CN102127554A - Japanese encephalitis particle vaccine and preparation method and application thereof - Google Patents
Japanese encephalitis particle vaccine and preparation method and application thereof Download PDFInfo
- Publication number
- CN102127554A CN102127554A CN2010105996877A CN201010599687A CN102127554A CN 102127554 A CN102127554 A CN 102127554A CN 2010105996877 A CN2010105996877 A CN 2010105996877A CN 201010599687 A CN201010599687 A CN 201010599687A CN 102127554 A CN102127554 A CN 102127554A
- Authority
- CN
- China
- Prior art keywords
- japanese encephalitis
- virus
- vaccine
- antigen
- epitope
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000710842 Japanese encephalitis virus Species 0.000 title claims abstract description 95
- 206010014596 Encephalitis Japanese B Diseases 0.000 title claims abstract description 59
- 201000005807 Japanese encephalitis Diseases 0.000 title claims abstract description 59
- 229960005486 vaccine Drugs 0.000 title claims abstract description 55
- 239000002245 particle Substances 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title claims abstract description 5
- 239000000427 antigen Substances 0.000 claims abstract description 34
- 102000036639 antigens Human genes 0.000 claims abstract description 34
- 108091007433 antigens Proteins 0.000 claims abstract description 34
- 241000700721 Hepatitis B virus Species 0.000 claims abstract description 16
- 230000003472 neutralizing effect Effects 0.000 claims abstract description 16
- 101710132601 Capsid protein Proteins 0.000 claims abstract description 14
- 230000014509 gene expression Effects 0.000 claims abstract description 11
- 241001465754 Metazoa Species 0.000 claims abstract description 9
- 241000588724 Escherichia coli Species 0.000 claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 239000002671 adjuvant Substances 0.000 claims description 14
- 241000700605 Viruses Species 0.000 claims description 13
- 241000894006 Bacteria Species 0.000 claims description 5
- 230000009465 prokaryotic expression Effects 0.000 claims description 5
- 210000004899 c-terminal region Anatomy 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 230000000890 antigenic effect Effects 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 238000005215 recombination Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 101710091045 Envelope protein Proteins 0.000 claims description 2
- 101710188315 Protein X Proteins 0.000 claims description 2
- 229920002684 Sepharose Polymers 0.000 claims description 2
- 108010003533 Viral Envelope Proteins Proteins 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 238000005119 centrifugation Methods 0.000 claims 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims 1
- 102100021696 Syncytin-1 Human genes 0.000 claims 1
- 238000005336 cracking Methods 0.000 claims 1
- 239000012528 membrane Substances 0.000 claims 1
- 239000002244 precipitate Substances 0.000 claims 1
- 238000001308 synthesis method Methods 0.000 claims 1
- 239000002504 physiological saline solution Substances 0.000 abstract description 5
- 241000699670 Mus sp. Species 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 230000002269 spontaneous effect Effects 0.000 abstract description 3
- 239000000568 immunological adjuvant Substances 0.000 abstract description 2
- 231100000433 cytotoxic Toxicity 0.000 abstract 1
- 230000001472 cytotoxic effect Effects 0.000 abstract 1
- 210000004698 lymphocyte Anatomy 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 25
- 238000011081 inoculation Methods 0.000 description 12
- 239000002574 poison Substances 0.000 description 9
- 231100000614 poison Toxicity 0.000 description 9
- 101000589010 Homo sapiens Myomesin-1 Proteins 0.000 description 8
- 101000589013 Mus musculus Myomesin-1 Proteins 0.000 description 8
- 102100032971 Myomesin-1 Human genes 0.000 description 8
- 230000036039 immunity Effects 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 229940031567 attenuated vaccine Drugs 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 230000008676 import Effects 0.000 description 4
- 230000000968 intestinal effect Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 241000710843 Japanese encephalitis virus group Species 0.000 description 3
- 102100030810 Tumor necrosis factor receptor superfamily member EDAR Human genes 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 102220023257 rs387907546 Human genes 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 229940124726 Japanese encephalitis vaccine Drugs 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 102220369446 c.1274G>A Human genes 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000006054 immunological memory Effects 0.000 description 2
- 229940031551 inactivated vaccine Drugs 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 231100000636 lethal dose Toxicity 0.000 description 2
- 238000011330 nucleic acid test Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 102220004457 rs11567847 Human genes 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 229940031626 subunit vaccine Drugs 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000238421 Arthropoda Species 0.000 description 1
- 208000031504 Asymptomatic Infections Diseases 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 101000886418 Drosophila melanogaster GATA-binding factor C Proteins 0.000 description 1
- 102100027723 Endogenous retrovirus group K member 6 Rec protein Human genes 0.000 description 1
- 101710204837 Envelope small membrane protein Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 235000016796 Euonymus japonicus Nutrition 0.000 description 1
- 240000006570 Euonymus japonicus Species 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101710145006 Lysis protein Proteins 0.000 description 1
