CN105884870B - 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 the designed foot-and-mouth disease virus polypeptide, the invention screens and identifies the foot-and-mouth disease virus polypeptide by adopting an ELISPOT method and a tetramer construction method, constructs the heavy chain tetramer of the pouch pig SLA-2, expresses the tetramer in a prokaryotic expression system pET-21a (+)/BL21 and extracts an inclusion body; foot-and-mouth disease virus polypeptide, screening polypeptide with specific CTL reaction by ELISPOT method, then renaturing and biotinylating the polypeptide with SLA-2 heavy chain and existing light chain beta 2m, reacting with FITC marked streptavidin to generate SLA-2-BSP/peptide tetramer, and then detecting the function of the tetramer by flow cytometry. According to the invention, a specific polypeptide epitope of the porcine O-type foot-and-mouth disease virus VP1 is identified by an ELISPOT method, a peptide tetramer is successfully constructed, a technical platform of the porcine tetramer is initially established, and a foundation is laid for researching the porcine specific CTL immune response and T epitope screening.
Description
Technical Field
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.
Background
Foot-and-Mouth Disease is an acute hot highly-contact infectious Disease caused by Foot and Foot Disease Virus (FMDV), mainly attacks artiodactyl animals, pigs are relatively susceptible groups, clinically, the Foot-and-Mouth Disease is mainly characterized by blister and ulceration of oral mucosa, hoof and breast skin, is the most highly infectious Disease in livestock epidemic diseases, and each outbreak of Foot-and-Mouth Disease brings great loss to agricultural economy. Therefore, the international animal epidemic Organization (OIE) always lists the animal epidemic diseases as the first list of the animal epidemic diseases of class A which must be reported for the disease. Therefore, the prevention and diagnosis of the foot-and-mouth disease of the pig are very important. Currently, the prevention and treatment of foot-and-mouth disease virus mainly depends on the traditional inactivated vaccine. The inactivated vaccine has the advantages of the inactivated vaccine, good immune effect, low cost and the like. However, there are some potential disadvantages that inactivated vaccines still have the risk of spreading virulence due to incomplete inactivation or gene recombination with external wild strains. In addition, for foot-and-mouth disease virus, its epidemic strains are constantly changing, that is, the development of vaccine strains always lags behind the emergence of the variant strains. In response to the above problems, a new foot-and-mouth disease vaccine needs to be searched. At present, the polypeptide vaccine is a research hotspot, because the vaccine is formed by combining different epitopes of viruses, the vaccine not only can cause immune response, but also overcomes the problem of virus dispersion of the vaccine. Therefore, how to isolate and identify the foot-and-mouth disease virus epitope is a key problem. Currently, the research on foot-and-mouth disease virus epitopes mostly focuses on B cell epitopes and T helper (Th) epitopes, which are mainly related to the induction of humoral immune response; cytotoxic T Lymphocyte (CTL) epitopes that elicit a cellular immune response are currently lacking. Foot and mouth disease virus is a strict intracellular parasite that must elicit a cellular immune response upon infection of host cells. Therefore, how to screen and identify foot-and-mouth disease virus CTL epitopes in vitro is a very valuable topic for research. At present, there are various methods for in vitro screening and identification of viral CTL epitopes, including cell killing experiments, ELISPOT and tetramer methods, etc. Among them, the combination of ELISPOT and tetramer is a common method for screening and identifying CTL epitopes of viruses at present.
The ELISPOT technique, also known as enzyme-linked immunospot technique, was first reported in 1983 by Czerkinsky et al (J Clin Microbiol,1983,17(6):965-969), which is an extension of the enzyme-linked immunosorbent assay (ELISA) technique, and is currently used to determine the function of cytokines or CTLs, and is used to determine the production of heat-sensitive toxins in E.coli. Herr et al (J Immunol Methods,1996,191(2):131-142) began in 1996 by applying this technique to the determination of cell killing by human HLA-A2-bound HIV epitope polypeptides based on the ability of HIV epitope peptides to stimulate CD8+T Lymphocytes (CTL) release interferon-gamma, so that CTL activity of the polypeptide can be indirectly measured. According to the research report (Wangyirei et al, Chinese AIDS, 2005,11(1):70-72), ELISPOT can detect single secretory cell from 100 ten thousand cells and can accelerate the secretion of cell factor<The detection is carried out on cells with 100 molecules per second, and the detection has higher reliability, repeatability and sensitivity.
MHC I/peptide tetramer technology was established by Altman et al (Science,1996,274(2584):94-96) in 1996 for the quantitative detection of specific T cells. The technology enables the detection of the activity of the antigen-specific CTL to reach the specific, high-efficiency and direct quantitative degree, and the sensitivity of the detection is 3-5 times of the ELISPOT detection result. The rationale behind this technique is that the affinity of binding of a single MHC-peptide complex to a TCR is low and the rate of dissociation is fast. Altman, however, found that 4 molecules of pMHC bound TCR with strong affinity and biological effect in the study. According to the specific binding relationship between Biotin and avidin 4:1, Biotin-protein ligase (Biotin-protein ligand A, BirA) is utilized to identify the specific sequence of protein (Liqing, etc., journal of cellular immunology 2005, 2005,21(5): 557) 560), 1 BirA substrate peptide sequence of Biotin is added to the carboxyl end of the heavy chain of MHC molecule, and BirA enzyme can bind Biotin to pMHC, so that 4 biotinylated pMHC monomers and 1 avidin-labeled streptavidin form a tetramer complex, further the overall affinity of pMHC and TCR is improved, and antigen-specific T lymphocytes can be rapidly detected and separated by a flow cytometer (FACS) with higher sensitivity.
