CN117304273A - Foot-and-mouth disease nanometer vaccine based on self-assembled nanoparticle protein and preparation method thereof - Google Patents
Foot-and-mouth disease nanometer vaccine based on self-assembled nanoparticle protein and preparation method thereof Download PDFInfo
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- CN117304273A CN117304273A CN202310065245.1A CN202310065245A CN117304273A CN 117304273 A CN117304273 A CN 117304273A CN 202310065245 A CN202310065245 A CN 202310065245A CN 117304273 A CN117304273 A CN 117304273A
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- 241000710198 Foot-and-mouth disease virus Species 0.000 title claims abstract description 100
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 title claims abstract description 87
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 58
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 57
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 57
- 229960005486 vaccine Drugs 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 238000001814 protein method Methods 0.000 title description 2
- 239000000427 antigen Substances 0.000 claims abstract description 70
- 102000036639 antigens Human genes 0.000 claims abstract description 70
- 108091007433 antigens Proteins 0.000 claims abstract description 70
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 25
- 229940031348 multivalent vaccine Drugs 0.000 claims description 13
- 230000001900 immune effect Effects 0.000 claims description 11
- 208000007407 African swine fever Diseases 0.000 claims description 3
- 241000589562 Brucella Species 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 2
- 229940031626 subunit vaccine Drugs 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 5
- 238000000746 purification Methods 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 210000003071 memory t lymphocyte Anatomy 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 229940031551 inactivated vaccine Drugs 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 230000007969 cellular immunity Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 2
- 230000004727 humoral immunity Effects 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 108010042365 Virus-Like Particle Vaccines Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940031416 bivalent vaccine Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 235000021439 fasting mimicking diet Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 210000001806 memory b lymphocyte Anatomy 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 229940023041 peptide vaccine Drugs 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
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- A61K39/385—Haptens or antigens, bound to carriers
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- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
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- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention belongs to the technical field of nanometer vaccines, and relates to self-assembled nanoparticle protein, foot-and-mouth disease nanometer vaccines, and a preparation method and application thereof. The invention provides a nanoparticle protein, and the amino acid sequence of the nanoparticle protein is shown as SEQ ID NO. 1. The nanoparticle protein can be used for preparing novel monovalent, bivalent or multivalent subunit vaccine of FMD, and the nanoparticle protein and foot-and-mouth disease characteristic antigen can be directionally loaded, so that the immunoprotection effect of the vaccine on different serotypes of FMDV can be obviously improved.
Description
Technical Field
The invention belongs to the technical field of nanometer vaccines, and particularly relates to self-assembled nanoparticle protein, foot-and-mouth disease nanometer vaccines, and a preparation method and application thereof.
Background
Foot and Mouth Disease (FMD) is an acute, febrile, highly contagious infectious disease caused by Foot and Mouth Disease Virus (FMDV), which has occurred for over 100 years and has not been destroyed so far. FMD has 7 serotypes (O, A, C, asian-1 and SAT 1-3), with the exception of serotype C, which are prevalent, with types A and O being the most prevalent, distributed predominantly in Asia, africa and the middle east, and no immunological cross-protection between the different sera. Vaccination is a reliable and effective means of specifically preventing FMD, a safe and effective vaccine being a prerequisite for successful prevention, control so as to eventually destroy FMD.
Currently, vaccine immunization remains the primary method of preventing and controlling FMDV, and commercial inactivated vaccines remain the most widely used vaccine. However, inactivated vaccines also have some disadvantages, such as the risk of virus transmission during production and use, and high manufacturing costs. Furthermore, the european union and the united states have limited the use of inactivated vaccines against FMDV. In addition, with the development of genetic engineering technology, substantial progress has been made in the development of novel FMDV subunit vaccines (e.g., subunit vaccines, synthetic peptide vaccines, multi-epitope vaccines, virus-like particle vaccines), but the current vaccines still have the problem of poor immune effects.
Disclosure of Invention
The invention aims to provide self-assembled nanoparticle protein, foot-and-mouth disease nanometer vaccine, and a preparation method and application thereof. The nanoparticle protein can be used for preparing novel monovalent, bivalent or multivalent subunit vaccine of FMD, and the nanoparticle protein and foot-and-mouth disease characteristic antigen can be directionally loaded, so that the immunoprotection effect of the vaccine on different serotypes of FMDV can be obviously improved.
