CN102028943A - Respiratory syncytial virus-like particle vaccine and preparation method thereof - Google Patents

Respiratory syncytial virus-like particle vaccine and preparation method thereof Download PDF

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CN102028943A
CN102028943A CN 201010589315 CN201010589315A CN102028943A CN 102028943 A CN102028943 A CN 102028943A CN 201010589315 CN201010589315 CN 201010589315 CN 201010589315 A CN201010589315 A CN 201010589315A CN 102028943 A CN102028943 A CN 102028943A
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rsv
ndv
vlp
cell
vaccine
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王希良
曹政
杨鹏辉
王鋮
高啸
赵忠鹏
罗德炎
段跃强
邢丽
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Institute of Microbiology and Epidemiology of AMMS
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Institute of Microbiology and Epidemiology of AMMS
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Abstract

The invention discloses a virus-like particle vaccine (NDV/RSV VLP vaccine) of which the centre is from NVD (Newcastle disease virus) and the surface protein is from RSV (respiratory syncytial virus), and also discloses a preparation method of the virus-like particle vaccine. The RSV VLP vaccine provided by the invention comprises a VLP composed of four structural proteins of respiratory syncytial virus M, F, NP and G; and the NDV/RSV VLP vaccine comprises four structural proteins of Newcastle disease virus M, F, NP and NH and VLP, wherein the VLP is formed by two surface proteins which are a NDV F/RSV F fusion protein composed of Newcastle disease virus F protein and respiratory syncytial virus F protein and an NDV HN/RSV G fusion protein composed of Newcastle disease virus HN protein and respiratory syncytial virus G protein. The test of pesticide effectiveness shows that RSV infection can be safely and effectively prevented after various dosage forms prepared by the VLP protein antigen formed with the method by adding or not adding adjuvants are used for processing immunization for different animals or the crowd, and the vaccine for processing immune prevention for the RSV inflection is supplied for the crowd with different ages.

Description

A kind of respiratory syncytial virus sample particle vaccines and preparation method thereof
Invention field
The invention belongs to field of biological pharmacy, relate to a kind of respiratory syncytial virus sample particle vaccines (RS VLP vaccine) and preparation method thereof, particularly a kind of core from Avian pneumo-encephalitis virus, surface protein from virus sample particle vaccines (NDV/RSV VLP vaccine) of syncytial virus and preparation method thereof.
Background technology
Respiratory syncytial virus (Respiratory syncytial virus, RSV), it is the one of the main reasons that has and cause the infant lower respiratory infection at world wide, rsv infection can cause a series of respiratory tract disease, from simple cold symptoms to serious lower respiratory infection and complication.The pilosity of infection first of RSV is born in 2 monthly age babies, and infection rate is up to 83%, and can not produce persistent immunity after infecting, have 50% infant can be at metainfective 1~2 year first subinfection again.Simultaneously, RSV also is a kind of common and important pathogen in teenager, old people and immunodeficiency crowd.RSV belongs to the non-segmental RNA viruses of sub-thread minus strand of Paramyxoviridae, pneumonitis virus genus, forms from host's lipid bilayer and the transmembrane protein that passes through wherein when RNA, the skin that its core is wrapped up by nucleocapsid is coated with and sprouts.The RNA of the RSV 10 kinds of protein of encoding altogether: three transmembrane proteins (G, F, SH), two stromatins (M, M2), three nucleocapsid proteins (N, P, L) and two non-structural proteins (NS1, NS2).Wherein three kinds of transmembrane glycoproteins and immunoprotection and the virulence of causing a disease are closely related, are respectively fusion rotein (F), adhesion protein (G) and little hydrophobin (SH).Studies show that anti-F antibody can stop RSV and host cell to merge, and anti-G antibody can stop the attaching of RSV to host cell, this makes F albumen and G albumen be considered to the main protection antigen of RSV.F albumen is the important protective antigen of RSV, and it is an important target site of neutrality antibody and protectiveness.Evidence suggests that simultaneously F albumen still is an important target site of CD8+T cell.G albumen has a hydrophobic N-terminal, by this N-terminal G albumen is anchored on the viral lipid peplos, according to the difference of the extracellular region protein sequence that is exposed, RSV can be divided into A and two hypotypes of B.Also there is another secreting type G albumen in G albumen simultaneously, and secreting type G albumen has lacked 65 amino acid residues of N-terminal, only stays the extracellular region of high glycosylation, there are some researches show that this secreting type segment might cause immunne response to develop to the Th2 direction.Thus, the rsv infection host to bring out the unusual natural immunity and obtain immunne response be the danger signal that cause the Th1/Th2 dysequilibrium and cause immunologic injury.
As everyone knows, vaccine is the most effective means of prevention and control infectious disease, does not also have approved vaccine to be used for the RSV immunoprophylaxis so far.In recent years studies show that, rsv infection to the inherent immunity responsibility a little less than, make that the Th2 immunne response is strong, cause the Th1/Th2 dysequilibrium, produce a large amount of inflammatory molecules and the interestization factor, and it is immature to produce neutrality antibody, causes that immunologic injury is the problem in science of RSV vaccine research.In the seventies in last century, the formalin deactivation RSV vaccine (F1-RSV vaccine) that the U.S. releases first, inoculate 2~7 months infant, having 80% infant the state of an illness to occur increases the weight of, and there are two examples dead, find that thus a large amount of oxyphil cell's infiltrations and inflammatory molecule and chemotactic factor have appearred in the dead pulmonary, make the disorder of Th1/Th2 balance.Because FI-RSV has caused too high Th2 immunne response; CD4+T cell-stimulating and some inflammatories of propagation back secretion and the interestization factor; and the generation of eosinophilic granulocyte, neutrophilic granulocyte of pulmonary and infiltration, generation low-affinity protection antibody have been quickened; CD8+T cytoactive and function have been damaged in addition; finally caused the RSV reset procedure slowly, pulmonary lesion and the virus beginning is a large amount of propagates, the F1-RSV vaccine ends in failure thus.Overcoming this several difficult points thus, is the basic principle of exploitation RSV vaccine.In recent years the weak malicious Seedling of a kind of responsive to temperature type RSV of American scholar report and the proteic recombined human of F/bovine parainfluenza virus live vaccine of a kind of RSV of carrying enter I phase clinical research, RSV recombinant subunit vaccine that the scholar carries out is arranged in the preclinical study stage, all face the potential danger of potential immunopathogenesis damage.Therefore, seeking new technical tactic, research and development RSV vaccine, is the great science prerequisite of safety, effective immune prevention and control RSV.
