 albumen, and it all has stronger binding ability with SLA-I albumen deriving from different lines pig, and Swine PBMC can be induced extremely significantly to discharge IFN-γ.
O type foot and mouth disease CTL epitope peptide (Q01) of the present invention is applied to the preparation of foot and mouth disease polypeptide vaccine.
The screening method of O type foot and mouth disease CTL epitope peptide (Q01) as above, it comprises the steps:
(1) biological information software netMHCpan2.4 (http://www.cbs.dtu.dk/services/NetMHCpan) is utilized, input FMDV VP1 protein sequence, select nonapeptide, under SLA gene prediction obtain can with SLA-I protein binding, and offer the CTL analogue epi-peptide that cell surface causes cytotoxic immune to reply, synthesize with artificial chemistry method the CTL analogue epi-peptide that above-mentioned prediction obtains;
(2) CTL analogue epi-peptide step (1) obtained and the external association reaction of SLA-I albumen, reaction mixture obtains solid powdery SLA-I-polypeptide complex after C-18 post desalination, refining, lyophilize;
(3) SLA-I-polypeptide complex screens the CTL analogue epi-peptide that can be combined with SLA-I through mass spectroscopy;
(4) with can with the protein bound CTL analogue epi-peptide of SLA-I for antigen, the pig spleen lymphocyte of the infection of foot-and-mouth disease obtained with separation is cell to be measured, carry out ELISPOT detection, determine to induce the CTL analogue epi-peptide producing T cell immunne 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 SEQIDNo: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-I dietary protein origin.
Utilize the combination of mass spectrometric determination SLA-I single chain molecule and polypeptide, point-devicely can filter out the polypeptide that can be combined with SLA-I molecule, specificity is high.Applicant of the present invention has built Ba-Ma mini pig SLA-I single chain molecule by early-stage Study, and has carried out external combination screening foot-and-mouth disease virus polypeptide, thus demonstrates the science of the method.But because SLA-I quasi-molecule exists restricted, the SLA-I deriving from different lines pig may 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 in conjunction with common polypeptide epitope.If energy Isolation and screening is adapted to the polypeptide of multiple strain pig SLA-I jointly, then can contribute to developing the foot-and-mouth disease virus multi-epitope vaccine with prevention effect.The present invention by aforesaid method screening and identification go out with derive from landrace, Yorkshire Pigs, EmilZatopek pig, Hebao pig, Yantai pig or Laiwu Black Pigs SLA-I albumen all there is good binding ability, and Swine PBMC can be induced significantly to discharge the CTL epitope peptide (Q01) 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. O type foot and mouth disease CTL epitope peptide (Q01) of the present invention all has stronger binding ability with SLA-I albumen deriving from different lines pig and cytotoxic immune can be caused to reply, be applicable to the preparation of the control vaccine of multiple strain swine foot-and-mouth disease virus, applied range.2. the present invention is that a large amount of screening and identification foot and mouth disease virus CTL epi-position will provide method from now on, and lays a good foundation for developing 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 studying the molecule prevented and treated in foot and mouth disease, cytosis mechanism.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE electrophorogram of the fusion protein S LA-I-MBP obtained through Amylose affinity chromatography, wherein: M, represent low molecular weight protein (LMWP) standard substance, 1-6, represent the black and Laiwu Black Pigs SLA-I-MBP purifying protein in landrace, Yorkshire Pigs, EmilZatopek pig, Hebao pig, Yantai respectively;
Fig. 2 is the SDS-PAGE electrophorogram of the SLA-I-MBP after FactorXa cutting, wherein: M, represent low molecular weight protein (LMWP) standard substance, 1-6, represent respectively the black and Laiwu Black Pigs SLA-I in landrace, Yorkshire Pigs, EmilZatopek pig, Hebao pig, Yantai cut after albumen, wherein SLA-I albumen size is 41.6kDa, and the size of fusion rotein is 84.1kDa;
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, represent the black and Laiwu Black Pigs SLA-I purifying protein in landrace, Yorkshire Pigs, EmilZatopek pig, Hebao pig, Yantai respectively;
Fig. 4 is the external in conjunction with mass spectrum measurement result of Q01 and six strain pig SLA-I;
Fig. 5 is the external in conjunction with mass spectrum measurement result of Q02 and six strain pig SLA-I;
Fig. 6 is the external in conjunction with mass spectrum measurement result of AS3 and six strain pig SLA-I;
In described Fig. 4 ~ Fig. 6, described A, B, C, D, E and F represent respectively, corresponding polypeptide and landrace SLA-I external in conjunction with mass spectrum measurement result, Yorkshire Pigs SLA-I external in conjunction with mass spectrum measurement result, EmilZatopek pig SLA-I external in conjunction with mass spectrum measurement result, Yantai black pig SLA-I external in conjunction with mass spectrum measurement result and Laiwu Black Pigs SLA-I external in conjunction with mass spectrum measurement result;
Fig. 7 is that ELISPOT detects polypeptid induction CD8 +t lymphocyte produces IFN-γ ability, wherein: * * represents candidate polypeptide difference extremely remarkable (P < 0.01) compared 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.Described six strain pigs comprise 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 getting six strain pigs is respectively seeded to 500mLLB substratum, and shaking culture in 37 degree of incubators of 170rpm, detects its OD every for some time 600, OD to be grown to 600time between 0.6 to 0.8, add IPTG to 0.5mmol/L, 37 degree of inductions 5 hours.