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101900205473 Plasmodium falciparum Circumsporozoite protein Proteins 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 241000710914 Totivirus Species 0.000 description 1
- 108010087302 Viral Structural Proteins Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005267 amalgamation Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 102220369447 c.1352G>A Human genes 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000007771 core particle Substances 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 229940124735 malaria vaccine Drugs 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000003360 nephrocyte Anatomy 0.000 description 1
- 210000004279 orbit Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000001206 photoelectron extended fine structure spectroscopy Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of biotechnology, and relates to a vaccine embedded with virus-like particles expressing multi-epitope antigen for Japanese encephalitis, and a preparation method and application thereof. The antigen of the vaccine is virus-like particles formed through spontaneous assembly of hepatitis B virus core antigen embedded with neutralizing antigen epitope expressing Japanese encephalitis virus and cytotoxic lymphocyte (CTL) antigen epitope, and is prepared through soluble expression of escherichia coli and purification. The Japanese encephalitis virus-like particle antigen is properly diluted with physiological saline, or is compatible with immunologic adjuvants to be prepared into the Japanese encephalitis particle vaccine. Animal experiments show that: the vaccine is safe and high-efficiency; mice inoculate with the vaccine generate high-level neutralizing antigen for the Japanese encephalitis virus to protect the mice against the attack of strong Japanese encephalitis virus by 100 percent.
Description
Technical field
The present invention relates to virus sample particle vaccines of a kind of chimeric expression japanese encephalitis virus multi-epitope antigen and its production and application, belong to biological technical field, be exclusively used in the immunoprophylaxis of the Japanese encephalitis of people and susceptible animal.
Background technology
Japanese encephalitis (Japanese Encephalitis, JE) be a kind of by japanese encephalitis virus (Japanese Encephalitis Virus, JEV) the infecting both domestic animals and human arthropod borne infection that causes, people and Ma present the encephalitis symptom, pig mainly shows as breeding difficulty, the most inapparent infection of other animal.This disease mainly betides south east asia, and China is one of important epidemic-stricken area of Japanese encephalitis, and annual human morbidity number is only second to India.In recent years, the Japanese encephalitis distribution range enlarges, the popular time is elongated along with global warming and variation of ecology and environment cause, and hazard rating strengthens day by day.
Though there are 5 genotype in japanese encephalitis virus, this virus has only a serotype.Especially cause of disease specificity neutralizing antibody level is consistent with the Japanese encephalitis infection level height of humans and animals for a large amount of humoral immunizatioies that studies show that, cellular immune level has determined to infect the removing ability of body to this virus.
Vaccination is one of important measures of people and susceptible animal prevention Japanese encephalitis in the epidemic-stricken area.The Japanese encephalitis vaccine that commercialization is used has two kinds, a kind of is the Japanese encephalitis inactivated vaccine, early stage is mouse brain purified virus inactivated vaccine, a few peoples have negative reaction after using, recently use Chinese hamster nephrocyte (BHK21) or African green monkey kidney cell (VERO) instead and be production matrix, generally need inoculation 2 to 3 times; Another kind is the Japanese encephalitis attenuated live vaccines; by China's biological products assay institute development; strain is JEV SA14-14-2; this vaccine one shot immunity can produce immune protective effect preferably; but because there is the anti-high wind of potential virulence danger in the weak poison of Japanese encephalitis vaccine, the World Health Organization does not ratify this vaccine so far in global widespread use.
Genetic engineering subunit vaccine since security good, be subjected to maternal antibody disturb little, can the matched reagent box distinguish advantages such as vaccine immunity and wild virus infection and have broad application prospects.Yet gene engineered subunit antigen immune originality is relatively weak, antibody response is slow, immunity system for better excitating organism, make it enough resistibilitys be arranged, be necessary to seek the immune effect of the genetic engineering subunit vaccine of method raising safely and efficiently japanese encephalitis virus.
(Virus-like particle is the protein grain of the highly structural that formed by the virus structural protein being made by manufacturers or users VLP) to virus-like particle, lacks viral nucleic acid, does not have infectious.Some VLP surfaces can be repeated high-density and be showed the exogenous antigen epi-position, thereby induce at the efficient immunne response of exogenous antigen epi-position.Recent studies show that, some VLP can effectively pass through MHC I class and MHC II classpath submission antigen, induce the CTL cell response, excite Th1 and the immune response of Th2 type, some viral VLP can induce that dendritic cell and monokaryon are huge has a liking for cell mass maturation and cytokine expression in vivo and in vitro.Therefore, using chimeric virus-like particle antigen prepd vaccine has characteristics safely and efficiently.