(Virus-like pratical VLP) forms the virion ghost that does not contain viral nucleic acid by viral primary structure albumen to virus-like particle.Because VLP size, structure and surface antigen and polypeptide epitope are almost as broad as long with real virus, so can be discerned by the host antigen presenting cells, cause immunne response and effect, and do not have potential side effect.(Virosome) compares with the virion that public's praise is arranged, and VLP is not by the live virus preparation, does not have potential danger; Compare with subunit vaccine, VLP is that a plurality of antigen proteins are formed, and has very strong immunogenicity, and the VLP vaccine is described as the new milestone of virus type vaccine development now thus.Present hepatitis B VLP vaccine (Recombivax HB, Merck), papillary tumor VLP vaccine (Gardisil, Merck) and influenza VLP vaccine go on the market, a plurality of kinds such as penta type VLP vaccine are in clinical research.There are some researches show that the VLP of RSV formation so far machine is not clear, have the scholar to utilize rsv protein to carry out VLP and form research that it is low that the result shows that oneself protein VLP forms efficient, is difficult to realize scale preparation.Thus, as using other VLP formation molecule, and carry one or more surface antigen molecule of RSV, is the growth point that innovation and development RSV forms VLP.Known to the crowd, (Newcastle Disease Virus NDV) belongs to attached myxovirus section with RSV to Avian pneumo-encephalitis virus, and main host is a birds.NDV can infect the mankind, but can be in interpersonal propagation.Form the mechanism of VLP for NDV, studied clearlyer now, the stromatin of NDV (M), nucleocapsid protein (NP), fusion rotein (F) and hemagglutinin-four kinds of primary structure albumen of neuraminic acid pheron (NH) can form pick-up rate height, Avian pneumo-encephalitis virus sample granule (ND VLP) that 26S Proteasome Structure and Function is complete.In addition, ND VLP also shows many ideal characteristics: at first, ND VLP can excite ideal body fluid and the dual immunne response of cell; Secondly, the antibody of generation mainly is sophisticated neutrality antibody, and ND VLP might become a good ideal carrier that carries the exogenous antigen molecule thus; The 3rd, NDV is a kind of poultry disease, and the mankind do not have immune background, if utilize ND VLP to carry RSV immunoprotection antigen molecule, will develop into new milestone to the RSV vaccine.
Summary of the invention
The object of the invention is to disclose a kind of respiratory syncytial virus sample particle vaccines (RS VLP vaccine), particularly core is from the respiratory syncytial virus sample particle vaccines (NDV/RSV VLP vaccine) of Avian pneumo-encephalitis virus, and the object of the invention also is to disclose the preparation method of this vaccine.
The present invention seeks to be achieved by the following scheme:
RS VLP vaccine of the present invention contains the VLP of syncytial virus M, F, four kinds of structural protein compositions of NP, G.
RS VLP vaccine of the present invention relates to following nucleotide sequence: the 1. proteic nucleotide sequence of RSV M; 2. the proteic nucleotide sequence of RSV NP; 3. the proteic nucleotide sequence of RSV G; 4. the proteic nucleotide sequence of RSV F; Wherein, described G protein nucleic acid sequence comprises from RSV A hypotype Ga and RSV B hypotype Gb.
RS VLP vaccine of the present invention relates to following aminoacid sequence: the 1. proteic aminoacid sequence of RSV M; 2. the proteic aminoacid sequence of RSV NP; 3. the proteic aminoacid sequence of RSV G; 4. the proteic aminoacid sequence of RSV F; Wherein, described G protein nucleic acid sequence comprises from RSV A hypotype Ga and RSV B hypotype Gb.
RS VLP vaccine of the present invention contains respiratory syncytial virus sample granule (RS VLP), and carries following surface protein: the 1. G albumen of RSV; 2. the F albumen of RSV; Wherein, described RSV G albumen comprises from the Ga of RSV A hypotype with from the Gb of B hypotype.
RS VLP vaccine of the present invention comprises one and carries RSV and the proteic recombinant eukaryon expression vector of NDV primary structure, chick fibroblast, mammalian cell, insect cell; Wherein, described carrier for expression of eukaryon is selected from: 1. pHWD2000; 2. pOPI3CAT; 3. pCAGGS; 4. pcDNA6/TR; 5. pCMV-HA; Described chick fibroblast is selected from: 1. UMNSAH/DF-1 cell; 2. separate 9-10 fowl in age fibroblast; Described mammalian cell is selected from: 1. VERO cell; 2. 293 cells; 3. COS cell; 4. human diploid cell; 5.; Bhk cell; 6. Chinese hamster ovary celI; 7. mdck cell; 8. Hep-G2 cell; Described insect cell is selected from: 1. Tn5 cell; 2. SF9 Cell cell.
Wherein, pHWD2000 carrier of the present invention is to be obtained by the transformation of pHW2000 carrier, comprise the pCMV promoter of removing pHW2000 carrier upstream, and replace with chicken β-actin protein promoter, insert the hCMV-IE enhancer in chicken β-actin protein promoter word upstream simultaneously, improved pCWD2000 can be in eukaryotic system efficiently expressing exogenous gene, pCWD2000 has less molecular weight simultaneously, can carry bigger exogenous genetic fragment.
NDV/RSV VLP vaccine of the present invention contains Avian pneumo-encephalitis virus M, F, four kinds of structural protein of NP, NH, and the VLP of two kinds of surface proteins compositions of NDV HN/RSV G fusion rotein of the NDV F/RSV F fusion rotein of Avian pneumo-encephalitis virus F albumen and syncystial virus F composition, newcastle disease virus HN albumen and syncytial virus G albumen composition.
NDV/RSV VLP vaccine of the present invention relates to following nucleotide sequence: the 1. proteic nucleotide sequence of NDV M; 2. the proteic nucleotide sequence of NDV NP; 3. the proteic nucleotide sequence of NDV HN; 4. the proteic nucleotide sequence of NDV F; 5. NDV HN/RSV G fusion rotein nucleotide sequence; 6. NDV F/RSV F fusion rotein nucleotide sequence; Wherein, described G protein nucleic acid sequence comprises from RSV A hypotype Ga and RSV B hypotype Gb.
NDV/RSV VLP vaccine of the present invention relates to following aminoacid sequence: the 1. proteic aminoacid sequence of NDV M; 2. the proteic aminoacid sequence of NDV NP; 3. the proteic aminoacid sequence of NDV HN; 4. the proteic aminoacid sequence of NDV F; 5. the aminoacid sequence of NDV HN/RSV G fusion rotein; 6. the aminoacid sequence of NDV F/RSV F fusion rotein; Wherein, described G protein nucleic acid sequence comprises from RSV A hypotype Ga and RSV B hypotype Gb.