(3) fusion rotein MBP-SLA-I affinity purification
Will 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 [20mmol/LTris-HCl(pH7.4), 200mmol/LNaCl, 1mmol/LEDTA (pH8.0), 1mmol/L sodium azide, 1mmol/LDTT] 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.Fusion protein S LA-I-MBP containing solubility in supernatant liquor, utilize the character that maltose binding protein (MBP) ligands specific in pillar is combined, carry out Amylose affinity chromatography, fusion protein S LA-I-MBP is adsorbed on Amylose pillar, remove 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 of 10mmol/L maltose is contained 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, the stripe size of inspection target protein and purity.The results are shown in Figure 1.Fig. 1 result shows, six strain pig SLA-I-MBP purity about 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 comparatively large, the specific binding of target protein molecule and antigen peptide may be interfered with, therefore need the MBP excision in fusion rotein.On pMAL-p2X carrier between malE gene and multiple clone site, there is one by the aminoacid sequence of Xa factor identification, Ile-Glu-Gly-Arg, can MBP be excised.
Concrete cutting method is:
MBP-SLA-I fusion rotein FactorXa step (3) obtained cuts, and cutting condition is: 1mol/LNaCl100 μ L, 100mmoL/LCaCl 220 μ L, 1mol/LTris-HCl20 μ L, the SLA-I-MBP protein concentrated solution 400 μ L(that step (3) obtains is about 1mgSLA-I-MBP albumen), FactorXa(1U enzyme cutting 50mg) 20U, add sterilizing deionized water to 1mL.21 DEG C of cutting 20h, carry out SDS-PAGE detection fusion Protein cleavage situation, the results are shown in Figure 2 after cutting.Fig. 2 result shows, and at 43kDa place, six strain pig SLA-I-MBP all cut out a treaty 41kDa band, consistent with target protein SLA-I size 41.6kDa.
Egg white mixture after cutting, first through Amylose column purification, collected the protein sample of post buffer A wash-out.The protein sample that collection obtains is again through DEAECeramicHyperDF anionresin column separating purification albumen, with excessively post damping fluid (20mmoL/LTris-HCl(pH8.0 of 0.15moL/LNaCl) of 10 column volumes containing 0.15mol/LNaCl) wash-out, detect according to spectrophotometer, collect elution peak, after concentrated, SDS-PAGE testing goal Protein S LA-I, the results are shown in Figure 3.Fig. 3 result shows, and finally collect the albumen obtained and present single band, size is 41.6kDa, and purity reaches more than 95%, meets the requirement carrying out protein function research.
The design of embodiment 2 foot and mouth disease virus analogue epi-peptide
Utilize biological information software netMHCpan2.4 (http://www.cbs.dtu.dk/services/NetMHCpan), input O type FMDV VP1 aminoacid sequence (AJ539138) and Asia1 type FMDV VP1 aminoacid sequence (EF149009), select nonapeptide, under SLA gene prediction obtain can with SLA-I protein binding, and offer the CTL analogue epi-peptide that cell surface causes cytotoxic immune to reply, devise 9 peptides that 3 derive from described FMDV VP1 albumen altogether, called after Q01 respectively, Q02, AS3, its aminoacid sequence and other essential informations are in table 1.Wherein Q01 and Q02 all 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 Asia1/Jiangsu/China/2005.In addition, design and synthesized irrelevant control peptide Co, its relevant information is in table 1.Each epitope peptide is stored in-80 DEG C with lyophilised state, for subsequent use.
