CN105884870A - Method for preparing tetramer from O type foot and mouth disease virus polypeptide - Google Patents
Method for preparing tetramer from O type foot and mouth disease virus polypeptide Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for preparing tetramer from O type foot and mouth disease virus polypeptide. Aiming at designed foot and mouth disease virus polypeptide, two methods of ELISPOT and tetramer construction are adopted for screening and identifying the foot and mouth disease virus polypeptide, a Hebao pig SLA-2 heavy chain tetramer is constructed, expression is carried out on a prokaryotic expression system pET-21a(+)/BL21, and an inclusion body is extracted; in the foot and mouth disease virus polypeptide, polypeptide causing a specific CTL reaction is screened out through an ELISPOT method, then the polypeptide, the SLA-2 heavy chain, and existing light chains beta 2m are subjected to renaturation, biotinylation and FITC label streptavidin reactions to generate the SLA-2-BSP/peptide tetramer, and then the function of the tetramer is detected through flow cytometry. Through an ELISPOT method, a specificity polypeptide epitope of O type foot and mouth disease viruses VP1 is obtained through identification, the peptide tetramer is successfully constructed, a pig tetramer technology platform is preliminarily built, and a foundation is laid for study of pig specificity CTL immune responses and T epitope screening.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of O type foot-and-mouth disease virus polypeptide and prepare tetrameric method.
Background technology
Foot and mouth disease is the acute febrile high degree in contact caused by foot and mouth disease virus (Foot and Mouth Disease Virus, FMDV)
Sexually transmitted disease, mainly encroaches on artiodactyls, and Swine is relatively vulnerable groups, clinically with oral mucosa, hoof and skin of breast
Vesicle occurring and festers for principal character, being the one that in disease of domestic animals, infectiousness is the strongest, the outburst of foot and mouth disease each time all can be given
Agricultural economy brings loss greatly.Therefore International Office of Epizootics (OIE) is classified as the A class animal that morbidity must be reported always
First of epidemic disease list.So prevention and diagnosis to the foot and mouth disease of pig are just particularly important.At present, the preventing and treating of foot and mouth disease virus
Rely primarily on traditional inactivated vaccine.Inactivated vaccine has the advantage of himself, good immune effect, and cost is low.But there is also
Some potential deficiencies, inactivated vaccine still have because inactivation thoroughly or due to extraneous street strain's gene recombinaton thus produce
The danger of raw virulence diffusion.It addition, for foot and mouth disease virus, its epidemic isolates is always in constantly variation, say, that vaccine strain
Development always lag behind the appearance of variation strain.For problem above, need to explore new foot-and-mouth disease vaccine.At present, polypeptide
Vaccine is a study hotspot, because this vaccine is to be combined by the different epi-positions of virus, can cause immunne response, again
Overcome the problem that vaccine itself dissipates poison.Therefore, how to separate and identify that foot and mouth disease virus epi-position is a crucial problem.Mesh
Before, the research of foot and mouth disease virus epi-position focuses mostly in B cell epi-position and helper T lymphocyte (T helper, Th) epi-position, and these are two years old
Class epi-position is mainly with to cause humoral immunoresponse(HI) relevant；Lack the cytotoxic T lymphocyte causing cellullar immunologic response at present
(Cytotoxic T Leukocyte, CTL) epi-position.Foot and mouth disease virus is strict cytozoon, after its host cells infected
Cellullar immunologic response will certainly be caused.Therefore, screen the most in vitro and identify that foot and mouth disease virus CTL epi-position is one and has very much
The problem of researching value.At present, the method for in-vitro screening and identifying virus CTL epi-position has multiple, tests including cell killing,
ELISPOT and tetramer method etc..Wherein, ELISPOT and the tetramer combine is screening at present and identifying virus CTL table
The method that position is conventional.
ELISPOT technology, is also called elispot assay, be nineteen eighty-three Czerkinsky etc. (J Clin Microbiol, 1983,
17 (6): 965-969) reported first, for measuring the heat-sensitive toxin that escherichia coli produce, is similar to enzyme-linked immunosorbent assay
(ELISA), it is the extension of elisa technique, is commonly used at present measure cytokine or the function of CTL.(the J Immunol such as Herr
Methods, 1996,191 (2): 131-142) started to be applied to this technology measure the HIV that people HLA-A2 combines in 1996
The cell killing reaction that epitope polypeptide causes, its ultimate principle is exactly that HIV epitope peptide can stimulate CD8+T lymphocyte (CTL)
Release gamma interferon, such that it is able to the CTL activity of indirect determination polypeptide.(Wang Yirui etc., China's AIDS according to research reports
Disease, 2005,11 (1): 70-72), ELISPOT can detect single secretory cell from 1,000,000 cells, can be thin to secretion
< cell of 100 molecules per second detects intracellular cytokine speed, has higher reliability, repeatability and susceptiveness.
MHC I/ peptide tetramer technology is to be set up in 1996 years by (Science, 1996,274 (2584): 94-96) such as Altman
For detection by quantitative specific T-cells.This technology makes Peptide-specific CTL Activity determination reach special, efficiently and directly determine
The degree of amount, its sensitivity is 3-5 times of ELISPOT testing result.The ultimate principle of this technology is that single MHC-peptide is combined
The affinity that thing is combined with TCR is the lowest, and speed of dissociating is fast.And Altman find under study for action the pMHC of 4 molecules with
TCR combines has stronger affinity and biology effect.According to the specific binding relation of biotin Yu Avidin 4:1, profit
(Li Qing etc., cell is exempted to identify the distinguished sequence of albumen with biotin-protein ligase (Biotin-protein ligase A, BirA)
Epidemiology magazine, 2005,2005,21 (5): 557-560), the heavy chain c-terminus of MHC molecule adds the BirA of 1 biotin
Substrate peptide sequences, biotin can be attached on pMHC by BirA enzyme, thus 4 biotinylated pMHC monomers and 1
The Streptavidin of individual Avidin labelling forms tetramer complex, and then the overall affinity of raising pMHC Yu TCR, and
By sensitivity higher flow cytometer (FACS) antigenspecific T lymphocyte can quickly be detected and separate.