The Major Histocompatibility Complex (MHC) of pigs is also known as leukocyte antigen (SLA) of pigs, whose class I molecules mediate cellular immune responses. The heavy chain and light chain of SLA I molecules and virus polypeptide can form a complex in cells, and the complex is presented to the cell surface by CD8+T Cell Receptor (TCR) recognition, which causes a killer T Cell (CTL) immune response, is beneficial to the body to eliminate viruses and resist diseases. These functional viral polypeptides are potential vaccine epitopes candidates. In vitro, how to identify and screen functional viral epitopes is a very critical technology.
At present, the Tetramer technology is not applied to the identification of the functions of foot-and-mouth disease virus polypeptides combined with pig SLA I molecules in China. The invention aims to combine ELISPOT technology and Tetramer technology to identify and screen the functions of foot-and-mouth disease virus polypeptide on the basis of researching the SLA-2 binding polypeptide of the pig in the early stage, and lays a foundation for developing a novel polypeptide vaccine containing a CTL epitope of the foot-and-mouth disease virus in the future.
Disclosure of Invention
Aiming at the designed foot-and-mouth disease virus polypeptide, the invention screens and identifies the foot-and-mouth disease virus polypeptide by adopting an ELISPOT method and a tetramer construction method, constructs the heavy chain tetramer of the pouch pig SLA-2, expresses the tetramer in a prokaryotic expression system pET-21a (+)/BL21 and extracts an inclusion body; foot-and-mouth disease virus polypeptide, screening polypeptide with specific CTL reaction by ELISPOT method, then renaturing and biotinylating the polypeptide with SLA-2 heavy chain and existing light chain beta 2m, reacting with FITC marked streptavidin to generate SLA-2-BSP/peptide tetramer, and then detecting the function of the tetramer by flow cytometry.
The technical characteristics of the invention are as follows:
1. porcine foot-and-mouth disease virus SLA I restrictive CTL epitope design
The method comprises the steps of taking NetMHCpan 2.8Version as a template, submitting cloned SLAI molecules including the pig SLA-2 to an online website template (www.cbs.dtu.dk/services/NetMHCpan /), and forming a prediction online tool of a porcine virus CTL epitope together with the original SLA I molecules of the website. VP1 and 3D non-structural protein sequences of three strains, namely 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 1/Jiangsu/China/2005, are respectively input into a prediction template to predict CTL epitopes, and polypeptide candidate epitopes with high affinity are selected.
2. ELISPOT test:
(1) diluting the coated IFN-gamma monoclonal antibody to 10 mug/mL by sterile PBS, and carrying out pH7.4;
(2) taking out the ELISPOT special plate, adding 50 μ L70% ethanol into each hole, treating for no more than 1min (generally tens of seconds, and the membrane is thoroughly moistened), discarding ethanol solution, adding 200 μ L sterile water into each hole, washing for 5 times, adding 100 μ L diluted IFN- γ monoclonal antibody solution into each hole, and incubating overnight at 4-8 deg.C;
(3) the next day, the antibody solution was aspirated and washed 2 times with 200 μ L sterile PBS per well;
(4) adding 20 μ L of blocking solution into each well, blocking at 37 deg.C for 2 hr, removing blocking solution by suction, adding 200 μ L of complete culture medium into each well, and incubating at room temperature for at least 30 min;
(5) the medium was aspirated off and 100. mu.L of prediluted polypeptide and PBMC cell suspension (diluted to 2.5X 10 cell suspension) was added to each well5cells/mL), 3 replicates per sample were set up;
(6) cover the plate cover, 37 deg.C, 5% CO2Incubating and culturing for 18-20 h under the condition (the optimal culture time needs to be tested and found), wherein cells cannot be disturbed during the culture period;
(7) aspirating the cell suspension, washing 5 times with 200 μ L sterile PBS per well, diluting the detection antibody to 0.5 μ g/mL with PBS containing 0.5% FBS, adding 100 μ L per well, incubating at 37 ℃ for 2h, and completing washing according to step (3);
(8) streptavidin (streptavidin-HRP) was diluted in PBS containing 0.5% FBS, 100. mu.L per well. Incubating at 37 ℃ for 1h, and finishing washing according to the step (3);
(9) adding 100 mu L of color development liquid into each hole, placing in the dark, reacting for 5-30 min according to the spot formation condition, washing each hole with deionized water to stop the substrate color development reaction, air-drying for 2h at room temperature or staying overnight for complete drying, and storing in a sealed plastic bag for a long time. Cellular Technology Ltd (CTL) for ELISPOT spots
Counting by an Analyzer plate reader, wherein image acquisition is respectively completed by using ImmunoCapture software, and analyzing, counting and quality control are performed by using ImmunoSpot 5.0 software.
3. Tetramerization of heavy chain SLA-2-BSP with light chain s beta 2m
1) And culturing of the cells
A: heavy chain SLA-2-HB-BSP and light chain beta 2m expression assay
Activating heavy chain recombinant bacteria SLA-2-HB-BSP/BL21 and light chain recombinant bacteria beta 2m/BL21, respectively inoculating 5mL of culture medium containing Amp (100 mu g/mL), culturing at 37 ℃ to OD600When reaching 0.4-0.6, adding IPTG to the final concentration of 1mmol/L, and taking thalli SDS-PAGE to detect the protein expression.