The invention provides a nanoparticle protein, and the amino acid sequence of the nanoparticle protein is shown as SEQ ID NO. 1.
The invention also provides application of the nanoparticle protein in preparation of vaccines; the vaccine comprises foot-and-mouth disease vaccine, brucella nano multivalent vaccine, novel crown multivalent vaccine or african swine fever multivalent vaccine.
The invention also provides application of the nanoparticle protein in improving the immune effect of the foot-and-mouth disease vaccine.
The invention also provides a foot-and-mouth disease vaccine, which comprises the nanoparticle protein and the foot-and-mouth disease characteristic antigen directionally loaded with the nanoparticle protein according to the technical scheme.
Preferably, the foot-and-mouth disease characteristic antigen comprises an O-type foot-and-mouth disease characteristic antigen VP1 and/or an a-type foot-and-mouth disease characteristic antigen VP1.
Preferably, the amino acid sequence of the antigen VP1 characteristic of the O-type foot-and-mouth disease is shown as SEQ ID NO. 2; the amino acid sequence of the antigen VP1 characteristic of the foot-and-mouth disease A is shown as SEQ ID NO. 3.
Preferably, the directional loading is performed by the SpyCatcher and SpyTag systems.
Preferably, the nanoparticle protein is coupled to SpyCatcher and the antigen characteristic of foot-and-mouth disease is coupled to SpyTag prior to directional loading.
Preferably, the amino acid sequence of the nanoparticle protein coupled with SpyCatcher is shown in SEQ ID NO. 4; foot-and-mouth disease characteristic antigens coupled to SpyTag include a type a foot-and-mouth disease characteristic antigen VP1 coupled to SpyTag and/or a type O foot-and-mouth disease characteristic antigen VP1 coupled to SpyTag; the amino acid sequence of the A-type foot-and-mouth disease characteristic antigen VP1 coupled with the SpyTag is shown as SEQ ID NO.5, and the amino acid sequence of the O-type foot-and-mouth disease characteristic antigen VP1 coupled with the SpyTag is shown as SEQ ID NO. 6.
The invention also provides a preparation method of the vaccine, which comprises the step of directionally loading nanoparticle proteins and foot-and-mouth disease characteristic antigens to obtain the foot-and-mouth disease vaccine.
The invention provides nanoparticle proteins. The nanoparticle protein can be used for preparing novel monovalent, bivalent or multivalent subunit vaccine of FMD, and the nanoparticle protein and foot-and-mouth disease characteristic antigen can be directionally loaded, so that the immunoprotection effect of the vaccine on different serotypes of FMDV can be obviously improved. The test result shows that the vaccine of the invention has stronger immune effect compared with the conventional subunit vaccine.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the structures of X-M, Y-A and Y-O provided by the invention.
FIG.2isadiagramshowingSDS-PAGEresultsofX-M,Y-A,Y-O,M-A,M-OandM-A+Oprovidedbythepresentinvention;
FIG.3isagraphofthetransmissionelectronmicroscoperesultsofX-M,M-A,M-OandM-A+Oprovidedbytheinvention;
FIG.4isagraphshowingELISAdetectionresultsofY-A,Y-O,Y-A+Y-O,M-A,M-OandM-A+O,whereintheleftgraphshowstheresultofVP1-Aspecificantibodytiter,andtherightgraphshowstheresultofVP1-Ospecificantibodytiter;
FIG.5isagraphofFCMdetectionresultsofY-A,Y-O,Y-A+Y-O,M-A,M-OandM-A+Oprovidedbythepresentinvention;
FIG.6showsICCSdetectionresultsofY-A,Y-O,Y-A+Y-O,M-A,M-OandM-A+Oprovidedbythepresentinvention; wherein, the left graph is a memory CD4+ T cell ratio result graph, and the right graph is a memory CD8+ T cell ratio result graph;
FIG.7showsELISAdetectionresultsofY-A,Y-O,Y-A+Y-O,M-A,M-OandM-A+Oprovidedbytheinvention; wherein, the left graph is a VP1-A specific antibody titer result graph, and the right graph is a VP1-O specific antibody titer result graph;
FIG.8isagraphshowingtheresultsofmonitoringorganinvolvementandsurvivalofmicewithY-A,Y-O,Y-A+Y-O,M-A,M-OandM-A+O,whereintheupperleftgraphshowstheresultsoforganinvolvementunderA-typechallenge,theupperrightgraphshowstheresultsofsurvivalofmiceunderA-typechallenge,thelowerleftgraphshowstheresultsoforganinvolvementunderO-typechallenge,andthelowerrightgraphshowstheresultsofsurvivalofmiceunderO-typechallenge.