The major histocompatibility complex (major histocompatility complex, MHC) of pig is also referred to as the leukocyte of pig and resists
Former (swine leukocyte antigen, SLA), its I quasi-molecule mediate cellular immunne response.The heavy chain of SLA I quasi-molecule with
Light chain and virus polypeptide can form complex intracellular, submission to cell surface by CD8+φt cell receptor (TCR) identifies,
Killer T cell (Cytotoxic T leukocyte, CTL) immunne response, beneficially body is caused to remove virus, resist the disease.
These have the virus polypeptide of function to be i.e. potential vaccine candidate peptide.In vitro, how to differentiate and screen the viral table having function
Position, is a technology the most crucial.
At present, domestic the most Tetramer technology is not applied to the foot-and-mouth disease virus polypeptide function that pig SLA I quasi-molecule combines
In qualification.The present invention intends on the basis of early-stage Study Hebao pig SLA-2 Binding peptide, by ELISPOT technology and Tetramer
Technology combines, and identifies and screen the function of foot-and-mouth disease virus polypeptide, comprises the novel of foot and mouth disease virus CTL epi-position for developing from now on
Polypeptide vaccine lays the foundation.
Summary of the invention
The present invention is directed to the foot-and-mouth disease virus polypeptide of design, ELISPOT will be used and build two kinds of method screenings of the tetramer and identify
Foot-and-mouth disease virus polypeptide, builds the Hebao pig SLA-2 heavy chain tetramer, and prokaryotic expression system pET-21a (+)/BL21 carries out
Expressing, inclusion body extracts；Foot-and-mouth disease virus polypeptide, is had by the screening of ELISPOT method and causes specific CTL to react
Polypeptide, then polypeptide and SLA-2 heavy chain, existing light chain β 2m are carried out renaturation, biotinylation and with FITC labelling
Streptavidin reaction generates SLA-2-BSP/ peptide tetramer, then by the tetrameric function of Flow cytometry.
The technical characteristic of the present invention is as follows:
1, swine foot-and-mouth disease virus SLA I restricted CTL epitope design
With NetMHCpan 2.8Version as template, by each merit of SLAI quasi-molecule including Hebao pig SLA-2 of clone
Can be filed in online website form (www.cbs.dtu.dk/services/NetMHCpan/) by gene, SLA I class original with website is divided
The prediction online tool of son each functional gene composition pig source virus CTL epitope.By O type foot and mouth disease virus
Tibet/CHA/99 (HuBHK99), A type foot and mouth disease virus A-HuBWH (1) 2009 and Asia I type foot and mouth disease virus
VP1 and the 3D nonstructural protein sequences of tri-kinds of strains of 1/Jiangsu/China/2005 input prediction template respectively, carries out CTL table
The prediction of position, selects polypeptide candidate's epi-position with high-affinity.
2, ELISPOT test:
(1) aseptic PBS dilution is coated IFN-γ monoclonal antibody to 10 μ g/mL, pH7.4；
(2) taking out ELISPOT personality board, every hole adds 50 μ L 70% Ethanol Treatment, and the process time does not at most exceed 1min (
As tens seconds, film runs through), discard ethanol solution, every hole adds the 200 aseptic washing of μ L 5 times, and every hole adds 100 μ L
The IFN-γ monoclonal antibody solution diluted, 4-8 DEG C of night incubation；
(3) next day, sucking antibody-solutions, every hole 200 aseptic PBS of μ L washes 2 times；
(4) every hole adds 20 μ L confining liquids, closes 2h, sucks confining liquid for 37 DEG C, and every hole adds 200 μ L complete mediums, room temperature
Hatch at least 30min；
(5) sucking culture medium, every hole adds the polypeptide of 100 μ L beforehand dilutions and PBMC cell suspension, and (cell suspension is diluted to
2.5×105Cells/mL), each sample arranges 3 repetitions；
(6) plate lid, 37 DEG C, 5%CO are built2Under the conditions of hatch cultivation 18～20h (optimal incubation time needs trial test to grope),
During cultivation, can not disturbance cell；
(7) sucking cell suspension, every hole 200 aseptic PBS of μ L washes 5 times, dilutes detection antibody extremely with the PBS containing 0.5%FBS
0.5 μ g/mL, every hole adds 100 μ L, hatches 2h for 37 DEG C, completes to clean according to step (3)；
(8) diluting streptavidin (streptavidin-HRP) with the PBS containing 0.5%FBS, every hole adds 100 μ L.Hatch for 37 DEG C
1h, completes to clean according to step (3)；
(9) every hole adds 100 μ L nitrite ion lucifuges placements, reacts 5～30min depending on spot formation situation, is sure not overresponse and causes relatively
High background develops the color, and terminates substrate chromogenic reaction with the every hole of deionized water wash, and room temperature air-dries 2h or overnight treats that it is complete
White drying, in the medium-term and long-term preservation of sealed plastic bag.ELISPOT speckle Cellular Technology Ltd. (CTL)
CompanyAnalyzer reads plate instrument counting, completes image with ImmunoCapture software the most respectively and obtains
Take, with Immunospot 5.0 software analysis statistics Quality Control.
3, heavy chain SLA-2-BSP and light chain s β 2m tetramerization
1), yeast culture
A: heavy chain SLA-2-HB-BSP and light chain β 2m detection of expression
Activation heavy chain recombinant bacterium SLA-2-HB-BSP/BL21 and light chain recombinant bacterium β 2m/BL21, inoculation 5mL contains Amp respectively
The culture medium of (100 μ g/mL), cultivates to OD for 37 DEG C600When reaching 0.4-0.6, add IPTG to final concentration of 1mmol/L,
Take thalline SDS-PAGE and detect protein expression situation.