The recombinant bacterium SLA-2-HB-BSP/pET-21a (+) -BL21 is detected by 12% SDS-PAGE and is normally expressed and is about 34 kD. The light chain beta 2m/pET-21a (+) is detected by induction and SDS-PAGE, and the protein is confirmed to be normally expressed, and the protein position is about 12 kD. Both heavy and light chains are suitable for the next tetramer preparation.
The correctly expressed heavy chain SLA-2-HB-BSP/pET-21a recombinant bacteria and the light chain s beta 2m/pET-21a recombinant bacteria are cultured at 37 ℃ overnight by SDS-PAGE detection.
B: the overnight cultures were diluted to 1L LB at 1:100 dilutions, respectively, and cultured to OD at 37 deg.C600IPTG was added to a final concentration of 0.5mmol/L, and the culture was continued for 4 hours to harvest bacteria (1 liter each of heavy and light chains).
2) Preparation of inclusion body
A: the bacteria were suspended in 30mL PBS and disrupted by 650W sonication (5sec on/10sec off, 10min).
B: inclusion bodies were washed 2 times with 25mmol/L Tris-HCl, pH8.0, 100mmol/L NaCl, 1% Triton-X100 wash.
C: inclusion bodies were washed 1 time with 25mmol/L Tris-HCl, pH8.0, 100mmol/L NaCl wash.
D: inclusion bodies were solubilized with 25mmol/L Tris-HCl, pH8.0, 100mmol/L NaCl containing 8mol/L urea to a protein concentration of 10 mg/mL.
3) And renaturation thereof
To 500mL of 100mmol/L Tris-HCl, pH8.0, 400mmol/L Arginine, 5mmol/L reduced glutamthione, 0.5mmol/L oxidized glutamthione was added 15mg of heavy chain, 10mg of light chain and 5mg of Hu62 epitope peptide at 4 ℃ and stirred for 48 hours.
4) Biotinylation of MHC monomers
500mL of the renaturation solution was concentrated to 5mL and buffer exchange was carried out with a PD10 column, the new buffer being 10mmol/L Tris pH8.0. The components of the kit were added to the protein solution described above according to the instructions of the BirA kit (identification) and treated at room temperature for 16 hours.
5) Isolation of biotinylated monomers
The overnight protein solution was subjected to AKTA FPLC separation: separating column Hiload 26/600Supdex 75pg, separation buffer 20mmol/L Tris-HCl pH8, 100mmol/L NaCl; the flow rate was 1 mL/min. The protein peak was collected at an elution volume of 163mL and concentrated to 1 mg/mL.
6) Identification of monomers
A: SDS-PAGE, the FPLC separated monomer, sample 8 u g, if seen heavy chain and light chain two bands, indicate the renaturation of monomer has been formed.
B: ELISA detection, FPLC separation of monomers, 4 degrees overnight coating, each hole 2 u g, two holes, at the same time adding non biotinylated monomer as control, HRP labeled streptavidin (biolegend) for detecting monomer biotinylation.
The PE-labeled tetramer was formed by mixing 0.312mg extravidine-PE per 1mg biotinylated monomer.
4. SLA-2/HB/Hu62Tetramer flow assay
(1) Peripheral blood mononuclear cells from the Tibet/CHA/99(HuBHK99) strain challenge group of pigs were isolated, centrifuged at 1500rmp for 3min at room temperature, the supernatant discarded, the cells washed once with PBS (pH 7.2), and centrifuged again.
(2) Cell pellets were resuspended in FACS washes (containing 0.1% NaN)3And 0.1% BSA in 1 × PBS).
(3) mu.L of PE-labeled SLA-2-HB-BSP/Hu62 tetramer was added to the cell suspension, and the mixture was left to react for 20min at room temperature with light.
(4) Adding 2mLFACS washing solution to resuspend the cells, centrifuging at 500 Xg room temperature for 3min, discarding the supernatant, and resuspending the cell pellet in 100 mu LFACS washing solution.
(5) mu.L (2.5. mu.g) FITC-labeled Anti-pig CD8a antibody was added and the reaction was continued at room temperature with exclusion of light for 20 min.
(6) The cells were washed once by adding FACS wash, centrifuged at 1500 rpm for 3min at room temperature, the supernatant was discarded, and the cell pellet was resuspended in an appropriate amount (300-400. mu.L) of FACS wash.
(7) Cell suspension samples were analyzed by flow cytometry and sorted.
Hu62 belongs to type O foot-and-mouth disease strain, Peripheral Blood Mononuclear Cells (PBMC) of pigs infected with type O virus are taken, and the PBMC are stained by using FITC-labeled Anti-Pig CD8a monoclonal antibody. PBMC are double-stained by using a PE-labeled SLA-2-HB-BSP-Hu62-s beta 2m tetramer, namely CTL aiming at a characteristic type of the Hu62 polypeptide epitope accounts for about 5.7 percent of total cells, and forms a very significant difference (P <0.01) with 0.1 percent of a control group (cells of an unvoiced group), thereby proving that the SLA-2-HB-Hu62-s beta 2m tetramer is successfully prepared, and simultaneously proving that Hu62 is a CTL epitope specific to the foot-and-mouth disease virus.
The result shows that the ELISPOT successfully screens the O-type foot-and-mouth disease virus polypeptide Hu62, and has obvious CTL function. The SLA-2-/Hu62 tetramer prepared from the Hu62 polypeptide was able to react with specific CTL cells. The invention identifies and obtains a specific polypeptide epitope of the porcine foot-and-mouth disease virus VP1 by an ELISPOT method, successfully constructs SLA-2/peptide tetramer of the polypeptide epitope, detects specific CTL aiming at the epitope, preliminarily establishes a porcine tetramer technical platform, and lays a foundation for researching porcine specific CTL immune response and T cell epitope screening.