Detailed Description
The invention provides a nanoparticle protein, the amino acid sequence of which is shown in SEQ ID NO. 1: MGIVASRFNHTNLSLELLVERLVEGAKLYEGHLKAEGFGSEDNIELARVPGSW EIPLIVKGKPVSFGVVTADTLEQAVEVLVRRAGTKQGNKGWDANMANLLLL KEDVEGVIALGGQTPHFEYVSLEASEVSEGLMLSTIEMANLFKKLG. The nanoparticle protein can be used for preparing novel monovalent, bivalent or multivalent subunit vaccine of FMD, and the nanoparticle protein and foot-and-mouth disease characteristic antigen can be directionally loaded, so that the immunoprotection effect of the vaccine on different serotypes of FMDV can be obviously improved. The nanoparticle protein can enhance antigen stability, enhance antigen delivery capability, enhance antigen recognition in an immune system, prolong residence time of the antigen in a lymphatic system, further enhance immune effects (cell and humoral immunity), simultaneously form multivalent vaccines by various antigens coupled on the surface of particles, activate wide cross protection of an immune system of an organism, and resist invasion of different pathogens. The nano particles are biological proteins, and have higher biocompatibility and safety. High yield, simple and convenient production, and can be mass-produced in a prokaryotic system. And has certain universality and is used for different antigen delivery.
The invention also provides application of the nanoparticle protein in preparation of an antibody detection kit, a multivalent antibody or a vaccine; the vaccine comprises foot-and-mouth disease vaccine, brucella nano multivalent vaccine, novel crown multivalent vaccine or african swine fever multivalent vaccine and the like.
The invention also provides application of the nanoparticle protein in improving the immune effect of the foot-and-mouth disease vaccine. The nanoparticle protein and the foot-and-mouth disease antigen are directionally loaded, so that the foot-and-mouth disease vaccine with improved immune effect can be obtained.
The invention also provides a foot-and-mouth disease vaccine, which comprises the nanoparticle protein and the foot-and-mouth disease characteristic antigen directionally loaded with the nanoparticle protein according to the technical scheme. In the present invention, the foot-and-mouth disease characteristic antigen preferably includes an O-type foot-and-mouth disease characteristic antigen VP1 and/or an a-type foot-and-mouth disease characteristic antigen VP1.
In the invention, the amino acid sequence of the antigen VP1 which is characteristic of the O-type foot-and-mouth disease is preferably shown as SEQ ID NO. 2: MQTSTGESADPVTATVENYGGETQVQRRQHTDVSFILDRFVKVTPKDQINVLDLMQTPAHTLVGALLRTATYYFADLEVAVKHEGNLTWVPNGAPETALDNTTNPTAYYRAPLTRLALPYTAPHRVLATVYN; the amino acid sequence of the antigen VP1 characteristic of the foot-and-mouth disease A is preferably shown as SEQ ID NO. 3: MPTAYHKQPFTRLALPYTAPHRVLATVYNGVNKYSATGGGRRGDLGSPAARVAAQLPSSFNFGAIRATTIHELLVRMKRAELYCPRPLLAVEVLSHDRHKQKIIAPTKQLL.
In the present invention, the directional loading is preferably performed by SpyCatcher and SpyTag systems. In the present invention, the nanoparticle protein is preferably coupled to SpyCatcher and the antigen characteristic of foot-and-mouth disease is preferably coupled to SpyTag prior to directional loading. In the invention, the C-terminal of the nanoparticle protein and the antigen characteristic of foot-and-mouth disease is preferably coupled with a purification tag, which is beneficial to purification after expression. In the present invention, the amino acid sequence of the nanoparticle protein (abbreviated as X-M) coupled to Spycatcher is preferably as shown in SEQ ID NO. 4:
wherein the single underlined part is the amino acid sequence of SpyCatcher, the thickened part is flexible amino acid, the italic part is the amino acid sequence of nanoparticle protein, and the double underlined partIs a histidine tag; the foot-and-mouth disease characteristic antigen coupled with SpyTag preferably comprises a type A foot-and-mouth disease characteristic antigen VP1 coupled with SpyTag and/or a type O foot-and-mouth disease characteristic antigen VP1 coupled with SpyTag; the amino acid sequence of the antigen VP1 (Y-A for short) coupled with SpyTag is preferably shown as SEQ ID NO.5:
wherein, the single underlined part is the amino acid sequence of SpyTag, the thickened part is flexible amino acid, the italic part is the amino acid sequence of the antigen VP1 characteristic of A-type foot-and-mouth disease, and the double underlined part is histidine tag; the amino acid sequence of the antigen VP1 (Y-O for short) coupled with SpyTag is preferably shown in SEQ ID NO. 6:
wherein, the single underlined part is the amino acid sequence of SpyTag, the thickened part is flexible amino acid, the italic part is the amino acid sequence of the antigen VP1 characteristic of O-type foot-and-mouth disease, and the double underlined part is histidine tag.