Recombinant bacterium SLA-2-HB-BSP/pET-21a (+)-BL21 is through inducing, 12%SDS-PAGE detects, and is finding that it is just being expressed
Often, position is about at 34kD.Light chain β 2m/pET-21a (+) detect through induction and SDS-PAGE, it was demonstrated that this protein expression is just
Often, protein positions is about at about 12kD.Heavy chain and light chain are suitable for carrying out next step the tetramer to be prepared.
By correct for the SDS-PAGE detection heavy chain SLA-2-HB-BSP/pET-21a recombinant bacterium expressed and light chain
S 37 DEG C of incubated overnight of β 2m/pET-21a recombinant bacterium.
B: respectively with in the dilution overnight culture of 1:100 to 1L LB, cultivate OD for 37 DEG C600=1, add IPTG extremely
Final concentration, to 0.5mmol/L, continues to cultivate 4 hours, results antibacterial (each 1 liter of heavy chain light chain).
2), prepared by inclusion body
A: antibacterial 30mL PBS suspends, 650W carrying out ultrasonic bacteria breaking (5sec on/10sec off, 10min).
B: inclusion body 25mmol/L Tris-HCl, pH8.0,100mmol/L NaCl, 1%Triton-X100 cleaning mixture washes 2 times.
C: inclusion body 25mmol/L Tris-HCl, pH8.0,100mmol/L NaCl cleaning mixture washes 1 time.
D: the inclusion body 25mmol/L Tris-HCl containing 8mol/L carbamide, pH8.0,100mmol/L NaCl dissolve, to albumen
Matter concentration is 10mg/mL.
3), renaturation
Under the conditions of 4 DEG C, toward the 100mmol/L Tris-HCl of 500mL, pH8.0,400mmol/L Arginine, 5mmol/L
Reduced Glutathione, 0.5mmol/L oxidised Glutathione adds 15mg heavy chain, 10mg light chain and 5mg
Hu62 epitope peptide, stirs 48 hours.
4), the biotinylation of MHC monomer
The renaturation solution of 500mL, is concentrated to 5mL, carries out buffer-exchanged with PD10 post, and new buffer is 10mmol/L Tris
pH8.0.According to the explanation of BirA test kit (Avidity), composition in test kit is joined in above-mentioned protein solution, room
Temperature processes 16 hours.
5), the separation of biotinylated monomers
Above-mentioned protein solution overnight is carried out AKTA FPLC separation: detached dowel Hiload 26/600Supdex 75pg, point
It is 20mmol/L Tris-HCl pH8,100mmol/L NaCl from buffer；Flow velocity 1mL/min.Collection elution volume is 163ml
Protein peak, be concentrated to 1mg/mL.
6), the qualification of monomer
A:SDS-PAGE, the monomer that above-mentioned FPLC is separated, point sample 8 μ g, if seeing heavy chain and light chain two band, show
After renaturation, monomer has been formed.
B:ELISA detects, and 4 DEG C are overnight coated the monomer that FPLC separates, every hole 2 μ g, holes, are simultaneously introduced the most biotinylated
Monomer, as comparison, carries out detecting the biotinylation situation of monomer with the strepavidin (Biolegend) of HRP labelling.
According to every 1mg biotinylated monomers add 0.312mg extravidine-PE amount mixing, formed PE labelling good four gather
Body.
4, SLA-2/HB/Hu62Tetramer flow cytometer detection
(1) separating the peripheral blood lymphocytes of Tibet/CHA/99 (HuBHK99) strain counteracting toxic substances group pig, 1500rmp room temperature is centrifuged 3
Min, abandons supernatant, with PBS (pH 7.2) washed cell once, and recentrifuge.
(2) cell precipitation is resuspended in FACS cleaning mixture (containing 0.1%NaN31 × PBS with 0.1%BSA).
(3) in cell suspension, add the SLA-2-HB-BSP/Hu62 tetramer of 5 μ L PE labellings, room temperature lucifuge reaction 20min.
(4) adding 2mLFACS cleaning mixture re-suspended cell, 500 × g room temperature is centrifuged 3min, abandons supernatant, and cell precipitation is resuspended in 100 μ L
FACS cleaning mixture.
(5) add the Anti-pig CD8a antibody of 5 μ L (2.5 μ g) FITC labelling, continue lucifuge reaction 20min in room temperature.
(6) adding FACS cleaning mixture washed cell once, 1500rmp room temperature is centrifuged 3min, abandons supernatant, and cell precipitation is resuspended in
In right amount in (300～400 μ L) FACS cleaning mixture.
(7) cell suspension sample through flow cytometry and sorts.
Hu62 belongs to O type foot and mouth disease strain, takes the peripheral blood lymphocytes (PBMC) of the pig attacking O type poison, uses FITC
PBMC is dyeed by the Anti-Pig CD8a monoclonal antibody of labelling.Re-use PE labelling
The SLA-2-HB-BSP-Hu62-s β 2m tetramer carries out double dyeing to PBMC, is i.e. directed to the characteristic type of Hu62 polypeptide epitope
CTL, account for the 5.7% of total cell, form extremely significantly difference (P < 0.01) with matched group 0.1% (non-counteracting toxic substances group cell),
Prove that the SLA-2-HB-Hu62-s β 2m tetramer is successfully prepared, prove that Hu62 is foot and mouth disease virus specific CTL table simultaneously
Position.
Result shows, ELISPOT successfully filters out an O type foot-and-mouth disease virus polypeptide Hu62, has significant CTL function.
The SLA-2-/Hu62 tetramer prepared by Hu62 polypeptide can be with specific CTL cell effect.The present invention passes through ELISPOT
Method identifies the specific polypeptide epi-position having obtained swine foot-and-mouth disease virus VP1, and successfully constructs this polypeptide epitope
SLA-2/ peptide tetramer, have detected the specific CTL being directed to this epi-position, tentatively establishes pig Tetramer technology platform, for grinding
Study carefully pig specific CTL immunity response and t cell epitope screening lays the foundation.