Drawings
FIG. 1 shows the expression detection of recombinant SLA-2-HB-BSP/pET-21a (+) protein, wherein M represents low molecular weight protein Marker, 1: uninduced group, 2: SLA-2-HB-BSP/pET-21a (+) protein induced for expression;
FIG. 2 is a diagram showing the detection of recombinant β 2M/pET-21a (+) protein expression, wherein M represents a low molecular weight protein Marker, 1, the expression of non-induced β 2M/pET-21a (+) recombinant bacteria, 2, the expression of induced β 2M/pET-21a (+) recombinant bacteria, and the size of the expressed protein is about 12 kD;
FIG. 3 is ELISPOT detecting the ability of each foot-and-mouth disease polypeptide to stimulate CTL to secrete IFN-gamma, # # represents a dominant polypeptide epitope;
FIG. 4 is a graph of representative ELISPOT results generated by stimulation of CTLs by each foot-and-mouth disease polypeptide, wherein: a, blank control group, no challenge; b, negative control group, after toxin counteracting, no polypeptide stimulation is added; c, Hu62 stimulation group; d, WX96 stimulated group; e, 3D314 stimulation group; f, AHu63 stimulation group; g, WH94 stimulation group; h, INT3 stimulation group; i, Q10 stimulation group; j, As3 stimulation groups, the number in the lower right hand corner of each group representing the number of plaques;
FIG. 5 is a graph of isolated biotinylated SLA-2-HB-BSP-Hu62- β 2m monomer FPLC, in order, from left to right, multimer (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 shows the preparation of SDS-PAGE quality control monomers, M, molecular weight standards; 1, SLA-2-BSP heavy chain; 2, β 2m light chain; 3, concentrated biotinylated and FPLC isolated monomers;
FIG. 7 is a Tetramer analysis of flow cytometry to detect the formation of SLA-2-BSP-Hu62- β 2 m;
FIG. 8A representative profile of tetramer detection by flow cytometry, wherein: a, taking pig PBMC of an uninmmunized group to detect tetramer; b, flow-through assay of tetramers by Immunity porcine PBMC, Q1 representing FITC Single-labeled CD8+The proportion of T cells; q2, representing FITC and PE labeled double positive cells, i.e., CD8 capable of recognizing tetramers+A T cell; q3, double negative cell; q4, PE singly labeled cells, i.e. other lymphocytes capable of recognizing tetramers.
Detailed Description
The present invention will now be further illustrated with reference to examples.
First, the reagents involved in the present invention are explained;
IPTG, DNA Marker and protein molecular weight Marker are all products of TaKaRa company; 4-chloro-1-naphthol, Tris (Tris-base), glycine, N, -dimethylallyl bisacrylamide and propionamide, PBS solution, NH4Cl, DMSO, Tris-base, L-arginine, EDTA, reduced Glutathione (GSH), oxidized glutathione (GSSG), PMSF, ammonium persulfate, TEMED, DTT, Coomassie brilliant blue R250 were purchased from Biotechnology engineering, Inc.; the BCA protein concentration determination kit, the western blot developing solution and the fixing solution are purchased from Biyuntian corporation; protein concentration ultrafiltration tubes were purchased from MilliPore corporation; Anti-His Tag Mouse Monoclonal Antibody and HRPConjugated Goat Anti-Mouse IgG from Emericco; ELISpotPLUSfor Porcine IFN-. gamma.kit was purchased from Mabtech; the porcine peripheral blood lymphocyte separation medium Kit is purchased from TBD company; Con-A (Canavanin A) was purchased from SIGMA; fetal bovine serum and RPMI 1640 medium were purchased from Gabicol; penicilin Streptomycin Solution dual antibody was purchased from Hyclone; hiload10/600 Superdex 200pg prepacked column from GE (Amersham biosciences); avidly company Biotin-Protein ligand-BIRA 500 Kit; fluorescein isothiocyanate-R-phytoerythrin, Sigma; PE Mouse An of BD companyti-Pig CD8a monoclonal antibody; the BD company PerCP Mouse Anti-Pig CD3a monoclonal antibody.
Second, the test animals are prepared.
Selecting one month-old pure breed of the pig, the boar and 8 pigs. (6 heads are used as test groups, 2 heads are used as controls), 2mL of pig blood is extracted, the supernatant is obtained by centrifugation at 5000rmp for 30min, and the antibody titer is detected.
The 3-day-old suckling mice are inoculated with 200 mu L of suckling mouse poison (10: 1 of suckling mouse poison), and the average death time of the suckling mice is 27 h. Treatment of dead suckling mice: shearing off the head of the suckling mouse, removing limbs and internal organs, adding sterile quartz sand and PBS (phosphate buffer solution) to 5mL, and grinding to obtain the 1:10 suckling mouse poison.
The resulting suckling mouse venom was passaged further: the supernatant was collected by centrifugation at 3000rpm for 20min, and 200. mu.L of the virus was administered to each suckling mouse. The strain is continuously transmitted for 4 generations until the average death time of the suckling mouse is 17 hours, the toxicity is stronger at the moment, and the selected strain is shown in Table 1.
TABLE 1 strains of foot-and-mouth disease Virus used in the experiments
The offending pigs are bred in a virulent animal house in a Lanzhou veterinary institute P3 laboratory, and the non-offending pigs are bred in a common animal house. And (4) taking the suckling mouse poison, diluting the suckling mouse poison to 300:1 by PBS (phosphate buffer solution), and performing intramuscular injection on the root of the ear for 1 mL.