NDV/RSV VLP vaccine of the present invention includes a kind of Avian pneumo-encephalitis virus core, and carries following surface protein: 1. NDV HN/RSV G fusion rotein; 2. NDV F/RSV F fusion rotein; Wherein, described RSV G albumen comprises from the Ga of RSV A hypotype with from the Gb of B hypotype.
NDV/RSV VLP vaccine of the present invention comprises one and carries RSV and the proteic recombinant eukaryon expression vector of NDV primary structure, chick fibroblast, mammalian cell, insect cell; Wherein, described carrier for expression of eukaryon is selected from: 1. pHWD2000; 2. pOPI3CAT; 3. pCAGGS; 4. pcDNA6/TR; 5. pCMV-HA; Described chick fibroblast is selected from: 1. UMNSAH/DF-1 cell; 2. separate 9-10 fowl in age fibroblast; Described mammalian cell is selected from: 1. VERO cell; 2. 293 cells; 3. COS cell; 4. human diploid cell; 5.; Bhk cell; 6. Chinese hamster ovary celI; 7. mdck cell; 8. Hep-G2 cell; Described insect cell is selected from: 1. Tn5 cell; 2. SF9 Cell cell.
Wherein, pHWD2000 carrier of the present invention is to be obtained by the transformation of pHW2000 carrier, comprise the pCMV promoter of removing pHW2000 carrier upstream, and replace with chicken β-actin protein promoter, insert the hCMV-IE enhancer in chicken β-actin protein promoter word upstream simultaneously, improved pCWD2000 can be in eukaryotic system efficiently expressing exogenous gene, pCWD2000 has less molecular weight simultaneously, can carry bigger exogenous genetic fragment.
Wherein, RS VLP vaccine of the present invention and NDV/RSV VLP vaccine can not contain adjuvant, also can contain in the following adjuvant one or more: 1. aluminum salt: aluminium hydroxide or aluminum phosphate; 2. SP01 adjuvant: form the oil-in-water adjuvant by 2% Squalene, 0.1% polyoxyethylene castor oil, 0.1% polyethers; 3. SP02 adjuvant: in the SP01 adjuvant, add a certain amount of (20~60ug) recombinant bacteria flagellins; P3BSK4 (N-palmitoyl-S-[2,3-bis (palmitoyloxy)-(2RS)-propul]-[R]-propyl-[R]-cysteinyl-[S]-seryl-[S]-(lysyl) 3-lysine); 5. hydrogel adjuvant: quaternary ammoniated chitosan, 6. rLT: reorganization thermolability enterotoxin.
RS VLP vaccine of the present invention and NDV/RSV VLP vaccine can be made into the dosage form that various clinical is suitable for, including but not limited to injection type, nasal spray type, sublingual lozenge, transdermal patch or micropin dosage form.
RS VLP vaccine of the present invention and NDV/RSV VLP vaccine comprise following several: RS VLP vaccine, NDV-HNF/RSV-GF VLP vaccine, NDV-NH/RSV-G VLP vaccine, NDV-F/RSV-F VLP vaccine, NDV-NH/RSV-G+NDV-F/RSV-F VLP vaccine.
The preparation method of RS VLP vaccine of the present invention comprises the steps:
1. make up the pHWD2000 carrier for expression of eukaryon; 2. the recombinant eukaryon expression vector that contains coding RSV M protein nucleic acid sequence; 3. the recombinant eukaryon expression vector that contains the proteic nucleotide sequence of coding RSV NP; 4. the recombinant eukaryon expression vector that contains the proteic nucleotide sequence of coding recombinant RSV incorporate G; 5. the recombinant eukaryon expression vector that contains the proteic nucleotide sequence of coding recombinant RSV incorporate F; 6. a kind of cell line that is used to express is provided, after transfection, can discharges RS VLP; 7. be provided for the method for purification RS VLP, method comprises sucrose gradient centrifugation purification process and column chromatography purification method.
The preparation method of NDV/RSV VLP vaccine of the present invention comprises the steps:
To contain the recombinant eukaryon expression vector and the common cell line that is used to express that transforms of the recombinant eukaryon expression vector of the nucleotide sequence that contains NDV-HN/RSV-G fusion rotein, NDV F/RSV F fusion rotein of the nucleotide sequence of coding NDV M, NDV NP, form the NDV-NHF/RSV-GF VLP that has the NDV core and carry RSV surface antigen G, F;
Maybe will contain the recombinant eukaryon expression vector and the common cell line that is used to express that transforms of the recombinant eukaryon expression vector of the nucleotide sequence that contains the NDV-HN/RSV-G fusion rotein of coding NDV M, NDV NP, the proteic nucleotide sequence of NDV F, form the NDV-NH/RSV-G VLP that has the NDV core and carry RSV surface antigen G;
Maybe will contain the recombinant eukaryon expression vector and the common cell line that is used to express that transforms of the recombinant eukaryon expression vector of the nucleotide sequence that contains NDV F/RSV F fusion rotein of coding NDV M, NDV NP, the proteic nucleotide sequence of NDV HN, form the NDV-F/RSV-F VLP that has the NDV core and carry RSV surface antigen F;
Wherein the described NDV-HNF/RSV-GF VLP of this method, NDV-NH/RSV-G VLP, NDV-F/RSV-F VLP and NDV-NH/RSV-G+NDV-F/RSV-F VLP all can excite the dual immunne response of body fluid and cell; Wherein said RSV G albumen comprises from RSV A hypotype Ga with from B hypotype Gb.
Wherein, this method is described is that the VLP (NDV-HNF/RSV-GF VLP, NDV-NH/RSV-G VLP, NDV-F/RSV-F VLP) of core comprises with NDV: 1. NDV M albumen; 2. NDV NP albumen; 3. NDV NH albumen; 4. NDV F albumen; 5. the proteic fusion rotein NDV-F/RSV-F of NDV F albumen born of the same parents and RSV F; 6. NDV HN albumen and the proteic fusion rotein NDV of RSV G HN/RSV G.
The preparation method of NDV/RSV VLP vaccine of the present invention can also be following steps:
1. make up the pHWD2000 carrier for expression of eukaryon; 2. the recombinant eukaryon expression vector that contains coding NDV M protein nucleic acid sequence; 3. the recombinant eukaryon expression vector that contains the proteic nucleotide sequence of coding NDV NP; 4. the recombinant eukaryon expression vector that contains the proteic nucleotide sequence of coding reorganization NDV NH; 5. the recombinant eukaryon expression vector that contains the proteic nucleotide sequence of coding reorganization NDV F; 6. the recombinant eukaryon expression vector that contains the nucleotide sequence of coding reorganization NDV HN/RSV G fusion rotein; 7. the recombinant eukaryon expression vector that contains the nucleotide sequence of coding reorganization NDV F/RSV F fusion rotein; 8. a kind of cell line that is used to express is provided, after transfection, can high efficiency discharges NDV/RSV VLP; 9. be provided for the method for purification NDV/RSV VLP, method comprises sucrose gradient centrifugation purification process and column chromatography purification method.