The invention also provides a preparation method of the vaccine, which comprises the step of directionally loading nanoparticle proteins and foot-and-mouth disease characteristic antigens to obtain the foot-and-mouth disease vaccine. The definition of nanoparticle proteins and antigens characteristic of foot and mouth disease is preferably as described above. accordingtothespecificsequencesabove,afterobtainingX-M,Y-AandY-O,theinventionpreferablymixesX-MwithY-Aand/orY-O,andreactsinneutralbuffertoobtaintypeAfoot-and-mouthdiseasevaccine(M-A),typeOfoot-and-mouthdiseasevaccine(M-O)ortypeA,Obivalentfoot-and-mouthdiseasevaccine(M-A+O). In the present invention, the neutral buffer preferably includes a neutral PBS buffer. In the summary of the invention, the reaction conditions are preferably 4℃for 16h.
The foot-and-mouth disease vaccine of the invention comprises full-length characteristic antigen, and has stronger capability of activating immune system than epitope peptide. The monovalent foot-and-mouth disease vaccine prepared based on the nanoparticle protein has strong immune effect. The invention prepares the novel bivalent subunit vaccine containing the two FMD serotype characteristic antigens based on the potential of preparing multivalent vaccine by carrying various antigens by the nanoparticle protein, and directionally loads the A-type and O-type characteristic antigens VP1 on the surface of the nanoparticle protein, thereby being capable of obviously improving the protection effect on the two serotypes FMDV. In addition, the nanoparticle protein can be directionally loaded with various characteristic antigens for preparing multivalent vaccines, and the prepared multivalent vaccines contain the characteristic antigens of a plurality of serotypes, so that the protective effect on FMDV of different serotypes can be remarkably improved.
For further explanation of the present invention, the self-assembled nanoparticle protein, foot-and-mouth disease nano vaccine, and the preparation method and application thereof provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The characteristic antigens VP1 of type A and/or type O are selected, and one or two VP1 are directionally loaded on the surface of nanoparticle protein (code M) for preparing novel monovalent and bivalent subunit vaccine.
The specific implementation steps are as follows:
1. construction, expression and characterization of novel FMD monovalent and bivalent subunit vaccines
1.1FMD monovalent and divalent subunit vaccine construction
The antigens VP1 characteristic of FMD serotypes A and O, designated VP1-A and VP1-O, were selected. Covalent bond (code X) is connected to the N end of nanoparticle protein through flexible amino acid to obtain X-M, covalent bond (code Y) is connected to the N end of VP1-A and VP1-O through flexible amino acid to obtain Y-A and Y-O, covalent bonding capability between two covalent bonds is utilized to load one or two antigens on the surface of nanoparticle protein in a directional way, and FMD monovalent or bivalent subunit vaccine is prepared.
FIG. 1 is a schematic structural diagram of X-M, Y-A and Y-O, wherein X and SC refer to SpyCatcher, Y and ST refer to SpyTag, M is the nanoparticle protein of the invention, A and VP1-A in Y-A refer to the antigen VP1 characteristic of type A foot-and-mouth disease, O and VP1-O in Y-O refer to the antigen VP1 characteristic of type O foot-and-mouth disease, and HIS refers to a histidine purification tag.