TABLE 2 challenge test arrangement
And taking a proper amount of fresh anticoagulation blood of the corresponding toxin-counteracting pig every day from 15 days after toxin-counteracting.
(1) Taking 2mL of fresh anticoagulation blood, and uniformly mixing the anticoagulation blood with the whole blood and tissue homogenate diluent in a volume of 1:1 in an equal volume.
(2) A15 mL centrifuge tube was taken, 3mL of the separation medium was added, and the mixture was placed at 18-22 ℃.
(3) The blood sample is carefully added to the surface of the separation fluid. Centrifuging at 18-22 deg.C and 2200rmp for 20 min. Centrifugation at low temperatures (e.g., 4 ℃) reduces cell yield.
(4) After centrifugation, the supernatant fraction of 0.5cm or less of the upper layer of the separated liquid (cell layer containing lymphocytes) was carefully aspirated with a pipette and discarded.
(5) The separated liquid layer and lymphocyte layer were carefully pipetted into another new centrifuge tube.
(6) And (5) adding 10mL of cell washing solution into the centrifuge tube obtained in the step (4) and uniformly mixing.
(7) The supernatant was discarded.
(8) The resulting cells were resuspended with a pipette in 5mL of cell wash.
(9) Centrifuge at 1600rmp for 10 min. The supernatant was discarded. Steps (7), (8), (9) were repeated, after which the cells were resuspended in complete culture with an appropriate volume of RPMI 1640.
(10) Cell count, adjusting the number of cells to 1X 107And storing for later use.
Finally, the preparation of the SLA-2-HB-BSP-As 63-s.beta.2m tetramer of the present invention was started.
1. Porcine foot-and-mouth disease virus SLA-I restrictive CTL epitope design
The method comprises the steps of taking NetMHCpan 2.8Version (www.cbs.dtu.dk/services/NetMHCpan /) as a template, submitting all cloned functional genes of SLA I molecules including the pig with the pig in a packet SLA-2 to an online website template, and forming a prediction online tool of the porcine virus CTL epitope together with all the original functional genes of the SLA I molecules of the website. VP1 and 3D non-structural protein sequences of three strains, namely 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 1/Jiangsu/China/2005, are respectively input into a prediction template to predict CTL epitopes, and polypeptide candidate epitopes with high affinity are selected as much as possible, and are shown in Table 3.
TABLE 3 prediction epitopes of the restricted porcine FMDV antigen
aBinding power of SLA-2-HB of polypeptide predicted by application of weight matrix in NetMHCpan 2.8Server (www.cbs.dtu.dk/services/NetMHCpan /)Number (high binding force polypeptide rank threshold of 0.5; weak binding force polypeptide rank threshold of 2.0)
bPolypeptide IC predicted by applying artificial neural network in NetMHCpan 2.8Server50Values (Strong binding/SB lower than 50 nM; Weak binding/WB lower than 500nM)
2. ELISPOT assay:
the ELISPOT is called Enzyme-linked Immunospot Assay (Enzyme-linked Immunospot Assay) and is a means which combines cell culture technology and ELISA technology and can detect the secretion condition of cytokines at the cellular level. The CTL epitope polypeptide with activity can stimulate specific CD8+T cells secrete IFN-gamma, and by utilizing the property, the predicted FMDV epitope peptide can be screened by the method. Separating pig peripheral blood mononuclear cells infected with the foot-and-mouth disease virus for ELSPOT specific T cell epitope peptide screening. Non-challenged PBMC is set as a blank control group, and challenged PBMC without polypeptide stimulus is set as a negative control group.
(1) Diluting the coated IFN-gamma monoclonal antibody to 10 mug/mL by sterile PBS, and carrying out pH 7.4;
(2) taking out the ELISPOT special plate, adding 50 μ L70% ethanol into each hole, treating for no more than 1min (generally tens of seconds, and the membrane is thoroughly moistened), discarding ethanol solution, adding 200 μ L sterile water into each hole, washing for 5 times, adding 100 μ L diluted IFN- γ monoclonal antibody solution into each hole, and incubating overnight at 4-8 deg.C;
(3) the next day, the antibody solution was aspirated and washed 2 times with 200 μ L sterile PBS per well;
(4) adding 20 μ L of blocking solution into each well, blocking at 37 deg.C for 2 hr, removing blocking solution by suction, adding 200 μ L of complete culture medium into each well, and incubating at room temperature for at least 30 min;
(5) the medium was aspirated off and 100. mu.L of prediluted polypeptide and PBMC cell suspension (diluted to 2.5X 10 cell suspension) was added to each well5cells/mL), 3 replicates per sample were set up;
(6) the plate cover is covered, the temperature is 37 ℃ and the CO content is 5 percent2Incubating and culturing for 18-20 h under the condition (the optimal culture time needs to be tested and found), wherein cells cannot be disturbed during the culture period;
(7) aspirating the cell suspension, washing 5 times with 200 μ L sterile PBS per well, diluting the detection antibody to 0.5 μ g/mL with PBS containing 0.5% FBS, adding 100 μ L per well, incubating at 37 ℃ for 2h, and completing washing according to step (3);
(8) streptavidin (streptavidin-HRP) was diluted in PBS containing 0.5% FBS, 100. mu.L per well. Incubating at 37 ℃ for 1h, and finishing washing according to the step (3);
(9) adding 100 mu L of color development liquid into each hole, placing in the dark, reacting for 5-30 min according to the spot formation condition, washing each hole with deionized water to stop the substrate color development reaction, air-drying for 2h at room temperature or staying overnight for complete drying, and storing in a sealed plastic bag for a long time. Cellular Technology Ltd (CTL) for ELISPOT spots
And counting by a plate reader, wherein the image acquisition is respectively completed by ImmunoCapture software, and the analysis, statistics and quality control are performed by ImmunoSpot 5.0 software.