Wherein, the method that makes up the recombinant eukaryon expression vector that contains reorganization NDV NH/RSV G and NDV F/RSV F protein nucleic acid sequence comprises the steps: 1. to pass through bioinformatics, analyzes NDV F and the proteic intracellular region of HN and strides film district nucleotide sequence; 2. by bioinformatics, analyze RSV F and the proteic extracellular region nucleotide sequence of G; 3. by the synthetic method of gene synthetic reorganization HN/G and F/F protein nucleic acid sequence; 4. on this method basis, utilize enzyme action and the means that are connected to make up and contain coding reorganization NDV NH/RSV G and the proteic carrier for expression of eukaryon of NDV F/RSV F.
Wherein, make up the method for recombinant expression carrier, comprise the steps: coding RSV M albumen, F albumen, NP albumen, the proteic dna segment of G are inserted in the pHWD2000 carrier, constitute reorganization pHWD2000-M, pHWD2000-F, pHWD2000-NP, pHWD2000-G expression vector; In another scheme, comprise with coding NDV M albumen, NP albumen, F albumen, HN albumen, reorganization F/F albumen, and the proteic dna segment of reorganization NH/G be inserted in the pHWD2000 carrier, constitute reorganization pHWD2000-M, pHWD2000-NP, pHWD2000-F, pHWD2000-HN, pHWD2000-F/F, pHWD2000-NH/G expression vector; Described reorganization NH is NDV-NH/RSV-G, i.e. pHWD2000-NH/G; Described reorganization F is NDV-F/RSV-F, i.e. pHWD2000-F/F.
Wherein, the inventive method relates to a RS VLP and NDV/RSV VLP expression host cell system, and wherein, institute's phalangeal cell is chick fibroblast or mammalian cell, and chick fibroblast has: 1. UMNSAH/DF-1 cell; 2. separate 9-10 fowl in age fibroblast; Mammalian cell is selected from: 1. VERO cell; 2. 293 cells; 3. COS cell; 4. human diploid cell; 5. bhk cell; 6. Chinese hamster ovary celI; 7. mdck cell; 8. Hep-G2 cell; 9. Tn5 cell; 10. SF9 Cell cell; Wherein, described NDV/RSV VLP expression host cell system comprises NDV-HNF/RSV-GF VLP, NDV-NH/RSV-G VLP, and NDV-F/RSV-F VLP.
Wherein, the present invention prepares RS VLP and NDV/RSV VLP cell culture processes comprises the steps: that adding concentration in cell culture medium is the glycosaminoglycans of 2-100 μ g/mL, preferred 10 μ g/mL; Wherein, the indication glycosaminoglycans is heparin sulfate, chondroitin sulfate B, sulfated hyaluronic acid or heparin, preferred heparin.
Wherein, the purification process when the present invention prepares RS VLP and NDV/RSV VLP comprises the steps: sucrose gradient centrifugation, and sucrose concentration is 30%, 45% and 60%; Or the column chromatography purification method, with the Sepharose6FFTM molecular sieve and carry out DEAE-SepharoseFFTM ion exchange.
Wherein, the step of sucrose gradient centrifugation is: 1. collect the RS VLP or the NDV/RSV VLP cell culture fluid of large-scale production, 4 ℃ of centrifugal 10min of 5000rmp remove cell and impurity; 2. get 4 ℃ of centrifugal 2h of supernatant 100000g, precipitation suspends with TNE buffer, and TNE buffer consists of 25mM Tris-HCl pH 7.4,150mM NaCl, 5mM EDTA; 3. suspension is carried out sucrose gradient centrifugation, sucrose adopts TNE buffer preparation, and system adopts 30% sucrose 1mL; 45% sucrose 1mL; 60% sucrose 4mL; 4 ℃ of centrifugal 2h of 100000g; 4. collect the intermediate layer, and use the refractometer density measurement; 5. the PBS balance Sepharose6FFTM medium with pH6.5-7.5 carries out gel chromatography, collects void volume stream and wears the peak; Foreign protein is removed and can be reached more than 95%; 6. adopt the DEAE-SepharoseFFTM medium to carry out ion-exchange chromatography, balance liquid is pH6.5-7.5, contains the PBS of 0.05-0.15M sodium chloride that eluent is the PBS that contains 0.2-0.5M sodium chloride, pH6.5-7.5; Foreign protein is removed and can be reached more than 99%, and the VLP stock solution behind the purification is vaccinogen liquid.
Percentage composition described in product of the present invention and the method is the quality volumn concentration.
The present invention relates to a kind of respiratory syncytial virus sample particle vaccines, comprise following several: RS VLP vaccine, NDV-HNF/RSV-GF VLP vaccine, NDV-NH/RSV-G VLP vaccine, NDV-F/RSV-F VLP vaccine or NDV-NH/RSV-G+NDV-F/RSV-F VLP vaccine.Add or do not add adjuvant through the VLP of this formation proteantigen, behind the injection type of preparation, nasal spray type, sublingual lozenge dosage form, transdermal dosage form immunity different animals or the crowd, can safely, effectively prevent rsv infection, for different age people immunity prevention and control rsv infection provides vaccine.Through effect experiment, humoral immunization and the cellullar immunologic response and the natural immunity that respiratory syncytial virus sample particle vaccines of the present invention is produced by approach immunity Balb/C mice, the responsive animals of Rhesus Macacus such as injection, spray nose, sublingual lozenge, transdermals respectively and acquired immunity is replied and the mucosal immune response effect, and the Th2/Th1 immunne response tends to balance, do not have the sign of immune injury of lung, have safety.With twice of respiratory syncytial virus sample particle vaccines immunity cotton mouse, the responsive animal of Rhesus Macacus; further by RSV wild strain counteracting toxic substances, all A type type strain RSV A2, Type B type strain G8537 and the clinical separation A in different regions and the popular wild strain of Type B RSV there is immune protective effect more than 90%.With respiratory syncytial virus sample particle vaccines inoculation all ages and classes section (below 2 years old, 3-17 year, 18-59 the year, more than 60 years old) crowd, twice of immunity, adopt injection, spray nose, sublingual lozenge, transdermal dosage form vaccine immunity respectively, demonstration discomfort do not occur to the each age group crowd, has safety.And can produce good dual immunne response and immune protective efficiency, its protective rate reaches more than 85%, shows that thus this vaccine has good immune protective effect to different age people.