The combination of embodiment 3SLA-I albumen and foot and mouth disease virus analogue epi-peptide and screening
Six strain pig SLA-I albumen each 1mL(protein content 1mg/mL that embodiment 1 purifying is obtained) mix with 5mLPBS 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 (Q01, Q02, AS3 or Co) respectively, and the mol ratio of peptide and target protein is 10:1, and 37 DEG C are spent the night.Mixture is added 30K super filter tube, the centrifugal 45min of 4500r/min.Add PBS, 37 DEG C of water-bath 2min, 4500r/min is centrifugal, is about 100uL to final volume.Join in mixed solution by 5mL citrate-phosphate salt buffer, room temperature places 2min.Proceed to 5K super filter tube, the centrifugal 5min of 4500r/min, collect filtrate and be about 5mL.Filtrate is respectively through C-18 post desalination, and method is as follows: Sep-PakC18 is connected into 5ml syringe, washes post with 2 ~ 3ml acetonitrile (acetonitrile); Post is washed with 2 ~ 3mL bi-distilled water (ddH2O); Add 1mL citric acid-Na 2hPO 4the polypeptide mixture that damping fluid washes out, under allowing it naturally rely on run by gravity; Post is washed with 5ml bi-distilled water (ddH2O); Finally use 0.5ml60% acetonitrile/40%ddH2O wash-out target protein (polypeptide), finally collect sample in 1mLeppendorf pipe, then put into freeze drier, lyophilize 12 hours, obtain solid powdery SLA-I-polypeptide complex.Get 0.5 μ g sample spot on sample target, be covered with 8mg/mLCHCA matrix (50% acetonitrile, 0.1%TFA).After to be dried, sample target is put into 4800MALDI-TOFTOF(FosterCity, AppliedBiosystems) mass spectrograph analyzes.First mass spectrometric laser intensity 3700, mass range 800-4000, shots value 750, acceleration voltage 20KV.
Q01 and six the external of strain pig SLA-I see Fig. 4 in conjunction with mass spectrum measurement result.
Q02 and six the external of strain pig SLA-I see Fig. 5 in conjunction with mass spectrum measurement result.
AS3 and six the external of strain pig SLA-I see Fig. 6 in conjunction with mass spectrum measurement result.
In Fig. 4 ~ Fig. 6, A, B, C, D, E and F represent respectively, corresponding polypeptide and landrace SLA-I external in conjunction with mass spectrum measurement result, Yorkshire Pigs SLA-I external in conjunction with mass spectrum measurement result, EmilZatopek pig SLA-I external in conjunction with mass spectrum measurement result, Yantai black pig SLA-I external in conjunction with mass spectrum measurement result and Laiwu Black Pigs SLA-I external in conjunction with mass spectrum measurement result.
Fig. 4 result shows, and Q01 and all strain pig SLA-I protein binding present stronger target peak, illustrate that Q01 can with all SLA-I protein binding.
Fig. 5 result shows, and Q02 presents very weak target peak, the target peak that can not detect with other strain pigs with long white and Yorkshire Pigs SLA-I albumen, illustrates that Q02 and all SLA-I albumen substantially can not be in conjunction with.
Fig. 6 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 Hebao pig and Yantai black pig SLA-I albumen, illustrate AS3 can with Hebao pig and Yantai black pig SLA-I protein binding, but very weak with the combination degree of other strain pig SLA-I albumen.
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, point-devicely can filter out the polypeptide that can be combined with SLA-I molecule, specificity is high.Applicant of the present invention has built Ba-Ma mini pig SLA-I single chain molecule by early-stage Study, and has carried out external combination screening foot-and-mouth disease virus polypeptide, thus demonstrates the science of the method.But because SLA-I quasi-molecule exists restricted, the SLA-I deriving from different lines pig may 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 in conjunction with common polypeptide epitope.If energy Isolation and screening is adapted to the polypeptide of multiple strain pig SLA-I jointly, then can contribute to developing the foot-and-mouth disease virus multi-epitope vaccine with prevention effect.Fig. 4 result shows, and Q01 and all strain pig SLA-I protein binding present stronger target peak, and illustrate that Q01 can with all SLA-I protein binding, Q01 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 that obtains for antigen, is cell to be measured, carries out ELISPOT detection to be separated the pig spleen lymphocyte of the infection of foot-and-mouth disease obtained.