The cAg of people's quasi-hepatitis B virus (HBc) can spontaneous assembling assembly virus-like particle in virus infection, the nucleic acid of parcel virus.The virus-like particle of the spontaneous formation of HBc has two kinds of sizes, is respectively by 180 and 240 and the icosahedron formed of core monomer antigen.The HBc total length is 183 Aa, and wherein 75-82Aa is the antigen visualization area, shows the virus like particle surface, spike district in the middle of constituting.39 Aa of the C-terminal of HBc are rich in arginine, have the function in conjunction with packaging virus nucleic acid, do not influence HBc after the removal and form virus-like particle.The HBc(1-149Aa that blocks) can in intestinal bacteria, efficiently express, and independently be assembled into virus-like particle, HBc(1-149Aa) the middle spike district of HBc and the C latter end that blocks all are exposed to the surface of virus particle, but epitope [the Birkett A of these zone amalgamation and expression external sources, Lyons K, Schmidt A, et al. A modified hepatitis B virus core particle containing multiple epitopes of the Plasmodium falciparum circumsporozoite protein provides a highly immunogenic malaria vaccine in preclinical analyses in rodent and primate hosts. Infect Immun 2002; 70 (12): 6860 – 6870. [PubMed:12438363].Some HBc that merge external source B cell antigen epi-position or T cell antigen epitope still can be assembled into chimeric VLP, and the induction of immunity animal produces the intensive immunne response, especially exogenous antigen epi-position inductive immune effect the best of inserting in HBc molecule intermediary spike district.As a kind of novel epitope submission system, HBc-VLP successfully has been used for the development of acquired immune deficiency syndrome (AIDS), hepatitis C and malaria new generation vaccine, and the clinical second phase that enters that has is studied.
Summary of the invention
Technical problem
The purpose of this invention is to provide a kind of novel Japanese encephalitis particle vaccines.This vaccine safety is efficient, does not contain infectious viral nuclei acid, and the antigen purity height can be inoculated the protective immune response that body produces infection with Japanese ence phalitis Viruses by rapid induction.Be used for the immunoprophylaxis of people and susceptible animal, prevent the generation of Japanese encephalitis safely, efficiently Japanese encephalitis.
Technical scheme
The invention provides a kind of virus sample particle vaccines that is used to prevent Japanese encephalitis, the antigen composition of this vaccine is among the chimeric JEV and the virus-like particle of the autonomous assembling of the hepatitis B virus core antigen of epitope and CTL epitope, by bacterium coli solubility expression, purifying preparation.Japanese encephalitis virus sample particulate antigen with physiological saline suitably dilution or with the immunological adjuvant compatibility after make Japanese encephalitis particle vaccines of the present invention.
Beneficial effect
Rapid induction produces cause of disease specificity neutralizing antibody behind the Japanese encephalitis particle vaccines inoculation mouse; two exempt from the resistibility that the back mouse acquisition of 2 weeks is attacked the lethal dose japanese encephalitis virus; and demonstrate stronger immunological memory; cause of disease specificity neutralizing antibody level significantly raises, and 100% protection mouse is to the attack of the strong poison of Japanese encephalitis.
Description of drawings
Fig. 1. expression vector PET-28a-VLP-con makes up synoptic diagram
Fig. 2. expression vector PET-28a-VLP-JEV makes up synoptic diagram
Fig. 3. HBV cAg (A:VLP-con) electromicroscopic photograph (* 150,000 times)
Virus-like particle (B:VLP-JEV) electromicroscopic photograph (* 150,000 times) of Fig. 4 chimeric expression japanese encephalitis virus epitope antigen
Fig. 5. the Japanese ence phalitis Viruses EDIII protein antibodies level that Japanese encephalitis particle vaccines inoculation mouse produces
Fig. 6. the Japanese ence phalitis Viruses antibody horizontal that Japanese encephalitis particle vaccines inoculation mouse produces
Fig. 7. the neutralizing antibody level that Japanese encephalitis particle vaccines inoculation mouse produces
Fig. 8. the Japanese encephalitis particle vaccines is inoculated the malicious protection situation of attacking of mouse.
Embodiment
Experimental technique that this embodiment relates to or step are with reference to " molecular cloning experiment guide " (second edition) 1 ~ 60 page, 672 ~ 681 pages and 822 ~ 847 pages.