Following experimental example and embodiment are used to further specify but are not limited to the present invention.
Experimental example 1 RS VLP vaccine of the present invention and NDV/RSV VLP vaccine calibrating experiment
The RS VLP vaccine of the embodiment of the invention 8 preparations and injection, nasal spray, sublingual lozenge, the transdermal dosage form outward appearance of NDV/RSV VLP vaccine are seen homogeneous, free from extraneous odour and microbiological contamination; By Cavia porcellus, rabbit test apyrogeneity, undue toxicity and acute toxic reaction; Measure protein content all at dosage range by the Lorry method; By the about 140-160 μ of electron microscopic observation VLP diameter m; ELISA measures dna content all below 50EU; Detecting the antigen protein composition by SDS-PAGE and WB exists; By 1 serum NAT of immune Balb/C mice more than 3200.
The intravital immunne response effect experiment of the Balb/C mice of experimental example 2 RS VLP vaccines of the present invention and NDV/RSV VLP vaccine
The injection, spray nose, sublingual lozenge, the transdermal vaccine dosage form that RS VLP vaccine, the NDV/RSV VLP vaccine of the embodiment of the invention 8 preparations are added or do not add corresponding adjuvant preparation, and be matched group with PBS, aluminum salt, SP01, SP02, P3BSK4, rLT, bacterial flagellum, press approach immunity 5-6 week Balb/C mice separately respectively, its immunizing dose is pressed embodiment 8 vaccine low dose group, twice (0,21 day) of immunity; The last immunity was plucked eyeball with each group mice in back 14 days and is got blood, separation of serum and immunocyte, gathered splenocyte, lung and nasal cavity irrigating solution; Measure serum antibody titer all more than 1: 6400 by the ELISA method, adopt mtt assay to measure NAT all more than 1: 3200, dynamic changes such as important cytokine, inflammatory molecule, chemotactic factor and eosinophilic granulocyte, neutrophilic granulocyte make TH2/Th1 reply balance in employing flow cytometer, ELISPOT instrument mensuration immunocyte and the serum, adopt the ELISA method to measure nasal cavity, lung-douching fluid, the SIgA antibody titer is all more than 1: 160.This explanation RS VLP vaccine of the present invention and NDV/RSV VLP vaccine add adjuvant and do not have adjuvant all can both produce satisfied dual immunne response no matter be, and present that test group has adjuvant to be better than not having the adjuvant group, high dose group is higher than low dose group immunne response effect, and feminine gender is not produced dual immunne response according to group; RS VLP vaccine of the present invention and NDV/RSV VLP vaccine are no matter be that injection or spray nose, sublingual lozenge, transdermal patch, micropin dosage form all can both produce satisfied dual immunne response, and the injection serum antibody titer is higher than micropin dosage form, spray nose, sublingual lozenge, transdermal patch type, and spray nose, sublingual lozenge, micropin dosage form mucosa and cell immunoreceptor are better than injection type; NDV-HNF/RSV-GF VLP vaccine immune response of the present invention is renderd a service suitable with RS VLP vaccine, but is better than NDV-NH/RSV-G VLP vaccine, NDV-F/RSV-F VLP vaccine, NDV-F/RSV-F VLP+NDV-NH/RSV-G VLP vaccine.Zhi Bei RS VLP vaccine and NDV/RSV VLP vaccine have good immunne response effect in the mice body thus.
The intravital immune protective effect experiment of the cotton mouse of experimental example 3 RS VLP vaccines of the present invention and NDV/RSV VLP vaccine
The injection, spray nose, sublingual lozenge, the transdermal vaccine dosage form that RS VLP vaccine, the NDV/RSV VLP vaccine of the embodiment of the invention 8 preparations are added or do not add corresponding adjuvant preparation are test group, and be matched group with PBS, aluminum salt, SP01, SP02, P3BSK4, rLT, bacterial flagellum and F1-RSV inactivated vaccine, press approach immunity 6-8 week cotton mouse separately respectively, its immunizing dose is pressed embodiment 8 vaccine low dose group, twice (0 of immunity, 21 days), back 14 days each test group of last immunity, control group mice are with 3 * 10 6The clinical separation RSV of pfu representative strains (A, Type B) aerosol nasal cavity counteracting toxic substances is respectively observed the protection effect.The result shows that RS VLP vaccine of the present invention and NDV/RSV VLP vaccine all can both produce the better protect effect; immune protective rate is more than 90%; and RS VLP vaccine protective rate reaches 98%, NDV-HNF/RSV-GF VLP vaccine protective rate reaches 97%, NDV-HN/RSV-G+NDV-F/RSV-F VLP vaccine protective rate reaches 96%, NDV-NH/RSV-G VLP vaccine protective rate reaches 94%, NDV-F/RSV-F VLP vaccine protective rate reaches 92%, is 0% and matched group FI-RSV inactivated vaccine protective rate reaches 60%, other each matched group protective rates reach.Zhi Bei RS VLP and NDV NH/RSV VLP vaccine have immune protective effect efficiently in the mice body thus.
The intravital immunne response of Rhesus Macacus of experimental example 4 RS VLP vaccines of the present invention and NDV/RSV VLP vaccine and immune protective effect experiment
The injection, spray nose, sublingual lozenge, the transdermal vaccine dosage form that RS VLP vaccine, the NDV/RSV VLP vaccine of the embodiment of the invention 8 preparations are added or do not add corresponding adjuvant preparation, and be matched group with PBS, aluminum salt, SP01, SP02, P3BSK4, rLT, bacterial flagellum and FI-RSV inactivated vaccine, press 3 years old Rhesus Macacus of approach immunity separately respectively, its immunizing dose is pressed embodiment 8 vaccine high dose group, twice (0,21 day) of immunity; The last immunity was plucked eyeball with each group mice in back 14 days and is got blood, separation of serum and immunocyte, gathered splenocyte, lung and nasal cavity irrigating solution; Measure serum antibody titer all more than 12800 by the ELISA method, adopt mtt assay to measure NAT all more than 6400, adopt dynamic changes such as flow cytometer, ELISPOT instrument mensuration immunocyte and serum important cytokine, inflammatory molecule, the interestization factor and eosinophilic granulocyte, neutrophilic granulocyte to make TH2/Th1 reply balance, adopt ELISA method nasal cavity, lung-douching fluid SIgA antibody titer more than 1: 200.This explanation NDV-NH/RSV VLP vaccine of the present invention adds adjuvant and does not have adjuvant all can both produce satisfied dual immunne response no matter be, and test group has the adjuvant group to be better than no adjuvant group, high dose is higher than low dose group immunne response effect, and matched group does not produce dual immunne response; RS VLP vaccine of the present invention and NDV/RSV VLP vaccine are no matter be that injection or spray nose, sublingual lozenge, transdermal patch, micropin dosage form all can both produce satisfied dual immunne response, and the injection serum antibody titer is higher than micropin dosage form, spray nose, sublingual lozenge, transdermal patch type, and spray nose, sublingual lozenge, micropin dosage form mucosa and cell immunoreceptor are better than injection type; NDV-HNF/RSV-GF VLP vaccine immune response of the present invention is renderd a service and is better than RSV VLP vaccine, NDV-NH/RSV-G+NDV-F/RSV-F VLP vaccine, NDV-NH/RSV-G VLP vaccine, NDV-F/RSV-F VLP vaccine.Zhi Bei RS VLP vaccine and NDV/RSV VLP vaccine have good immunne response effect in the Rhesus Macacus body thus.