Accompanying drawing explanation
Fig. 1 for restructuring SLA-2-HB-BSP/pET-21a (+) protein expression detection, M represent low molecular weight protein (LMWP) Marker, 1: do not lure
Lead group, 2: the SLA-2-HB-BSP/pET-21a of abduction delivering (+) albumen；
Fig. 2 be recombinant beta 2m/pET-21a (+) protein expression detection, M represent low molecular weight protein (LMWP) Marker, 1, do not induce
β 2m/pET-21a (+) recombinant bacterium expression, 2, the β 2m/pET-21a of abduction delivering (+) recombinant bacterium expression, expressing protein
Size about 12kD；
Fig. 3 is the ability that ELISPOT detects each foot and mouth disease polypeptide stimulation CTL secretion of gamma-IFN, and ## represents advantage polypeptide epitope；
Fig. 4 stimulates CTL to be produced representational ELISPOT result, wherein: A, blank group by each foot and mouth disease polypeptide, does not attacks
Poison；B, negative control group, not adding polypeptide after counteracting toxic substances stimulates；C, Hu62 stimulation group；D, WX96 stimulation group；E, 3D314
Stimulation group；F, AHu63 stimulation group；G, WH94 stimulation group；H, INT3 stimulation group；I, Q10 stimulation group；J, As3
Stimulation group, the speckle number that the digitized representation in each group of lower right corner is corresponding；
Fig. 5, for separating biotinylation SLA-2-HB-BSP-Hu62-β 2m monomer FPLC collection of illustrative plates, from left to right, is followed successively by polymer
(120.12), SLA-2-BSP-Hu62-β 2m monomer (163.41), heavy chain SLA-2-BSP (228.49) and light chain β 2m
(287.97)；
Fig. 6 is the preparation of SDS-PAGE Quality Control monomer, M, molecular weight standard；1, SLA-2-BSP heavy chain；2, β 2m light chains；
3, monomer after the biotinylation of concentration and FPLC separation；
Fig. 7 is that the Tetramer that Flow cytometry SLA-2-BSP-Hu62-β 2m is formed analyzes；
Fig. 8 Flow cytometry tetramer representativeness collection of illustrative plates, wherein: A, represents and takes the non-immune group Swine PBMC detection tetramer；
B, immune group Swine PBMC detects tetrameric streaming result, and Q1 represents the CD8 of the mono-labelling of FITC+The ratio of T cell；
Q2, represents double positive cells of FITC and PE labelling, can identify tetrameric CD8+T cell；Q3, double negative cells；
The cell of the mono-labelling of Q4, PE, i.e. other can identify tetrameric lymphocyte.
Detailed description of the invention
The present invention is further illustrated in conjunction with embodiment.
First, reagent involved in the present invention is described；
IPTG, DNA Marker and molecular weight of albumen Marker is TaKaRa Products；4-chloro-1-naphthols, three hydroxyl first
Base aminomethane (Tris-alkali), glycine, N, N ,-dimethyl fork bisacrylamide and propionic acid amide., PBS solution, NH4Cl、
DMSO, Tris-base, L arginine, EDTA, reduced glutathion (GSH), oxidized form of glutathione (GSSG),
PMSF, Ammonium persulfate., TEMED, DTT, coomassie brilliant blue R250 are purchased from Sheng Gong biological engineering company limited；BCA
Determination of protein concentration test kit, western blot developer solution and fixative solution are purchased from green skies company；Protein concentration super filter tube is purchased from
MilliPore company；Anti-His Tag Mouse Monoclonal Antibody and HRPConjugated Goat anti-Mouse
IgG is purchased from Amy Jie Ke company；ELISpotPLUSFor Porcine IFN-γ Kit is purchased from Mabtech company；Pig peripheral blood lymph
Cell separation liquid Kit is purchased from TBD company；Con-A (concanavalin A, Con A) is purchased from SIGMA company；Hyclone and RPMI 1640
Culture medium is purchased from Gabicol；Penicillin Streptomycin Solution is dual anti-purchased from Hyclone；Hiload10/600 Superdex
200pg prepacked column is purchased from GE (Amersham Biosciences) company；AVIDITY company Biotin-Protein
Ligase-BIRA500Kit；SIGMA company Fluorescein isothiocyanate-R-Phycoerythrin；BD company PE
Mouse Anti-Pig CD8a monoclonal antibody；BD company PerCP Mouse Anti-Pig CD3a monoclonal antibody.
Secondly, carry out experimental animal and prepare.
Select the purebred Hebao pig at a monthly age, boar, 8.(6 are tested group, and 2 compare), extracts pig blood 2mL,
5000rmp is centrifuged 30min and takes supernatant, detects antibody titer.
The neonatal rat of 3 ages in days, connects neonatal rat poison 200 μ L (the neonatal rat poison of 10:1), neonatal rat mean time to death 27h.Process death
Neonatal rat: cut neonatal rat head, removes extremity and internal organs, puts into aseptic quartz sand and PBS liquid to 5mL, grinds, obtain 1:10
Neonatal rat poison.
Continuing to pass on by the neonatal rat poison obtained: 3000rpm is centrifuged 20min and takes supernatant, every neonatal rat meets malicious 200 μ L.So connect
Resuming for 4 generations, to neonatal rat mean time to death at 17h, now toxicity is relatively strong, and selected strain is shown in Table 1.
The strain of foot and mouth disease virus used tested by table 1
Counteracting toxic substances pig is supported at Lanzhou veterinary institute P3 laboratory poison animal house by force, and non-counteracting toxic substances pig is supported in conventional animal room.Take neonatal rat poison,
PBS liquid is diluted to 300:1, root intramuscular injection 1mL after ear.
Table 2 challenge test arrangement
From the beginning of after counteracting toxic substances 15 days, take appropriate corresponding counteracting toxic substances pig fresh anticoagulation every day.
(1) take fresh anticoagulation 2mL, mix with whole blood and tissue homogenate diluent 1:1 equal-volume.
(2) take a 15mL centrifuge tube, add 3mL separation liquid and be placed in 18-22 DEG C.