One, the structure of hepatitis B virus core antigen skelemin (HBc-con) prokaryotic expression carrier PET28a-VLP-Con
With reference to synthetic hepatitis B virus core antigen skelemin (HBc-con) encoding gene (its nucleotides sequence is classified Seq NO.3 as) of the artificial design of hepatitis B virus gene sequence (AY707087.1) in the GenBank database.Compare with the encoding gene of hepatitis B virus core antigen 1-149Aa, the HBc-con encoding gene has 2 artificial constructed restriction enzyme site BamH I and EcoR I at 231 and 238, and HBc-con inserts four amino acid PEFS at the 78th of HBc and 79 Aa; Add Nco I and Xho I site respectively at the two ends of hepatitis B virus core antigen skelemin (HBc-con) encoding gene, so that hepatitis B virus core antigen skelemin (HBc-con) encoding gene imports the PET-28a carrier; Hepatitis B virus core antigen skelemin (HBc-con) encoding gene is not with terminator codon, so that utilize the his label in PET-28a expression cassette downstream.Hepatitis B virus core antigen skelemin (HBc-con) encoding gene imports PET-28a through Nco I and Xho I site, obtain hepatitis B virus core antigen skelemin (HBc-con) prokaryotic expression carrier, called after PET28a-VLP-Con, sequencing result show consistent with design.
Two, the structure of virus-like particle antigen (VLP-JEV) the prokaryotic expression carrier PET28a-VLP-JEV of chimeric japanese encephalitis virus epitope
Design three pairs of primers, by recombinant PCR method, enzyme cut with the method that is connected respectively with in two on the japanese encephalitis virus E albumen and epitope and a CTL epitope import the HBc skelemin.In two and epitope "
327 SYSGSDGPCKI 337 " [Wu S-C, Lin C-W. Neutralizing peptide ligands selected from phage-displayed libraries mimic the conformational epitope on domain III of the Japanese encephalitis virus envelope protein. Virus Res 2001; 76:59-69.] and "
384 VVGRGDKQI 392 " [Seif; S. A.; Morita; K.; Matsuo; S., Hasebe, F. and Igarashi, A., Finer mapping of neutralizing epitope (s) on the C-terminal of Japanese encephalitis virus E-protein expressed in recombinant Escherichia coli system. Vaccine 1995. 13:1515-1521.] be illustrated in spike district in the middle of the antigen of HBc, promptly between the 78th and 79 Aa of HBc, the CTL epitope "
60 CYHASVTDI 68 " [Takada; K.; Masaki; H., Konishi, E.; Takahashi; M. and Kurane, I., Definition of an epitope on Japanese encephalitis virus (JEV) envelope protein recognized by JEV-specific murine CD8+ cytotoxic T lymphocytes.
Arch Virol2000. 145:523-534.] insert the C latter end of HBc.
P1:5`-CGAT
CCATGGACATTGACCCTT-3` Nco I sees seq NO.4
P2:5`-TCT
GGATCCTCGATTTTGCACGGGCCATCGCTGCCGCTATAGCTCAAGTTATTACCCAC-3` BamH I sees seq NO.5
P3:5`-ATA
GAATTCGTGGTGGGCCGTGGCGATAAACAGATCTCCCCAGCATCTAGG-3` EcoR I sees seq NO6
P4:5`-CAA
CTCGAGAACCACAGTAGTTTCC-3` Xho I sees seq NO.7
P5:5`-CGAT
CCATGGACATTGACCCTT-3` Nco I sees seq NO.8
P6:5`-CAA
CTCGAGGATATCGGTCACGCTCGCATGATAGCAAACCACAGTAGTTTCC-3` Xho I sees seq NO.9
P1 and P2 introduce among the JEV and epitope "
327SYSGSDGPCKI
337"; See seq NO.10
P3 and P4 introduce among the JEV and epitope "
384VVGRGDKQI
392"; See seq NO11
P5 and P6 introducing JEV CTL epitope "
60CYHASVTDI
68".See seq NO.12.
The condition of described PCR is 94 ℃, 5min; 94 ℃, 30sec, 50 ℃, 30sec, 72 ℃, 30sec, 30 circulations; 72 ℃, 7 min.
Recombination imports PET-28a through Nco I and Xho I site, obtains virus-like particle antigen (VLP-JEV) prokaryotic expression carrier of chimeric japanese encephalitis virus epitope, and called after PET28a-VLP-JEV is correct through sequence verification.