The immunity of respiratory syncytial virus sample particle vaccines last back 14 days each test group, matched group Rhesus Macacus are observed the protection effect with the clinical separation RSV of 2 * 107pfu representative strains (A, Type B) difference aerosol nasal cavity counteracting toxic substances.The result shows; RS VLP vaccine of the present invention and NDV/RSV VLP vaccine all can both produce high-caliber protection effect; immune protective rate is more than 90%, and RS VLP vaccine and NDV-HNF/RSVGF VLP vaccine protective rate reach 100%, NDV-F/RSV-F+NDV-NH/RSV-G VLP vaccine protective rate reaches 98%, NDV-NH/RSV-G VLP vaccine protective rate reaches 97%, NDV-F/RSV-F VLP vaccine protective rate reaches 96% and matched group FI-RSV inactivated vaccine protective rate reaches 68%, other each matched group protective rates reach is 0%.Zhi Bei NDV NH/RSV VLP vaccine has immune protective effect efficiently in the Rhesus Macacus body thus.
The intragroup immunne response of people of experimental example 5 RS VLP vaccines of the present invention and NDV/RSV VLP vaccine and immune protective effect experiment
The RS VLP vaccine of embodiment 8 preparations, NDV/RSV VLP vaccine adds or does not add the injection of corresponding adjuvant, the spray nose, sublingual lozenge, transdermal vaccine dosage form, and use PBS, aluminum salt, SP01, SP02, P3BSK4, rLT, bacterial flagellum is a matched group, inoculate healthy different age people (below 2 years old by approach separately respectively, 2-17 year, 18-59 year, more than 60 years old) group, below 2 years old, 2-17 year age group immunizing dose is pressed embodiment 8 vaccine low dosages, 18-59 year, the age group immunizing dose is pressed embodiment 8 vaccine high doses more than 60 years old, inoculate twice (0,21 days), the last immunity was gathered and is respectively organized crowd's venous blood in back 14 days, separation of serum and immunocyte, gather the nasal cavity irrigating solution, and omnidistance viewing test vaccine group crowd changes of vital signs.Measure serum antibody titer more than 3200 by the ELISA method, adopt mtt assay to measure NAT more than 1: 1600, adopt dynamic change TH2/Th1 such as flow cytometer, ELISPOT instrument mensuration immunocyte and serum important cytokine, inflammatory molecule, chemotactic factor and eosinophilic granulocyte, neutrophilic granulocyte to reply and tend to balance, adopt ELISA method nasal cavity irrigating solution SIgA antibody titer more than 1: 160; Overall process is observed 90 days reaction symptoms such as no significant discomfort of vaccination crowd; Follow up a case by regular visits to its vaccine by on-the-spot epidemiology through 360 days tracking good immune protective effect is arranged; its protective rate reaches more than 85%; and RS VLP and NDV-HNF/RSV-GF VLP vaccine protective rate all reach 96%, NDV-NH/RSV-G+NDV-F/RSV-F VLP vaccine protective rate reaches 94%, NDV-NH/RSV-G VLP vaccine protective rate reaches 92%, NDV-F/RSV-F VLP vaccine protective rate reach 88% and each matched group protective rate to reach be 12%.Zhi Bei RS VLP vaccine and NDV/RSV VLP vaccine are no matter be that injection or spray nose or sublingual lozenge or transdermal immune are seeded in good immunne response and immune protective effect are arranged in the different age people thus.
The safety experiment of experimental example 6 RS VLP vaccines of the present invention and NDV/RSV VLP vaccine
(1) aseptic, mycoplasma test: with RS VLP vaccine, NDV/RSV VLP vaccination sulphur glycollate culture medium, nutrient agar slant medium and the improvement Martin culture medium culturing 14d of embodiment 8 preparations, and do negative control with physiological saline solution, cultivation temperature is 25 ℃, 35 ℃.The result shows that RS VLP vaccine, NDV/RSV VLP two class vaccines are not all seen bacterial growth.RS VLP vaccine and NDV/RSV VLP vaccine are used semifluid and broth bouillon respectively, 37 ℃ of initial culture 21 days, inferior being commissioned to train supported 21 days, and physiological saline solution is done negative control, and the result shows that RS VLP and NDV/RSV VLP two class vaccines all do not have the mycoplasma growth; With the DNA staining seed culture of viruses is inoculated the Vero cell culture 3 days, go down to posterity once, use the dibenzamide fluorescent dyeing.The result shows that RS VLP and NDV/RSV VLP two class vaccines all do not have the mycoplasma growth.
(2) hemolytic test: choose body weight and be the Cavia porcellus about 350g, gather fresh guinea pig blood 1ml,, again blood cell volume is recovered and dilute 10 times with PBS washing 3 times.PBS dilution RS VLP and NDV/RSV VLP vaccine (by embodiment 8 preparations), be respectively 2 times, 4 times, 8 times, the guinea pig blood cell joined in the adjuvant to be checked of dilution, after 8 hours, estimate haemocytolysis and be as the criterion, and detect absorbance at the 570nm place with range estimation or the detection of supernatant concentration.The result shows, blood cell does not take place break, no haemolysis.Illustrate that the composition in RS VLP and the NDV/RSV VLP vaccine can not make erythrocyte splitting.Therefore, RS VLP and NDV/RSV VLP two class vaccines all do not have hemolytic reaction.