(3) blood sample is carefully added on the liquid level separating liquid.18-22 DEG C, 2200rmp is centrifuged 20min.Low temperature (such as 4 DEG C)
It is centrifuged and can reduce cell yield.
(4) after centrifugal, with the supernatant of suction pipe careful sucking-off separation liquid upper strata (comprising the cellular layer of lymphocyte) below 0.5cm
Part, discards.
(5) separation liquid layer and buffy coat are carefully drawn with suction pipe as in another new centrifuge tube.
(6) gained centrifuge tube adds 10mL cell washing solution mixing in step 4.
(7) supernatant discarded.
(8) with suction pipe with 5mL cell washing solution resuspended gained cell.
(9) 1600rmp is centrifuged 10min.Abandon supernatant.Repeat step (7), (8), (9), after with the RPMI 1640 of proper volume
Complete medium re-suspended cell.
(10) cell counting, adjusting cell number is 1 × 107, save backup.
Finally start the SLA-2-HB-BSP-As63-s tetrameric preparation of β 2m in the present invention.
1, swine foot-and-mouth disease virus SLA-I restricted CTL epitope design
With NetMHCpan 2.8Version (www.cbs.dtu.dk/services/NetMHCpan/) as template, include lotus by clone
Bag pig SLA-2 is filed in online website form at the interior SLA each functional gene of I quasi-molecule, and SLA I class original with website is divided
The prediction online tool of son each functional gene composition pig source virus CTL epitope.By O type foot and mouth disease virus
Tibet/CHA/99 (HuBHK99), A type foot and mouth disease virus A-HuBWH (1) 2009 and Asia I type foot and mouth disease virus
VP1 and the 3D nonstructural protein sequences of tri-kinds of strains of 1/Jiangsu/China/2005 input prediction template respectively, carries out CTL table
The prediction of position, selects polypeptide candidate's epi-position with high-affinity as far as possible, is shown in Table 3.
Table 3 restricted pig FMDV antigen prediction epi-position
aThe polypeptide of application weight matrix prediction in NetMHCpan 2.8Server (www.cbs.dtu.dk/services/NetMHCpan/)
(strong adhesion polypeptide order threshold value is 0.5 with the adhesion mark of SLA-2-HB；Weak binding power polypeptide order threshold value is 2.0)
bNetMHCpan 2.8 Server applies the polypeptide IC of neural network prediction50(strong combination/SB is less than 50nM for value；Weak
In conjunction with/WB less than 500nM)
2, ELISPOT test:
ELISPOT full name be ELISpot test (Enzyme-linked Immunospot Assay) be that one combines carefully
Born of the same parents' culture technique and elisa technique, it is possible in the means of cell-based assay cytokine secretion profile.And due to active
CTL epitope polypeptide can stimulate specific CD8+T cell secretion of gamma-IFN, utilizes this character, can pass through the method
The FMDV epitope peptide obtaining prediction screens.Separate the pig peripheral blood mononuclear cell infecting foot and mouth disease virus, be used for
ELSPOT specific T-cells epitope peptide screens.Setting up non-counteracting toxic substances PBMC is blank group, and stimulates not add polypeptide
The counteracting toxic substances PBMC of thing is negative control group.
(1) aseptic PBS dilution is coated IFN-γ monoclonal antibody to 10 μ g/mL, pH7.4；
(2) taking out ELISPOT personality board, every hole adds 50 μ L 70% Ethanol Treatment, and the process time does not at most exceed 1min (
As tens seconds, film runs through), discard ethanol solution, every hole adds the 200 aseptic washing of μ L 5 times, and every hole adds 100 μ L
The IFN-γ monoclonal antibody solution diluted, 4-8 DEG C of night incubation；
(3) next day, sucking antibody-solutions, every hole 200 aseptic PBS of μ L washes 2 times；
(4) every hole adds 20 μ L confining liquids, closes 2h, sucks confining liquid for 37 DEG C, and every hole adds 200 μ L complete mediums, room temperature
Hatch at least 30min；
(5) sucking culture medium, every hole adds the polypeptide of 100 μ L beforehand dilutions and PBMC cell suspension, and (cell suspension is diluted to
2.5×105Cells/mL), each sample arranges 3 repetitions；
(6) plate lid, 37 DEG C, 5%CO are built2Under the conditions of hatch cultivation 18～20h (optimal incubation time needs trial test to grope),
During cultivation, can not disturbance cell；
(7) sucking cell suspension, every hole 200 aseptic PBS of μ L washes 5 times, dilutes detection antibody extremely with the PBS containing 0.5%FBS
0.5 μ g/mL, every hole adds 100 μ L, hatches 2h for 37 DEG C, completes to clean according to step (3)；
(8) diluting streptavidin (streptavidin-HRP) with the PBS containing 0.5%FBS, every hole adds 100 μ L.Hatch for 37 DEG C
1h, completes to clean according to step (3)；
(9) every hole adds 100 μ L nitrite ion lucifuges placements, reacts 5～30min depending on spot formation situation, is sure not overresponse and causes relatively
High background develops the color, and terminates substrate chromogenic reaction with the every hole of deionized water wash, and room temperature air-dries 2h or overnight treats that it is complete
White drying, in the medium-term and long-term preservation of sealed plastic bag.ELISPOT speckle Cellular Technology Ltd. (CTL)
CompanyRead plate instrument counting, complete image with ImmunoCapture software the most respectively and obtain
Take, with Immunospot 5.0 software analysis statistics Quality Control.
Each group is reacted according to ELISPOT standard test procedure, and result as shown in Figure 3 and Figure 4, utilizes CTL companyRead plate instrument, the effect speckle of each group of peptide read, representative peptide stimulation CTL after secernent one
Individual IFN-γ molecule, speckle is the most, and the ability of representative peptide stimulation CTL secretion of gamma-IFN is the strongest, stimulates the peptide that ability is the strongest selected
Make Dominant Epitopes peptide.From the figure 3, it may be seen that the ability that Hu62 polypeptide stimulates CTL is significantly larger than other polypeptide, secondly be INT3,
Ahu63 and Q10.Fig. 4 C-J is each polypeptide representational ELISPOT testing result, and A-B represents blank and the moon respectively
Property comparison.As seen from the figure, compared with matched group, each polypeptide all has certain stimulation ability, but prepared by the tetramer to be used for,
Must select the polypeptide of advantage, therefore Hu62 can carry out, as next step, the advantage polypeptide epitope that tetramer preparation selects.