Three, solubility expression and the purifying of Japanese encephalitis particulate antigen in intestinal bacteria
With PET28a-VLP-Con and PET28a-VLP-JEV transformed into escherichia coli (BL21 DE3), with positive strain in the LB substratum 37 ℃ to be cultured to bacterial concentration be OD600 ≈ 0.4, in 18 ~ 20 ℃ of environment, be 0.1mM isopropylthiogalactoside (IPTG) abduction delivering 12h ~ 15h then with final concentration.Centrifugal collection thalline, ultrasonic treatment, with intestinal bacteria lysate supernatant 12000rpm after centrifugal 30 minutes, with final concentration is that 0.22 μ m filters behind 20% ammonium sulphate precipitation particulate antigen, the physiological saline solution, use Sepharose CL-4B sieve chromatography purifying, obtain particulate antigen, its aminoacid sequence is that Seq NO.1, nucleotides sequence are classified Seq NO.2 as.HBc-con and VLP-JEV can express by highly-soluble in intestinal bacteria, and independently are assembled into virus-like particle (Fig. 1)
Four, Japanese encephalitis particle vaccines mouse immune protection test
Testing program
6 age in week BALB/C mice, be divided into 5 groups, 5 every group, respectively VLP-Con, VLP-JEV and the ISA206 adjuvant emulsion VLP-JEV of the dilution of immunization physiological saline, the particulate antigen content of every mouse immune is 50 μ g; The commercially available vaccine control group is that pig is used Japanese encephalitis attenuated vaccine (veterinary biological product company limited before the section of Wuhan), and every mouse inoculation 1/10 pig is used dosage; Every mouse inoculation of blank 100 μ L physiological saline.Head exempts from back 2 all booster immunizations once, and each is organized vaccine kind and dosage and just exempts from identical.Two exempt from back 2 all immune mouses counts lethal dose (LD with 10 sesquialters
50) (the JEV NJ08 strain) intracranial inoculation of the strong poison of Japanese encephalitis attack poison, observed for 3 weeks.Before the immunization, head exempts from the back and exempts from 1 week of back 1 week, two and attack 1 week of poison back eye socket venous blood collection respectively, separation of serum detects antibody.It is envelope antigen that the ELISA detection of antibodies adopts the reorganization ED3 albumen of purifying.The detection of neutralizing antibody adopts virus (JEV SA14-14-2) plaque to reduce test, virus plaque is reduced 50% maximum serum dilution factor and is defined as NAT, gets 3 sample determinations for every group.
Test-results
ELISA antibody
The Japanese encephalitis particle vaccines immune mouse of the Japanese encephalitis particle vaccines of no adjuvant and compatibility ISA206 adjuvant has all produced the antibody response (Fig. 2 and Fig. 3) at recombination Japanese encephalitis ED3 antigen and japanese encephalitis virus.1 week behind the initial immunization, the antibody against Japanese encephalitis level that the Japanese encephalitis particle vaccines group mouse of the no adjuvant of immunity produces a little more than with the Japanese encephalitis particle vaccines of ISA206 adjuvant, but two exempt from afterwards that 1 all adjuvant group antibody against Japanese encephalitis levels are higher than no adjuvant group.It is close with the Japanese encephalitis commercialized vaccine at the antigenic antibody horizontal of ED3 that Japanese encephalitis particle vaccines immune mouse produces, but the antibody horizontal particle vaccines at totivirus is lower than commercialized vaccine, infers that there is other dominance epitope in japanese encephalitis virus.
Neutralizing antibody
Inoculation Japanese encephalitis particle vaccines and commercialization attenuated vaccine mouse have all produced japanese encephalitis virus neutralizing antibody (Fig. 4), two NATs of exempting from the Japanese encephalitis particle vaccines immune mouse of back 1 week application ISA206 adjuvant are 1:40, close with commercialized vaccine, the neutralizing antibody of no adjuvant Japanese encephalitis particle vaccines group mouse is low slightly, is 1:20.Japanese encephalitis particle vaccines inoculation mouse all is significantly increased attacking poison back viral special neutralizing antibody level of one week, shows that the Japanese encephalitis particle vaccines can induce stronger immunological memory reaction.
Attack the poison protection
Mouse is attacked malicious protection test result (Fig. 5) and shows that inoculation ISA206 adjuvant Japanese encephalitis particle vaccines and commercialization attenuated vaccine mouse are with 10 times of LD
50The strong poison of japanese encephalitis virus (JEV NJ08 strain) all do not have death, 100% immunoprotection after attacking poison; The survival rate of no adjuvant Japanese encephalitis particle vaccines immune group mouse is 70%; The particulate antigen group mouse that inoculates no Japanese encephalitis epitope is all dead.The Japanese encephalitis particle vaccines can produce good immune protection by inducing mouse; the immune protective effect that adds ISA206 adjuvant Japanese encephalitis particle vaccines is better than not having adjuvant Japanese encephalitis particle vaccines; similar to the commercialization attenuated vaccine, inductive Japanese encephalitis specificity neutralizing antibody level is more consistent after this and the vaccine inoculation.