Each group was reacted according to the ELISPOT standard test procedure, and the results are shown in FIGS. 3 and 4, using CTL
And (3) reading a plate reader, wherein the effective spots of each group of peptides read represent one IFN-gamma molecule secreted by the peptides after stimulating CTL, the more spots, the stronger the ability of the peptides to stimulate CTL to secrete IFN-gamma, and the peptide with the strongest stimulating ability is selected as the dominant epitope peptide. As can be seen in fig. 3, the ability of the Hu62 polypeptide to stimulate CTLs is much higher than the other polypeptides, followed by INT3, Ahu63, and Q10. FIGS. 4C-J show representative ELISPOT assays for each polypeptide, and A-B show blank and negative controls, respectively. It can be seen that each polypeptide has a certain stimulatory capacity compared to the control group, but for tetramer preparation, the most dominant polypeptide must be selected, so Hu62 can be used as the dominant polypeptide epitope for the next tetramer preparation. 3. Tetramerization of heavy chain SLA-2-BSP with light chain s beta 2m
1) And culturing of the cells
A: the activated heavy chain recombinant bacterium SLA-2-HB-BSP/BL21 and the light chain recombinant bacterium beta 2m/BL21 are respectively inoculated with 5mL of active heavy chain recombinant bacterium SLA-2-HB-BSP/BL21 and light chain recombinant bacterium beta 2m/BL21Amp (100. mu.g/mL) in medium, incubated at 37 ℃ to OD600When reaching 0.4-0.6, adding IPTG to the final concentration of 1mmol/L, and taking thalli SDS-PAGE to detect the protein expression.
The recombinant bacterium SLA-2-HB-BSP/pET-21a (+) -BL21 is detected by 12% SDS-PAGE and is normally expressed and is about 34 kD. The light chain beta 2m/pET-21a (+) is detected by induction and SDS-PAGE, and the protein is confirmed to be normally expressed, and the protein position is about 12 kD. Both heavy and light chains are suitable for the next tetramer preparation.
The correctly expressed heavy chain SLA-2-HB-BSP/pET-21a recombinant bacteria and the light chain s beta 2m/pET-21a recombinant bacteria are cultured at 37 ℃ overnight by SDS-PAGE detection.
B: the overnight cultures were diluted to 1L LB at 1:100 dilutions, respectively, and cultured to OD at 37 deg.C600IPTG was added to a final concentration of 0.5mmol/L, and the culture was continued for 4 hours to harvest bacteria (1 liter each of heavy and light chains).
2) Preparation of inclusion body
A: the bacteria were suspended in 30mL PBS and disrupted by sonication at 650W (5sec on/10sec off, 10min).
B: inclusion bodies were washed 2 times with 25mmol/L Tris-HCl, pH8.0, 100mmol/L NaCl, 1% Triton-X100 wash.
C: inclusion bodies were washed 1 time with 25mmol/L Tris-HCl, pH8.0, 100mmol/LM NaCl wash.
D: inclusion bodies were solubilized with 25mmol/L Tris-HCl, pH8.0, 100mmol/L NaCl containing 8mol/L urea to a protein concentration of 10 mg/mL.
3) And renaturation thereof
To 500mL of 100mmol/L Tris-HCl, pH8.0, 400mmol/L Arginine, 5mmol/L reduced glutamthione, 0.5mmol/L oxidized glutamthione was added 15mg of heavy chain, 10mg of light chain and 5mg of epitope peptide at 4 ℃ and stirred for 48 hours.
4) Biotinylation of MHC monomers
500mL of the renaturation solution was concentrated to 5mL and buffer exchange was carried out with a PD10 column, the new buffer being 10mmol/L Tris pH8.0. The components of the kit were added to the protein solution described above according to the instructions of the BirA kit (identification) and treated at room temperature for 16 hours.
5) Isolation of biotinylated monomers
The overnight protein solution was subjected to AKTA FPLC separation: separating column Hiload 26/600Supdex 75pg, separation buffer 20mmol/L Tris-HCl pH8, 100mmol/L NaCl; the flow rate was 1 mL/min. The protein peak was collected at an elution volume of 163mL and concentrated to 1 mg/mL.
6) Identification of monomers
A: SDS-PAGE, the FPLC separated monomer, sample 8 u g, if seen heavy chain and light chain two bands, indicate the renaturation of monomer has been formed.
B: ELISA detection, FPLC separated monomer 2. mu.g per well coated overnight at 4 ℃, two wells simultaneously with the addition of non-biotinylated monomer as control, and detection of monomer biotinylation was carried out with HRP-labeled streptavidin (biolegend), as shown in FIG. 5, OD595Reading: biotinylation monomer hole 1 ═ 0.92, biotinylation monomer hole 2 ═ 0.86, and non-biotinylation monomer hole 1 ═ 0.12; non-biotinylated monomer well 2 ═ 0.09. Biotinylation was clearly compared to the non-biotinylated sample, indicating successful biotinylation.
The PE-labeled tetramer was formed by mixing 0.312mg extravidine-PE per 1mg biotinylated monomer as shown in FIG. 6.