1.2 expression and purification of FMD monovalent and bivalent subvaccines
Based on the sequences of SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, three constructs X-M, Y-A and Y-O (the C-terminal of the three constructs is introduced with a purification tag to facilitate purification) are obtained by expression and purification in a prokaryotic system E.coli. purifiedX-MandY-AaremixedinaneutralPBSbuffersolutionaccordingtoamolarratioof1:2,andreactedat4℃for16hourstoobtainthetypeAmonovalentfoot-and-mouthdiseasevaccine(markedasM-A). Purified X-M and Y-O are mixed in a neutral PBS buffer solution according to a molar ratio of 1:2, and after 16 hours of reaction at 4 ℃, the O-type monovalent foot-and-mouth disease vaccine (marked as M-O) is obtained. purifiedX-M,Y-AandY-OaremixedinaneutralPBSbuffersolutionaccordingtoamolarratioof1:2:2,andthemixtureisreactedfor16hoursatthetemperatureof4℃topreparethemultivalentsubunitvaccine(markedasM-A+O)withtwoantigensonthesurface.
SDS-PAGEtestswereperformedonX-M,Y-A,Y-O,M-A,M-OandM-A+O,transmissionelectronmicroscopyobservationtestswereperformedonX-M,M-A,M-OandM-A+O,andimmunologicalevaluationtestswereperformedbasedonY-A,Y-O,Y-A+Y-O,M-A,M-OandM-A+O.
Characterization of 1.3FMD monovalent and bivalent subunit vaccine
1.3.1SDS-PAGE
SDS-PAGEresults(FIG.2)showedthattheconstructedX-M,Y-A,Y-O,M-A,M-OandM-A+Owerehighlypurifiedandthemolecularweightswereexpected,indicatingsuccessfulconstructionofmultivalentsubunitvaccines.
1.3.2 Transmission Electron microscope
thetransmissionelectronmicroscoperesults(FIG.3)showthatX-M,M-A,M-OandM-A+Oshowuniformsphericalnanoparticlemorphology,indicatingthatthenanoparticlesurfaceisdirectionallyloadedwithantigenandhasnoinfluenceonthemorphology.
Fmd monovalent and bivalent nanovaccine immunological assessment
BALB/c mice were divided into 6 groups: groupY-A(freeantigen),groupY-O(freeantigen),groupY-A+Y-O(simplemixtureoftwofreeantigens),groupM-A(X-McoupledY-Ananovaccine),groupM-O(X-McoupledY-Onanovaccine)andgroupM-A+O(X-MbivalentvaccinecoupledY-AandY-Osimultaneously). theantigenamountsoftheY-AgroupandtheY-OgrouparerespectivelyconsistentwiththeantigenamountscarriedbytheM-AgroupandtheM-Ogroup,andtheantigenamountsoftheY-A+Y-OgroupandtheantigenamountscarriedbytheM-A+Ogroupareconsistent. All mice were inoculated subcutaneously at week 0 and week 4, respectively. Mice were euthanized at week 8 and mouse serum and spleen were collected for subsequent analysis.
2.1 humoral immune assessment
2.1.1ELISA detection of antibody levels against different antigens of FMD in mouse serum
ELISAdetectionresults(FIG.4)showedthatM-A+OproducedhighlevelsofspecificantibodiesagainstdifferentserotypesofFMDantigenascomparedtothemixedantigengroup(Y-A+Y-O). theM-AgroupproducedhigherlevelsofspecificantibodiesthantheY-Agroup,andtheM-OgroupproducedhigherlevelsofspecificantibodiesthantheY-Ogroup.
2.1.2FCM detection of memory B cell fraction in mouse spleen
theFCMtestresults(fig.5)showthattheM-a+omultivalentvaccinestimulatedmicetoproducemorememorybcellsthanthemixedantigengroup(Y-a+y-O). theM-AgroupstimulatedmicetoproducemorememoryBcellsthantheY-Agroup,andtheM-OgroupstimulatedmicetoproducemorememoryBcellsthantheY-Ogroup.
2.2 cellular immune assessment
Analysis of different T cell populations including CD4 in the spleen of mice after different vaccinations by intracellular cytokine staining (ICCS) + And CD8 + Central memory T cells.
ICCSassay(FIG.6)showedthatM-A+OmultivalentvaccinestimulatedmicetoproducemoreCD4thanthemixedantigengroup(Y-A+Y-O) + And CD8 + Central memory T cells. M-AstimulatedmiceproducedmoreCD4thanY-Astimulatedmice + And CD8 + Central memory T cells, M-O group stimulated mice to produce more CD4 than Y-O group + And CD (compact disc)8 + Central memory T cells.