SEQUENCE?LISTING
＜110〉Agricultural University Of Nanjing
＜120〉a kind of Japanese encephalitis particle vaccines and its production and application
<130>
<160> 12
<170> PatentIn?version?3.3
<210> 1
<211> 190
<212> PRT
＜213〉artificial sequence
<400> 1
Met?Asp?Ile?Asp?Pro?Tyr?Lys?Glu?Phe?Gly?Ala?Thr?Val?Glu?Leu?Leu
1 5 10 15
Ser?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Arg?Asp?Leu?Leu?Asp
20 25 30
Thr?Ala?Ser?Ala?Leu?Tyr?Arg?Glu?Ala?Leu?Glu?Ser?Pro?Glu?His?Cys
35 40 45
Ser?Pro?His?His?Thr?Ala?Leu?Arg?Gln?Ala?Ile?Leu?Cys?Trp?Gly?Glu
50 55 60
Leu?Met?Thr?Leu?Ala?Thr?Trp?Val?Gly?Asn?Asn?Leu?Ser?Tyr?Ser?Gly
65 70 75 80
Ser?Asp?Gly?Pro?Cys?Lys?Ile?Glu?Asp?Pro?Glu?Phe?Val?Val?Gly?Arg
85 90 95
Gly?Asp?Lys?Gln?Ile?Ser?Pro?Ala?Ser?Arg?Asp?Leu?Val?Val?Asn?Tyr
100 105 110
Val?Asn?Thr?Asn?Met?Gly?Leu?Lys?Ile?Arg?Gln?Leu?Leu?Trp?Phe?His
115 120 125
Ile?Ser?Cys?Leu?Thr?Phe?Gly?Arg?Glu?Thr?Val?Leu?Glu?Tyr?Leu?Val
130 135 140
Ser?Phe?Gly?Val?Trp?Ile?Arg?Thr?Pro?Pro?Ala?Tyr?Arg?Pro?Pro?Asn
145 150 155 160
Ala?Pro?Ile?Leu?Ser?Thr?Leu?Pro?Glu?Thr?Thr?Val?Val?Cys?Tyr?His
165 170 175
Ala?Ser?Val?Thr?Asp?Ile?Leu?Glu?His?His?His?His?His?His
180 185 190
<210> 2
<211> 573
<212> DNA
＜213〉artificial sequence
<400> 2
atggacattg?acccttataa?agaatttgga?gctactgtgg?agttactctc?gtttttgcct 60
tctgactttt?ttccttccgt?cagagatctc?ctagacaccg?cctcagctct?gtatcgggaa 120
gccttagagt?ctcctgagca?ttgctcacct?caccatactg?cactcaggca?agcaattctc 180
tgctgggggg?aattgatgac?tctagctacc?tgggtgggta?ataacttgag?ctatagcggc 240
agcgatggcc?cgtgcaaaat?cgaggatcca?gaattcgtgg?tgggccgtgg?cgataaacag 300
atctccccag?catctaggga?tctagtagtc?aattatgtta?atactaacat?gggtttaaag 360
atcaggcaac?tattgtggtt?tcatatatct?tgccttactt?ttggaagaga?gactgtactt 420
gaatatttgg?tctctttcgg?agtgtggatt?cgcactcctc?cagcctatag?accaccgaat 480
gcccctatct?tatcaacact?tccggaaact?actgtggttt?gctatcatgc?gagcgtgacc 540
gatatcctcg?agcaccacca?ccaccaccac?tga 573
<210> 3
<211> 471
<212> DNA
＜213〉artificial sequence
<400> 3
taccatggac?attgaccctt?ataaagaatt?tggagctact?gtggagttac?tctcgttttt 60
gccttctgac?ttttttcctt?ccgtcagaga?tctcctagac?accgcctcag?ctctgtatcg 120
ggaagcctta?gagtctcctg?agcattgctc?acctcaccat?actgcactca?ggcaagcaat 180
tctctgctgg?ggggaattga?tgactctagc?tacctgggtg?ggtaataact?tggaggatcc 240
agaattctcc?ccagcatcta?gggatctagt?agtcaattat?gttaatacta?acatgggttt 300
aaagatcagg?caactattgt?ggtttcatat?atcttgcctt?acttttggaa?gagagactgt 360
acttgaatat?ttggtctctt?tcggagtgtg?gattcgcact?cctccagcct?atagaccacc 420
aaatgcccct?atcttatcaa?cacttccgga?aactactgtg?gttctcgagc?a 471
<210> 4
<211> 22
<212> DNA
＜213〉artificial sequence
<400> 4
cgatccatgg?acattgaccc?tt 22
<210> 5
<211> 59
<212> DNA
＜213〉artificial sequence
<400> 5
tctggatcct?cgattttgca?cgggccatcg?ctgccgctat?agctcaagtt?attacccac 59
<210> 6
<211> 51
<212> DNA
＜213〉artificial sequence
<400> 6
atagaattcg?tggtgggccg?tggcgataaa?cagatctccc?cagcatctag?g 51
<210> 7
<211> 25
<212> DNA
＜213〉artificial sequence
<400> 7
caactcgaga?accacagtag?tttcc 25
<210> 8
<211> 22
<212> DNA
＜213〉artificial sequence
<400> 8
cgatccatgg?acattgaccc?tt 22
<210> 9
<211> 52
<212> DNA
＜213〉artificial sequence
<400> 9
caactcgagg?atatcggtca?cgctcgcatg?atagcaaacc?acagtagttt?cc 52
<210> 10
<211> 11
<212> PRT
＜213〉artificial sequence
<400> 10
Ser?Tyr?Ser?Gly?Ser?Asp?Gly?Pro?Cys?Lys?Ile
1 5 10
<210> 11
<211> 9
<212> PRT
＜213〉artificial sequence
<400> 11
Val?Val?Gly?Arg?Gly?Asp?Lys?Gln?Ile
1 5
<210> 12
<211> 9
<212> PRT
＜213〉artificial sequence
<400> 12
Cys?Tyr?His?Ala?Ser?Val?Thr?Asp?Ile
1 5