3, heavy chain SLA-2-BSP and light chain s β 2m tetramerization
1), yeast culture
A: activation heavy chain recombinant bacterium SLA-2-HB-BSP/BL21 and light chain recombinant bacterium β 2m/BL21, inoculation 5mL contains respectively
The culture medium of Amp (100 μ g/mL), cultivates to OD for 37 DEG C600When reaching 0.4-0.6, add IPTG to final concentration of 1mmol/L,
Take thalline SDS-PAGE and detect protein expression situation.
Recombinant bacterium SLA-2-HB-BSP/pET-21a (+)-BL21 is through inducing, 12%SDS-PAGE detects, and is finding that it is just being expressed
Often, position is about at 34kD.Light chain β 2m/pET-21a (+) detect through induction and SDS-PAGE, it was demonstrated that this protein expression is just
Often, protein positions is about at about 12kD.Heavy chain and light chain are suitable for carrying out next step the tetramer to be prepared.
By correct for the SDS-PAGE detection heavy chain SLA-2-HB-BSP/pET-21a recombinant bacterium expressed and light chain
S 37 DEG C of incubated overnight of β 2m/pET-21a recombinant bacterium.
B: respectively with in the dilution overnight culture of 1:100 to 1L LB, cultivate OD for 37 DEG C600=1, add IPTG extremely
Final concentration, to 0.5mmol/L, continues to cultivate 4 hours, results antibacterial (each 1 liter of heavy chain light chain).
2), prepared by inclusion body
A: antibacterial 30mL PBS suspends, 650W carrying out ultrasonic bacteria breaking (5sec on/10sec off, 10min).
B: inclusion body 25mmol/L Tris-HCl, pH8.0,100mmol/L NaCl, 1%Triton-X100 cleaning mixture washes 2 times.
C: inclusion body 25mmol/L Tris-HCl, pH8.0,100mmol/LM NaCl cleaning mixture washes 1 time.
D: the inclusion body 25mmol/L Tris-HCl containing 8mol/L carbamide, pH8.0,100mmol/L NaCl dissolve, to albumen
Matter concentration is 10mg/mL.
3), renaturation
Under the conditions of 4 DEG C, toward the 100mmol/L Tris-HCl of 500mL, pH8.0,400mmol/L Arginine, 5mmol/L
Reduced Glutathione, 0.5mmol/L oxidised Glutathione adds 15mg heavy chain, 10mg light chain and 5mg
Epitope peptide, stirs 48 hours.
4), the biotinylation of MHC monomer
The renaturation solution of 500mL, is concentrated to 5mL, carries out buffer-exchanged with PD10 post, and new buffer is 10mmol/L Tris
pH8.0.According to the explanation of BirA test kit (Avidity), composition in test kit is joined in above-mentioned protein solution, room
Temperature processes 16 hours.
5), the separation of biotinylated monomers
Above-mentioned protein solution overnight is carried out AKTA FPLC separation: detached dowel Hiload 26/600Supdex 75pg, point
It is 20mmol/L Tris-HCl pH8,100mmol/L NaCl from buffer；Flow velocity 1mL/min.Collection elution volume is 163ml
Protein peak, be concentrated to 1mg/mL.
6), the qualification of monomer
A:SDS-PAGE, the monomer that above-mentioned FPLC is separated, point sample 8 μ g, if seeing heavy chain and light chain two band, show
After renaturation, monomer has been formed.
B:ELISA detects, and 4 DEG C are overnight coated the monomer that FPLC separates, every hole 2 μ g, holes, are simultaneously introduced the most biotinylated
Monomer, as comparison, carries out detecting the biotinylation situation of monomer with the strepavidin (Biolegend) of HRP labelling, as
Shown in Fig. 5, OD595Reading: biotinylated monomers hole 1=0.92, biotinylated monomers hole 2=0.86, non-biotinylated monomers
Hole 1=0.12；Non-biotinylated monomers hole 2=0.09.Biotinylation and non-biotinylation sample form significantly contrast, show raw
Thing element chemical conversion merit.
According to every 1mg biotinylated monomers add 0.312mg extravidine-PE amount mixing, formed PE labelling good four gather
Body, as shown in Figure 6.
4, SLA-2/HB/Hu62Tetramer flow cytometer detection
(8) separating the peripheral blood lymphocytes of Tibet/CHA/99 (HuBHK99) strain counteracting toxic substances group pig, 1500rmp room temperature is centrifuged 3
Min, abandons supernatant, with PBS (pH 7.2) washed cell once, and recentrifuge.
(9) cell precipitation is resuspended in FACS cleaning mixture (containing 0.1%NaN31 × PBS with 0.1%BSA).
(10) in cell suspension, add the SLA-2-HB-BSP/Hu62 tetramer of 5 μ L PE labellings, room temperature lucifuge reaction 20min.
(11) adding 2mLFACS cleaning mixture re-suspended cell, 500 × g room temperature is centrifuged 3min, abandons supernatant, and cell precipitation is resuspended in 100 μ L
FACS cleaning mixture.
(12) add the Anti-pig CD8a antibody of 5 μ L (2.5 μ g) FITC labelling, continue lucifuge reaction 20min in room temperature.
(13) adding FACS cleaning mixture washed cell once, 1500rmp room temperature is centrifuged 3min, abandons supernatant, and cell precipitation is resuspended in
In right amount in (300～400 μ L) FACS cleaning mixture.
(14) cell suspension sample through flow cytometry and sorts.