(3) acute toxicity test: the RS VLP and the NDV/RSV VLP vaccine 0.5ml lumbar injection body weight of getting embodiment 8 preparations are 12~18g Balb/C mice, every group 10, establish the PBS negative control group simultaneously, active state, body weight change and survival rate that continuous 2 weeks are observed mice.The result shows, experiment mice is all survived, ill symptoms such as perpendicular hair, lethargy do not occur, be slow in action, and body weight presents increase, prove that thus RS VLP and NDV/RSV VLP two class vaccines are safe to animal under the concentration of test, and put to death after 14 days and carry out the gross anatomy inspection, do not see that internal organs have pathological change.In body weight is the intravital acute toxicity result of Beagle Canis familiaris L. of 8~10kg: the RS VLP and the NDV/RSV VLP vaccine intramuscular injection 15mL that get embodiment 8 preparations, every group 10, establish the PBS negative control group simultaneously, continuous 2 all observed behaviors, body weight and survival rate change.The result as seen, the Beagle Canis familiaris L. does not see toxic reaction, behavior is normal, does not have death, with matched group Canis familiaris L. zero difference relatively, each Canis familiaris L. body weight increases to some extent, and puts to death gross anatomy and do not see that internal organs have tangible pathological change.Therefore, RS VLP and NDV/RSV VLP two class vaccines all do not have acute toxic reaction, and use is safe.
(4) hypersensitive test research: the RS VLP and the NDV/RSV VLP vaccine subcutaneous vaccination body weight respectively of getting embodiment 8 preparation are 250~350g Hartley Cavia porcellus, 5 of each sample inoculation Cavia porcelluss, every inoculation 0.5ml, the next day once, totally 3 times.Back 21 days of the 3rd injection, ear vein gives identical RS VLP and NDV/RSV VLP vaccine 0.5ml respectively, and inoculates 3 Cavia porcelluss respectively as positive, negative control with human albumin and normal saline with same method.Inject and observed animal in back 30 minutes and 3 days, positive, negative control is all set up, and RS VLP and NDV/RSV VLP two class vaccine group Cavia porcelluss do not have death, and do not have allergic symptoms such as rhinocnesmus, sneeze, dysphoria, dyspnea, shock, spasm.Therefore, RS VLP and NDV/RSV VLP two class vaccines all do not have irritated reaction in animal body.
(5) rabbit thermal source matter test: getting the qualified body weight of preliminary examination is that 3 of 2~3kg rabbit are fixing, and take temperature after 30 minutes is surveyed 2 times altogether, 30 minutes at interval, and require 2 temperature difference to be not more than 0.2 ℃, 2 mean temperatures of each rabbit are at 38.6-39.5 ℃.The RS VLP and the NDV/RSV VLP vaccine of embodiment 8 preparations are preheated to 38 ℃, and in 15 minutes, oneself rabbit ear limit vein only slowly injects 0.5ml/ respectively behind the 2nd thermometric.Injection back is every 30 minutes take temperatures 1 time, tie-in 6 times.The result shows: RS VLP and NDV/RSV VLP vaccine give individual intensification of rabbit and all do not surpass 0.2 ℃, and 3 rabbit intensification summations do not surpass 0.4 ℃, do not cause the exothermic reaction of rabbit.Therefore, the RS VLP of preparation and NDV/RSV VLP two class vaccines all do not have thermal source matter.
(6) immunopathogenesis damage test: RS VLP and NDV/RSV VLP vaccine by embodiment 8 preparations pass through detections such as mice, Rhesus Macacus and crowd's peripheral blood antibody subtype, the interestization factor, inflammatory factor, eosinophilic granulocyte, neutrophilic granulocyte, basophil and trachea, lungs, liver, spleen, kidney.Result of the test shows that no matter RS VLP and NDV/RSV VLP two class vaccine virus immunization are at big toy, or the Th2/Th1 immunne response tends to balance in human body, does not have the sign of immune organ damage, therefore has safety.
The stability experiment of experimental example 7 RS VLP vaccines of the present invention and NDV/RSV VLP vaccine
RS VLP and NDV/RSV VLP vaccine by embodiment 8 preparations, place 2-8 ℃, room temperature (20-25 ℃), 37 ℃ of 1 week, 2 weeks, 1 month, 3 months, 6 months, 12 months, 18 months and 24 months, sampling to observe outward appearance, pH value, aseptic, electron microscopic observation particle diameter respectively, immune animal is observed safety.The result shows: RS VLP and NDV/RSV VLP vaccine are placed 2-8 ℃ does not all have phenomenon such as variable color layering in 24 months, pH value no change between 7.0-7.2, and the electron microscopic observation size is consistent, and injection or collunarium or transdermal act normally for the Balb/c mice; RS VLP and NDV/RSVVLP vaccine are placed 25 ℃ of room temperatures all has good stability in 3 months; RS VLP and NDV/RSV VLP vaccine are placed 37 ℃ of room temperatures all has good stabilizing effect in 1 month.By presentation of results, RS VLP and NDV/RSV VLP two class vaccines are placed 2-8 ℃ of physicochemical property, biology performance is stable, at least 24 months effect duration.
Following embodiment all can realize the described effect of above-mentioned experimental example.
The specific embodiment
Embodiment 1: the gene of respiratory syncytial virus sample particle vaccines of the present invention and albumen constitute
F protein nucleic acid sequence and protein sequence with the HN protein nucleic acid sequence of the NP protein nucleic acid sequence of the M protein nucleic acid sequence of the proteic nucleic acid of G of the proteic nucleic acid of F of the proteic nucleic acid of NP of the M protein nucleic acid of RSV and protein sequence, RSV and protein sequence, RSV and protein sequence, RSV and protein sequence, NDV and protein sequence, NDV and protein sequence, NDV and protein sequence, NDV see Table shown in the 1-20.
The expression vector establishment of embodiment 2. respiratory syncytial virus sample particle vaccines of the present invention
To make up reorganization pHWD2000 expression vector is example: utilize method amplification RSV M gene white nucleus acid sequence, F protein gene sequence, G protein gene sequence, the NP protein gene sequence of PCR and be inserted in the pHWD2000 carrier formation reorganization pHWD2000-M, pHWD2000-F, pHWD2000-G, pHWD2000-NP expression vector.NDVM gene white nucleus acid sequence, F protein gene sequence, NP protein gene sequence and be inserted in the pHWD2000 carrier in another scheme constitute reorganization pHWD2000-M, pHWD2000-F, pHWD2000-NP expression vector.
PHWD2000-NH/G expression vector establishment: pass through bioinformatics method, analyze NDV HN protein gene sequence intracellular region (CT) and stride film district (TM), analyze RSV G protein gene sequence extracellular region simultaneously, by the synthetic reorganization of the synthetic method of gene HN/G protein gene sequence, utilize enzyme action and the method that is connected to make up recombinant expression carrier pHWD2000-NH/G then, and to comprise Bsa I enzyme action be a little.