Claims (5)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105996877A CN102127554B (en) | 2010-12-22 | 2010-12-22 | Japanese encephalitis particle vaccine and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105996877A CN102127554B (en) | 2010-12-22 | 2010-12-22 | Japanese encephalitis particle vaccine and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102127554A true CN102127554A (en) | 2011-07-20 |
| CN102127554B CN102127554B (en) | 2012-07-25 |
Family
ID=44265828
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105996877A Expired - Fee Related CN102127554B (en) | 2010-12-22 | 2010-12-22 | Japanese encephalitis particle vaccine and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102127554B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102357246A (en) * | 2011-11-02 | 2012-02-22 | 江苏省中医药研究院 | EGFR and HER2 combined polypeptide epitope vaccine |
| CN105688202A (en) * | 2016-03-03 | 2016-06-22 | 四川农业大学 | Japanese encephalitis (JE) vaccine composition and preparing method thereof |
| WO2017032303A1 (en) * | 2015-08-25 | 2017-03-02 | 厦门大学 | Polypeptide carrier for presenting target polypeptide and uses thereof |
| US10987418B2 (en) | 2017-02-17 | 2021-04-27 | Xiamen University | Polypeptide carrier for presenting target polypeptide and uses thereof |
| KR20220021007A (en) * | 2013-08-28 | 2022-02-21 | 애피바디 에이비 | Binding polypeptides having a mutated scaffold |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1609204A (en) * | 2003-03-20 | 2005-04-27 | 上海天甲生物医药有限公司 | Bivalent hepatitis B-encephalitis B vaccine |
| CN101899466A (en) * | 2010-06-29 | 2010-12-01 | 中国人民解放军第三军医大学 | Dengue virus and Japanese encephalitis virus embedded pseudo virus particle vaccine and preparation method thereof |
-
2010
- 2010-12-22 CN CN2010105996877A patent/CN102127554B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1609204A (en) * | 2003-03-20 | 2005-04-27 | 上海天甲生物医药有限公司 | Bivalent hepatitis B-encephalitis B vaccine |
| CN101899466A (en) * | 2010-06-29 | 2010-12-01 | 中国人民解放军第三军医大学 | Dengue virus and Japanese encephalitis virus embedded pseudo virus particle vaccine and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 《中国人兽共患病杂志》 20020630 吴玉水 日本脑炎病毒DNA疫苗和基因工程亚单位颗粒疫苗的动物保护实验研究 p3-6 1-5 第18卷, 第6期 2 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102357246A (en) * | 2011-11-02 | 2012-02-22 | 江苏省中医药研究院 | EGFR and HER2 combined polypeptide epitope vaccine |
| KR20220021007A (en) * | 2013-08-28 | 2022-02-21 | 애피바디 에이비 | Binding polypeptides having a mutated scaffold |
| KR102497083B1 (en) | 2013-08-28 | 2023-02-07 | 애피바디 에이비 | Binding polypeptides having a mutated scaffold |
| WO2017032303A1 (en) * | 2015-08-25 | 2017-03-02 | 厦门大学 | Polypeptide carrier for presenting target polypeptide and uses thereof |
| KR20180038557A (en) * | 2015-08-25 | 2018-04-16 | 시아먼 유니버시티 | Polypeptide carriers and their uses for presenting target polypeptides |
| EP3342866A4 (en) * | 2015-08-25 | 2019-02-27 | Xiamen University | SUPPORT POLYPEPTIDE FOR THE PRESENTATION OF A TARGET POLYPEPTIDE AND USES THEREOF |
| AU2016313660B2 (en) * | 2015-08-25 | 2019-11-07 | Xiamen University | Polypeptide carrier for presenting target polypeptide and uses thereof |
| US10548973B2 (en) | 2015-08-25 | 2020-02-04 | Xiamen University | Polypeptide carrier for presenting target polypeptide and uses thereof |
| KR102116320B1 (en) * | 2015-08-25 | 2020-05-29 | 시아먼 유니버시티 | Polypeptide carriers and uses thereof for presenting target polypeptides |
| CN105688202A (en) * | 2016-03-03 | 2016-06-22 | 四川农业大学 | Japanese encephalitis (JE) vaccine composition and preparing method thereof |
| CN105688202B (en) * | 2016-03-03 | 2019-07-19 | 四川农业大学 | A kind of Japanese encephalitis vaccine composition and preparation method thereof |