Hu62 belongs to O type foot and mouth disease strain, takes the peripheral blood lymphocytes (PBMC) of the pig attacking O type poison, uses FITC
PBMC is dyeed by the Anti-Pig CD8a monoclonal antibody of labelling.Re-use PE labelling
The SLA-2-HB-BSP-Hu62-s β 2m tetramer carries out double dyeing to PBMC, is i.e. directed to the characteristic type of Hu62 polypeptide epitope
CTL, account for the 5.7% of total cell, see Fig. 7, form extremely significantly difference with matched group 0.1% (non-counteracting toxic substances group cell)
(P < 0.01), it was demonstrated that the SLA-2-HB-Hu62-s β 2m tetramer is successfully prepared, proves that Hu62 is that foot and mouth disease virus is special simultaneously
The CTL epi-position of property.Representational tetramer flow cytometer detection is shown in Fig. 8.
This research, has successfully filtered out an O type foot-and-mouth disease virus polypeptide Hu62 by immunity and ELISPOT, and many with this
Hebao pig SLA-2-HB-BSP and s β 2m albumen that peptide builds with early stage construct the tetramer, utilize flow cytometry success
The tetramer containing polypeptide Hu62 of preparation detected.Research final certification polypeptide Hu62 has immunologic competence, is potential
O type foot and mouth disease virus CTL epi-position.
Claims (1)
1. an O type foot-and-mouth disease virus polypeptide prepares tetrameric method, it is characterised in that step is:
1), swine foot-and-mouth disease virus SLAI restricted CTL epitope design
With NetMHCpan 2.8Version as template, by each merit of SLAI quasi-molecule including Hebao pig SLA-2 of clone
Online website form can be filed in by gene, SLAI quasi-molecule each functional gene original with website composition pig source virus CTL epitope
Prediction online tool, by O type foot and mouth disease virus Tibet/CHA/99 (HuBHK99), A type foot and mouth disease virus
A-HuBWH (1) 2009 and the non-knot of VP1 and 3D of Asia I type tri-kinds of strains of foot and mouth disease virus 1/Jiangsu/China/2005
Structure protein sequence input prediction template respectively, carries out the prediction of CTL epi-position, selects polypeptide candidate's epi-position with high-affinity,
2), ELISPOT test:
(1) aseptic PBS dilution is coated IFN-γ monoclonal antibody to 10 μ g/mL, pH7.4；
(2) taking out ELISPOT personality board, every hole adds 50 μ L 70% Ethanol Treatment, and the process time does not at most exceed 1min film
Running through, discard ethanol solution, every hole adds the 200 aseptic washing of μ L 5 times, and every hole adds the IFN-γ that 100 μ L have diluted
Monoclonal antibody solution, 4-8 DEG C of night incubation；
(3) next day, sucking antibody-solutions, every hole 200 aseptic PBS of μ L washes 2 times；
(4) every hole adds 20 μ L confining liquids, closes 2h, sucks confining liquid for 37 DEG C, and every hole adds 200 μ L complete mediums, room temperature
Hatch at least 30min；
(5) sucking culture medium, every hole adds the polypeptide of 100 μ L beforehand dilutions and is diluted to 2.5 × 105The PBMC cell of cells/mL
Suspension, each sample arranges 3 repetitions；
(6) plate lid, 37 DEG C, 5%CO are built2Under the conditions of hatch cultivation 18～20h, during cultivation, can not disturbance cell；
(7) sucking cell suspension, every hole 200 aseptic PBS of μ L washes 5 times, dilutes detection antibody extremely with the PBS containing 0.5%FBS
0.5 μ g/mL, every hole adds 100 μ L, hatches 2h for 37 DEG C, completes to clean according to step (3)；
(8) diluting streptavidin with the PBS containing 0.5%FBS, every hole adds 100 μ L, hatches 1h for 37 DEG C, according to step (3)
Complete to clean；
(9) every hole adds 100 μ L nitrite ion lucifuges placements, reacts 5～30min depending on spot formation situation, with the every hole of deionized water wash
Terminating substrate chromogenic reaction, room temperature air-dries 2h or overnight treats that it is completely dried, in the medium-term and long-term preservation of sealed plastic bag,
To ELISPOT spot count, complete Image Acquisition, be analyzed statistics Quality Control；
3), heavy chain SLA-2-BSP and light chain s β 2m tetramerization
(1), yeast culture
A: heavy chain SLA-2-HB-BSP and light chain β 2m detection of expression
Activation heavy chain recombinant bacterium SLA-2-HB-BSP/BL21 and light chain recombinant bacterium β 2m/BL21, inoculates 5mL respectively, contains
The culture medium of 100 μ g/mL Amp, cultivates to OD for 37 DEG C600When reaching 0.4-0.6, add IPTG to final concentration of 1mmol/L,
Take thalline SDS-PAGE and detect protein expression situation,
Recombinant bacterium SLA-2-HB-BSP/pET-21a (+)-BL21 is through inducing, 12%SDS-PAGE detects, and is finding that it is just being expressed
Often, light chain β 2m/pET-21a (+) detect through induction and SDS-PAGE, it was demonstrated that this protein expression is normal, and heavy chain and light chain are equal
Prepared by the tetramer being appropriate to next step,
By correct for the SDS-PAGE detection heavy chain SLA-2-HB-BSP/pET-21a recombinant bacterium expressed and light chain
S 37 DEG C of incubated overnight of β 2m/pET-21a recombinant bacterium,
B: respectively with in the dilution overnight culture of 1:100 to 1L LB, cultivate OD for 37 DEG C600=1, add IPTG extremely
Final concentration, to 0.5mmol/L, continues to cultivate 4 hours, gathers in the crops antibacterial, each 1 liter of heavy chain SLA-2-HB-BSP and light chain β 2m；
(2), prepared by inclusion body