2.4 animal toxicity test
tofurtherverifythatthenovelsubunitmonovalentandmultivalentvaccinescanprotectmicefromFMDV,realFMDV(typeaandtypeO)challengeexperimentswereperformedonmicetoobservetheimmunoprotectioneffectsofM-A,M-OandM-a+oonmice.
2.4.1ELISA detection of specific antibody levels in mouse serum against different serotypes of FMD antigen
ELISAdetectionresults(FIG.7)showedthatM-A+OmultivalentvaccinestimulatedmicetoproducehighlevelsofspecificantibodiesagainstdifferentserotypesofFMDantigenascomparedtothemixedantigengroup(Y-A+Y-O). M-AgroupstimulatedmiceproducedhighlevelsofspecificantibodiestotypeAFMDantigencomparedtoY-AgroupstimulatedmiceproducedhighlevelsofspecificantibodiestotypeOFMDantigencomparedtoY-Ogroupstimulatedmice.
2.4.2 monitoring of organ involvement and survival in mice
theresultsofthemiceorganinvolvementandsurvivalmonitoring(FIG.8)showedthatthemiceoftheM-A+Omultivalentvaccinegrouphadminimalorganinvolvement,only9%and11%comparedtothemixedantigengroup(Y-A+Y-O); comparedwiththemixedantigengroup(Y-A+Y-O),thesurvivalrateofthemiceintheM-A+Omultivalentvaccinegroupishighest,ashighas95%and93%,whichshowsthatthenovelmultivalentsubunitvaccineiseffectiveinprotectingthemicefromFMDV.
In summary, by targeted loading of antigens VP1 of both serotype a and O FMDs onto the nanoparticle surface, a novel bivalent subunit vaccine against both serotypes of FMD was constructed. Compared with the mixed antigen group, the bivalent vaccine can obviously improve immune effects, including humoral immunity and cellular immunity, particularly induces specific antibody response against FMD antigens of two serotypes, and protects mice from true A-type and O-type FMDV. The novel FMDV multivalent subunit vaccine has higher market development and application values.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. The nanoparticle protein is characterized in that the amino acid sequence of the nanoparticle protein is shown as SEQ ID NO. 1.
2. Use of the nanoparticle protein of claim 1 for the preparation of a vaccine; the vaccine comprises foot-and-mouth disease vaccine, brucella nano multivalent vaccine, novel crown multivalent vaccine or african swine fever multivalent vaccine.
3. Use of the nanoparticle protein of claim 1 for improving the immune effect of foot-and-mouth disease vaccines.
4. A foot-and-mouth disease vaccine comprising the nanoparticle protein of claim 1 and an antigen characteristic of foot-and-mouth disease that is targeted to loading with the nanoparticle protein.
5. The vaccine of claim 4, wherein the foot-and-mouth disease characteristic antigen comprises type O foot-and-mouth disease characteristic antigen VP1 and/or type a foot-and-mouth disease characteristic antigen VP1.
6. The vaccine of claim 5, wherein the amino acid sequence of the antigen VP1 characteristic of type O foot-and-mouth disease is shown in SEQ ID NO. 2; the amino acid sequence of the antigen VP1 characteristic of the foot-and-mouth disease A is shown as SEQ ID NO. 3.
7. The vaccine of claim 4, wherein the directed loading is by the SpyCatcher and SpyTag system.
8. The vaccine of claim 7, wherein the nanoparticle protein is coupled to SpyCatcher and the antigen characteristic of foot-and-mouth disease is coupled to SpyTag prior to directional loading.
9. The vaccine of claim 8, wherein the nanoparticle protein coupled to SpyCatcher has an amino acid sequence shown in SEQ ID No. 4; foot-and-mouth disease characteristic antigens coupled to SpyTag include a type a foot-and-mouth disease characteristic antigen VP1 coupled to SpyTag and/or a type O foot-and-mouth disease characteristic antigen VP1 coupled to SpyTag; the amino acid sequence of the A-type foot-and-mouth disease characteristic antigen VP1 coupled with the SpyTag is shown as SEQ ID NO.5, and the amino acid sequence of the O-type foot-and-mouth disease characteristic antigen VP1 coupled with the SpyTag is shown as SEQ ID NO. 6.
10. The method for preparing the vaccine according to any one of claims 4 to 9, wherein the nanoparticle protein and the antigen characteristic of foot-and-mouth disease are directionally loaded to obtain the foot-and-mouth disease vaccine.
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