| US10987418B2 (en) | 2017-02-17 | 2021-04-27 | Xiamen University | Polypeptide carrier for presenting target polypeptide and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102127554B (en) | 2012-07-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Denis et al. | Immunogenicity of papaya mosaic virus-like particles fused to a hepatitis C virus epitope: evidence for the critical function of multimerization | |
| CN113666990B (en) | T cell vaccine immunogen for inducing broad-spectrum anti-coronavirus and application thereof | |
| KR101608121B1 (en) | Method for Preparing Virus Like Particle of Nodavirus, Yeast Strain producing the Same and Vaccine Composition Comprising the Same | |
| CN102675471A (en) | Pig foot-and-mouth disease virus O-type broad spectrum multi-epitope recombination antigen and application thereof | |
| Boutier et al. | Current knowledge and future prospects of vaccines against cyprinid herpesvirus 3 (CyHV-3) | |
| CN112679584A (en) | Swine fever epitope peptide and application thereof | |
| US12226472B2 (en) | Foot-and-mouth disease virus-like particle antigen, and vaccine composition, preparation method, and application thereof | |
| CN102127554A (en) | Japanese encephalitis particle vaccine and preparation method and application thereof | |
| CN105349562B (en) | Recombinant vector expressing porcine parvovirus VP2 protein, recombinant bacterium and application thereof | |
| CN106146626B (en) | A kind of Erysipelothrix rhusiopathiae subunit vaccine and its preparation method and application | |
| CN102168088A (en) | T cell immunogen gene TI and applications thereof in foot-and-mouth disease protein subunit vaccine and inactivated vaccine | |
| CN111556896A (en) | Cross-immunization antigen vaccine and preparation method thereof | |
| CN102512671A (en) | Anti-foot-and-mouth disease type O virus-like particle vaccine and preparation method thereof | |
| CN113862284A (en) | Gene for coding recombinant avian influenza virus HA protein, virus-like particle, vaccine, preparation and application | |
| JP7515611B2 (en) | Foot-and-mouth disease virus-like particle antigens, vaccine compositions thereof, methods of preparation and uses | |
| CN114634578A (en) | Vaccine composition against novel coronavirus infection | |
| CN115044562B (en) | Recombinant rabies virus with chimeric expression molecular adjuvant and preparation method and application thereof | |
| CN114805599B (en) | VLPs based on ADDOmer chimeric porcine O-type foot-and-mouth disease virus epitope and application | |
| WO2023207717A1 (en) | Development and use of broad-spectrum vaccine for h5n8 avian influenza | |
| KR102365464B1 (en) | Development of recombinant subunit Zika virus vaccine and preparing method thereof | |
| KR102451412B1 (en) | Novel immunopotent recombinant protein with simultaneous induction of cellular and humoral immune responses and broad spectrum of protective efficacy, and foot-and-mouth disease (FMD) vaccine composition comprising the same | |
| CN105085625A (en) | Genetically engineered vaccine against both Enterovirus 71 and Coxsackie virus A16 | |
| CN102657864A (en) | Immune adjuvant of foot-and-mouth disease vaccine and application of immune adjuvant | |
| CN102212120A (en) | Salmonella paratyphi A PagC subunit vaccine and preparation method thereof | |
| CN113827714A (en) | H7N9 subtype avian influenza virus-like particle vaccine preparation and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120725 Termination date: 20131222 |