4. SLA-2/HB/Hu62Tetramer flow assay
(8) Peripheral blood mononuclear cells from the Tibet/CHA/99(HuBHK99) strain challenge group of pigs were isolated, centrifuged at 1500rmp for 3min at room temperature, the supernatant discarded, the cells washed once with PBS (pH 7.2), and centrifuged again.
(9) Cell pellets were resuspended in FACS washes (containing 0.1% NaN)3And 0.1% BSA in 1 × PBS).
(10) mu.L of PE-labeled SLA-2-HB-BSP/Hu62 tetramer was added to the cell suspension, and the mixture was left to react for 20min at room temperature with light.
(11) Add 2mLFACS washing heavy suspension cells, 500 Xg room temperature centrifugation for 3min, abandon the supernatant, cell precipitation heavy suspension in 100L FACS washing.
(12) mu.L (2.5. mu.g) FITC-labeled Anti-pig CD8a antibody was added and the reaction was continued at room temperature with exclusion of light for 20 min.
(13) The cells were washed once by adding FACS wash, centrifuged at 1500 rpm for 3min at room temperature, the supernatant was discarded, and the cell pellet was resuspended in an appropriate amount (300-400. mu.L) of FACS wash.
(14) Cell suspension samples were analyzed by flow cytometry and sorted.
Hu62 belongs to type O foot-and-mouth disease strain, Peripheral Blood Mononuclear Cells (PBMC) of pigs infected with type O virus are taken, and the PBMC are stained by using FITC-labeled Anti-Pig CD8a monoclonal antibody. PBMC are double-stained by using a PE-labeled SLA-2-HB-BSP-Hu62-s beta 2m tetramer, namely specific CTLs aiming at the Hu62 polypeptide epitope account for about 5.7 percent of total cells, as shown in a figure 7, a very significant difference (P <0.01) is formed with 0.1 percent of cells of a control group (cells of an unvaccinated group), the preparation success of the SLA-2-HB-Hu62-s beta 2m tetramer is proved, and Hu62 is proved to be a specific CTL epitope of foot-and-mouth disease virus. A representative tetramer flow assay is shown in FIG. 8.
In the research, a O-type foot-and-mouth disease virus polypeptide Hu62 is successfully screened out through immunization and ELISPOT, a tetramer is constructed by the polypeptide, the pre-constructed pouch pig SLA-2-HB-BSP and the s beta 2m protein, and the prepared tetramer containing the polypeptide Hu62 is successfully detected by using flow cytometry. The research finally proves that the polypeptide Hu62 has immunological activity and is a potential O type foot-and-mouth disease virus CTL epitope.
Sequence listing
<110> university of Dalian
<120> method for preparing tetramer from O-type foot-and-mouth disease virus polypeptide
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Claims (1)
1. A method for preparing tetramer by O-type foot-and-mouth disease virus polypeptide is characterized by comprising the following steps:
1) porcine foot and mouth disease virus SLAI restrictive CTL epitope design
Using NetMHCpan 2.8Version as a template, submitting various cloned SLAI molecules including the SLA-2 of the pig to an online website template, forming a prediction online tool of a swine virus CTL epitope with various original SLAI molecules of the website, respectively inputting VP1 and 3D non-structural protein sequences of three strains of 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 1/Jiangsu/China/2005 into the prediction template to predict the CTL epitope, selecting a polypeptide candidate epitope with high affinity,
2) ELISPOT test:
(1) diluting the coated IFN-gamma monoclonal antibody to 10 mug/mL by sterile PBS, and carrying out pH 7.4;
(2) taking out the ELISPOT special plate, adding 50 μ L70% ethanol into each hole, treating for no more than 1min, discarding ethanol solution, adding 200 μ L sterile water into each hole, washing for 5 times, adding 100 μ L diluted IFN- γ monoclonal antibody solution into each hole, and incubating overnight at 4-8 deg.C;
(3) the next day, the antibody solution was aspirated and washed 2 times with 200 μ L sterile PBS per well;
(4) adding 20 μ L of blocking solution into each well, blocking at 37 deg.C for 2 hr, removing blocking solution by suction, adding 200 μ L of complete culture medium into each well, and incubating at room temperature for at least 30 min;
(5) the medium was aspirated off and 100. mu.L of prediluted polypeptide and diluent were added to each wellReleased to 2.5X 105cells/mL PBMC cell suspension, 3 replicates per sample set;
(6) the plate cover is covered, the temperature is 37 ℃ and the CO content is 5 percent2Incubating and culturing for 18-20 h under the condition, wherein cells cannot be disturbed during the culturing period;
(7) aspirating the cell suspension, washing 5 times with 200 μ L sterile PBS per well, diluting the detection antibody to 0.5 μ g/mL with PBS containing 0.5% FBS, adding 100 μ L per well, incubating at 37 ℃ for 2h, and completing washing according to step (3);
(8) diluting streptavidin with PBS containing 0.5% FBS, adding 100 μ L of streptavidin to each well, incubating at 37 ℃ for 1h, and completing washing according to the step (3);
(9) adding 100 mu L of color development liquid into each hole, placing in the dark, reacting for 5-30 min according to the spot formation condition, washing each hole with deionized water to stop the substrate color development reaction, air-drying for 2h at room temperature or staying overnight for complete drying, storing in a sealed plastic bag for a long time, counting ELISPOT spots, completing image acquisition, performing analysis statistics and quality control; screening out the epitope Hu62 with the most advantage for preparing tetramer;