A: antibacterial 30mL PBS suspends, 650W carrying out ultrasonic bacteria breaking, 5sec on/10sec off, 10min,
B: inclusion body 25mmol/L Tris-HCl, pH8.0,100mmol/L NaCl, 1%Triton-X100 cleaning mixture washes 2 times,
C: inclusion body 25mmol/L Tris-HCl, pH8.0,100mmol/L NaCl cleaning mixture washes 1 time,
D: the inclusion body 25mmol/L Tris-HCl containing 8mol/L carbamide, pH8.0,100mmol/L NaCl dissolve, to albumen
Matter concentration is 10mg/mL；
(3), renaturation
Under the conditions of 4 DEG C, toward the 100mmol/L Tris-HCl of 500mL, pH8.0,400mmol/L Arginine, 5mmol/L
Reduced Glutathione, 0.5mmol/L oxidised Glutathione adds 15mg heavy chain SLA-2-HB-BSP, 10mg
Light chain β 2m and 5mg Hu62 epitope peptide, stir 48 hours, obtain renaturation solution；
(4), the biotinylation of MHC monomer
The renaturation solution of 500mL, is concentrated to 5mL, carries out buffer-exchanged with PD10 post, and new buffer is 10mmol/L Tris
PH8.0, joins composition in test kit in above-mentioned protein solution, room temperature treatment 16 hours；
(5), the separation of biotinylated monomers
Above-mentioned protein solution overnight is carried out AKTA FPLC separation: detached dowel Hiload 26/600Supdex 75pg, point
It is 20mmol/L Tris-HCl pH8,100mmol/L NaCl from buffer；Flow velocity 1mL/min, collection elution volume is 163ml
Protein peak, be concentrated to 1mg/mL；
(6), the qualification of monomer
A:SDS-PAGE, the monomer separated by above-mentioned FPLC, point sample 8 μ g, if seeing heavy chain SLA-2-HB-BSP and light
Chain β 2m two band, after showing renaturation, monomer has been formed,
B:ELISA detects, and 4 DEG C are overnight coated the monomer that FPLC separates, every hole 2 μ g, holes, are simultaneously introduced the most biotinylated
Monomer, as comparison, carries out detecting the biotinylation situation of monomer with the strepavidin of HRP labelling,
According to every 1mg biotinylated monomers add 0.312mg extravidine-PE amount mixing, formed PE labelling good four gather
Body；
4), SLA-2/HB/Hu62Tetramer flow cytometer detection
(1) separating the peripheral blood lymphocytes of Tibet/CHA/99 (HuBHK99) strain counteracting toxic substances group pig, 1500rmp room temperature is centrifuged 3
Min, abandons supernatant, with the PBS washed cell of pH 7.2 once, and recentrifuge,
(2) cell precipitation is resuspended in FACS cleaning mixture
(3) adding the SLA-2-HB-BSP/Hu62 tetramer of 5 μ L PE labellings in cell suspension, room temperature lucifuge reacts 20min,
(4) adding 2mLFACS cleaning mixture re-suspended cell, 500 × g room temperature is centrifuged 3min, abandons supernatant, and cell precipitation is resuspended in 100 μ L
FACS cleaning mixture,
(5) add 5 μ L, the Anti-pig CD8a antibody of 2.5 μ gFITC labellings, continue lucifuge reaction 20min in room temperature,
(6) adding FACS cleaning mixture washed cell once, 1500rmp room temperature is centrifuged 3min, abandons supernatant, and cell precipitation is resuspended in
In 300～400 μ LFACS cleaning mixture,
FACS cleaning mixture therein contains 0.1%NaN3With the 1 × PBS of 0.1%BSA,
(7) cell suspension sample through flow cytometry and sorts,
It is directed to the CTL of the characteristic type of Hu62 polypeptide epitope, accounts for the 5.7% of total cell, formed the most notable with matched group 0.1%
Difference P < 0.01, it was demonstrated that the SLA-2-HB-Hu62-s β 2m tetramer is successfully prepared, simultaneously prove Hu62 be foot and mouth disease virus
Specific CTL epi-position.
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| CN109633145A (en) * | 2018-12-04 | 2019-04-16 | 常州市第二人民医院 | Use the research method of -1 aided assessment early stage rheumatoid arthritis of follistatin sample albumen in serum |
| CN111100199A (en) * | 2018-12-29 | 2020-05-05 | 北京百普赛斯生物科技有限公司 | Fluorescein labeled protein tetramer and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103242427A (en) * | 2012-02-10 | 2013-08-14 | 中国农业大学 | CTL epitope polypeptide of porcine reproductive and respiratory syndrome virus (PRRSV) and application thereof |
| CN103864905A (en) * | 2013-12-30 | 2014-06-18 | 大连大学 | CTL (Cytotoxic T Lymphocyte) epitope peptide of foot-and-mouth disease virus type O and screening method of CTL epitope peptide |
-
2016
- 2016-01-25 CN CN201610049172.7A patent/CN105884870B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103242427A (en) * | 2012-02-10 | 2013-08-14 | 中国农业大学 | CTL epitope polypeptide of porcine reproductive and respiratory syndrome virus (PRRSV) and application thereof |
| CN103864905A (en) * | 2013-12-30 | 2014-06-18 | 大连大学 | CTL (Cytotoxic T Lymphocyte) epitope peptide of foot-and-mouth disease virus type O and screening method of CTL epitope peptide |
Non-Patent Citations (2)
| Title |
|---|
| NIANZHI ZHANG等: "Crystal Structure of Swine Major Histocompatibility Complex Class I SLA-1*0401 and Identification of 2009 Pandemic Swine-Origin Influenza A H1N1 Virus Cytotoxic T Lymphocyte Epitope Peptides", 《JOURNAL OF VIROLOGY》 * |
| 张香春等: "荷包猪SLA-2 重链基因偶合底物肽及表达研究", 《生物技术》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109633145A (en) * | 2018-12-04 | 2019-04-16 | 常州市第二人民医院 | Use the research method of -1 aided assessment early stage rheumatoid arthritis of follistatin sample albumen in serum |
| CN111100199A (en) * | 2018-12-29 | 2020-05-05 | 北京百普赛斯生物科技有限公司 | Fluorescein labeled protein tetramer and preparation method and application thereof |
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