Concrete detection method is as follows:
Choose the purebred landrace (boar) 4 at a monthly age, antibody test is determined without FMDV antibody.Stay 2 for control group, all the other 2 every pigs inoculate O type and Asia1 type foot and mouth disease virus respectively, as experimental group.Pig attacks poison blood sampling separation peripheral blood lymphocyte (PBMC) afterwards in 10 days, after carrying out cell counting, utilizes ELISPOT test kit (MABTECHAB, Sweden) to measure the ability of candidate polypeptide induction PBMC release IFN-γ.By the Swine PBMC of control group and experimental group with 2 × 10 5the cell concn of cells/well is inoculated in ELISPOT test kit 96 orifice plate, adds each group of given the test agent by certain concentration simultaneously.Not add any sample for blank group (Blank), be 10 μ g/ml to add negative control PEPC o(final concentration) negative control group is set for stimulator, with sample peptide (Q01, Q02 and AS3, final concentration is 10 μ g/ml) for stimulator is as testing sample group, often organizes sample and establish 3 parallel holes.The ability that candidate polypeptide induction PBMC discharges IFN-γ is measured according to the method described in test kit.The measurement result of experimental group and control group is shown in Fig. 7.
Fig. 7 result shows, and in experimental group, Q01 stimulates the average blob produced 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 compared with experimental group control peptide Co, Q01 can induce PBMC extremely significantly to discharge IFN-γ, and Q02 and AS3 can not discharge IFN-γ by obvious stimulation PBMC.In addition, experimental group Q01 and control group Q01 stimulates the average blob number difference produced extremely remarkable p< 0.01), therefore Q01 can be considered to foot and mouth disease virus CTL epitope polypeptide.Although other polypeptide Q02 with AS3 and control group corresponding polypeptide stimulate significant difference compared with the spot number that produces ( p< 0.05), but due to compared with control peptide difference remarkable, therefore Q02 and AS3 can not be considered to effective CTL epitope peptide.Each experimental result at least repeats 3 times.Read plate instrument reads spot, statistical study.
ELISPOT technology is called ELISpot again, 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 secrete gamma-interferon under the stimulation of active epitope's peptide, i.e. IFN-γ.Each spot that ELISPOT produces represents the cell of a secretion of gamma-IFN, and spot number reflects polypeptide stimulates CD8 +t lymphocyte produces the ability of IFN-γ, thus the indirect proof CTL activity of polypeptide.According to the result of Fig. 7 display, in experimental group, compared with control peptide Co, Q01 can induce PBMC extremely significantly to discharge IFN-γ, and Q02 and AS3 can not discharge IFN-γ by obvious stimulation PBMC.Result illustrate Q01 be Dominant Epitopes peptide, other two foot and mouth disease epitope peptide QO2 and AS3 irritation cell immunne response ability more weak, be not Dominant Epitopes peptide.
Control peptide Co mensuration group and blank group also have small part spot to produce, this be due in lymphocyte except CD8 +beyond T lymphocyte, also have natural killer cell (NK), scavenger cell etc. when without the ability still having certain secretion of gamma-IFN when antigenic stimulation.

Claims (2)

1. an O type foot and mouth disease CTL epitope peptide, is characterized in that: the aminoacid sequence of described CTL epitope peptide is: Ala-Thr-Arg-Val-Thr-Glu-Leu-Leu-Tyr.
2. CTL epitope peptide as claimed in claim 1 is preparing the application prevented and treated in the polypeptide vaccine of foot and mouth disease.
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CN105461811B (en) * 2015-12-18 2018-12-18 大连大学 A kind of method of O-shaped foot-and-mouth disease virus polypeptide and SLA-2 heavy chain, the crystallization of light chain β 2m renaturation
CN105884870B (en) * 2016-01-25 2022-05-27 大连大学 Method for preparing tetramer from O-type foot-and-mouth disease virus polypeptide
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CN117304277B (en) * 2023-09-26 2024-03-08 中国农业科学院兰州兽医研究所 O-type foot-and-mouth disease virus VP4 protein T cell epitope polypeptide and application thereof

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CN101597327A (en) * 2008-10-28 2009-12-09 中国人民解放军第二军医大学 Molecular mimic peptide of a kind of O type foot and mouth disease virus antigen epitope and uses thereof
CN101659695A (en) * 2008-08-27 2010-03-03 中牧实业股份有限公司 O-type aftosa synthetic peptide vaccine

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