3) tetramerization of heavy chain SLA-2-BSP with light chain s beta 2m
(1) And culturing of the cells
A: heavy chain SLA-2-HB-BSP and light chain beta 2m expression assay
Activating heavy chain recombinant bacteria SLA-2-HB-BSP/BL21 and light chain recombinant bacteria beta 2m/BL21, respectively inoculating 5mL of culture medium containing 100 mu g/mL of Amp, and culturing at 37 ℃ to OD600When reaching 0.4-0.6, adding IPTG to the final concentration of 1mmol/L, taking thalli SDS-PAGE to detect the protein expression,
the recombinant bacterium SLA-2-HB-BSP/pET-21a (+) -BL21 is induced and detected by 12% SDS-PAGE to find that the expression is normal, the light chain beta 2m/pET-21a (+) is induced and detected by SDS-PAGE to confirm that the protein is normally expressed, and both the heavy chain and the light chain are suitable for the next tetramer preparation,
heavy chain SLA-2-HB-BSP/pET-21a recombinant bacteria and light chain s beta 2m/pET-21a recombinant bacteria which are detected by SDS-PAGE and correctly expressed are cultured at 37 ℃ overnight,
b: the overnight cultures were diluted to 1L LB at 1:100 dilutions, respectively, and cultured to OD at 37 deg.C600Add IPTG to final concentration ═ 1When the concentration reaches 0.5mmol/L, the culture is continued for 4 hours, and the bacteria are harvested, wherein the heavy chain SLA-2-HB-BSP and the light chain beta 2m are respectively 1 liter;
(2) preparation of inclusion body
A: the bacteria were suspended in 30mL PBS, disrupted by sonication at 650W, off at 5sec on/10sec, 10min,
b: the inclusion bodies were washed 2 times with 25mmol/L Tris-HCl, pH8.0, 100mmol/L NaCl, 1% Triton-X100 wash,
c: the inclusion bodies were washed 1 time with 25mmol/L Tris-HCl, pH8.0, 100mmol/L NaCl wash,
d: the inclusion bodies were solubilized with 25mmol/L Tris-HCl, pH8.0, 100mmol/L NaCl containing 8mol/L urea to a protein concentration of 10 mg/mL;
(3) renaturation of
Adding 15mg of heavy chain SLA-2-HB-BSP, 10mg of light chain beta 2m and 5mg of Hu62 epitope peptide into 500mL of 100mmol/L Tris-HCl, pH8.0, 400mmol/L Arginine, 5mmol/L reduced glutamthione and 0.5mmol/L oxidized glutamthione at the temperature of 4 ℃, and stirring for 48 hours to obtain a renaturation solution;
(4) biotinylation of MHC monomers
500mL of renaturation solution, concentrate to 5mL, exchange the buffer solution with PD10 column, the new buffer solution is 10mmol/L Tris pH8.0, add the ingredients in the kit to the above-mentioned protein solution, process for 16 hours at room temperature;
(5) isolation of biotinylated monomers
The overnight protein solution was subjected to AKTA FPLC separation: separating column Hiload 26/600Supdex 75pg, separation buffer 20mmol/L Tris-HCl pH8, 100mmol/L NaCl; the flow rate is 1mL/min, the protein peak with the elution volume of 163mL is collected and concentrated to 1 mg/mL;
(6) identification of monomers
A: SDS-PAGE, the monomers separated from the FPLC are spotted at 8 μ g, if two bands of heavy chain SLA-2-HB-BSP and light chain beta 2m are seen, the monomers are formed after renaturation,
b: ELISA detection, coating FPLC separated monomer at 4 ℃ overnight, 2 mug each in each hole, adding non-biotinylated monomer as a control, detecting biotinylation of monomer by HRP-labeled streptavidin,
mixing the biotinylated monomer in the amount of 0.312mg extravidine-PE to form PE labeled tetramer;
4) SLA-2/HB/Hu62Tetramer flow assay
(1) Separating peripheral blood mononuclear cells of the Tibet/CHA/99(HuBHK99) strain challenge group pigs, centrifuging at 1500rmp at room temperature for 3min, discarding the supernatant, washing the cells once with PBS of pH 7.2, centrifuging again,
(2) resuspending the cell pellet in FACS Wash
(3) Adding 5 μ L of SLA-2-HB-BSP/Hu62 tetramer marked by PE into the cell suspension, reacting for 20min at room temperature in dark,
(4) adding 2mLFACS washing solution to resuspend the cells, centrifuging at 500 Xg for 3min at room temperature, discarding the supernatant, resuspending the cell sediment in 100. mu.L FACS washing solution,
(5) mu.L of Anti-pig CD8a antibody labeled with FITC of 2.5. mu.g is added, and the mixture is reacted for 20min at room temperature in the dark,
(6) adding FACS washing solution to wash the cells once, centrifuging at 1500rmp for 3min at room temperature, discarding the supernatant, suspending the cell precipitate in 300-400 mu LFACS washing solution,
wherein the FACS wash contained 0.1% NaN3 and 0.1% BSA in 1 XPBS,
(7) cell suspension samples were analyzed and sorted by flow cytometry,
CTL aiming at the characteristic type of the Hu62 polypeptide epitope accounts for about 5.7 percent of total cells, forms a very significant difference P <0.01 with 0.1 percent of a control group, proves that the preparation of the SLA-2-HB-Hu62-s beta 2m tetramer is successful, and simultaneously proves that Hu62 is the specific CTL epitope of the foot-and-mouth disease virus.
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| 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 |
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| 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 |
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