PHWD2000-F/F expression vector establishment: pass through bioinformatics method, analyze NDV F protein gene sequence intracellular region (CT) and stride film district (TM), analyze RSV F protein gene sequence extracellular region simultaneously, by the synthetic reorganization of the synthetic method of gene F/F protein gene sequence, utilize enzyme action and the method that is connected to make up recombinant expression carrier pHWD2000-F/F then, and to comprise Bsa I enzyme action be a little.
Embodiment 3. cell culture and cell are criticized the storehouse and are set up
With the UMNSAH/DF-1 cell culture and to build the storehouse be example: the UMNSAH/DF-1 cell derives from American Type Culture Collection (ATCC).Culture medium adopts Dulbecco ' s modified Eagle medium (DMEM), add penicillin and streptomycin, not adding or add a certain amount of hyclone (10-2%) cultivates at cell factory or cell fermentation jar, set up cell work seed lot storehouse, and reach calibrating comprehensively such as the foreign aid source factor, oncogenicity and stability etc. after going down to posterity, cell uses algebraically to be controlled in 60 generations, all meets the requirement of production of vaccine medium.
Described cell culture and cell are criticized the storehouse and are set up and to include but not limited to: 1. UMNSAH/DF-1 cell; 2. human diploid cell; 3. Vero cell; 4. Chinese hamster ovary celI; 5. Hep-2 cell; 6. mdck cell.
Embodiment 4: plasmid transfection reaches to be stablized the screening of high expressing cell system and builds the storehouse
RS VLP vaccine: select UMNSAH/DF-1 cell in embodiment 3 cell banks for use, the expression plasmid transfection UMNSAH/DF-1 cell that adopts liposome method that embodiment 2 is made up, and the cell line of high expressed RS VLP is stablized in screening, specifically: 1. in the 35mm hole, add pHWD2000-NP 1.0 μ g, pHWD2000-M 1.0 μ g, pHWD2000-G 1.0 μ g, pHWD2000-F 0.75 μ g; 2. the liposome that adds 5 μ L OptiMEM configuration, incubated at room 45min.Join then by in the host cell of OptiMEM rinse, hatch 5h; 3. centrifugal removal carrier-liposome mixture, and adding DMEM cultivates in cell; 4. pHWD2000-M, pHWD2000-F, four kinds of recombinant vectors of pHWD2000-NP, pHWD2000-G coexpression in cell line, but self assembly RS VLP; 5. express the UMNSAH/DF-1 cell strain of RS VLP by the pressurization screening.The result shows, screens the cell strain of stablize high expressed RS VLP during 23 generations at the UMNSAH/DF-1 cell, through cell line stably express and output, VLP is big or small and structure, exogenous factor and oncogenicity, chromosome integrity etc. were passaged to for 40 generations and do not have phenotypic alternation.Thus, set up the UMNSAH/DF-1 cell line of stable high table RS VLP.Further set up three grades of cell seed lot storehouses,, preserve standby in the placement liquid nitrogen through comprehensive calibrating is met the production of vaccine requirement with RS VLP.
NDV/RSV VLP vaccine: select UMNSAH/DF-1 cell in embodiment 3 cell banks for use, the expression plasmid transfection UMNSAH/DF-1 cell that adopts liposome method that embodiment 2 is made up, and the cell line of high expressed NDV/RSV VLP is stablized in screening, specifically: 1. in the 35mm hole, add pHWD2000-NP 1.0 μ g, pHWD2000-M 1.0 μ g, pHWD2000-NH 0.75 μ g, pHWD2000-F 0.75 μ g, pHWD2000-F/F 0.75 μ g; 2. pHWD2000-NH/G 0.75 μ g adds the liposome of 5 μ LOptiMEM configuration, incubated at room 45min.Join then by in the host cell of OptiMEM rinse, hatch 5h; 3. centrifugal removal carrier-liposome mixture, and adding DMEM cultivates in cell; 4. pHWD2000-M, pHWD2000-NP, respectively with pHWD2000-F/F and pHWD2000-NH/G or pHWD2000-F and pHWD2000-NH/G or pHWD2000-F/F and pHWD2000-NH, recombinant vector coexpression in cell line, but self assembly NDV-NHF/RSV-GF VLP, NDV-NH/RSV-G VLP, NDV-F/RSV-F VLP; 5. express the UMNSAH/DF-1 cell strain of NDV/RSV VLP by the pressurization screening.The result shows, just screen the cell strain of stablizing high expressed NDV/RSVVLP during 21 generations at the UMNSAH/DF-1 cell, being passaged to for 40 generations through cell line stably express and output, VLP size and structure, exogenous factor and oncogenicity, chromosome integrity etc. does not have phenotypic alternation.Thus, set up the UMNSAH/DF-1 cell line of stable high table NDV/RSV VLP.Further set up three grades of cell seed lot storehouses,, preserve standby in the placement liquid nitrogen through comprehensive calibrating is met the production of vaccine requirement with NDV/RSV-G VLP.
The cell line of described stable high table RS VLP and NDV/RSV VLP includes but not limited to: 1. UMNSAH/DF-1 cell; 2. human diploid cell; 3. Vero cell; 4. Chinese hamster ovary celI; 5. Hep-2 cell; 6. mdck cell.
Embodiment 5:RS VLP or NDV/RSV VLP large-scale production
Get the UMNSAH/DF-1 work batch storehouse cell of stably express RS VLP or NDV/RSV VLP, make cell density reach 1 * 10 by 20L cell factory or the production of adding microcarrier 30L cell fermentation jar amplification culture with DMEM cell culture fluid (not adding or add the heparin of 10-2% hyclone, adding 10 μ g/mL) 7-8When above, collecting cell is cultivated RS VLP or NDV/RSV VLP vaccinogen liquid.
RS VLP or NDV/RSV VLP large-scale method for producing comprise following method:
1. human diploid cell: the storehouse cell is criticized in the diploid cell work of getting stably express RS VLP or NDV/RSV VLP, by 20L cell factory amplification culture during to suitable cell density, collecting cell is cultivated RS VLP or NDV/RSV VLP vaccinogen liquid with DMEM cell culture fluid (do not add or add the 10-2% hyclone, add the heparin of 10 μ g/mL).
2. Vero cell: the Vero cell work of getting stably express RS VLP or NDV/RSV VLP is criticized the storehouse cell, by 20L cell factory or the 30L cell fermentation jar amplification culture that adds microcarrier during to suitable cell density, collecting cell is cultivated RS VLP or NDV/RSV VLP vaccinogen liquid with DMEM cell culture fluid (do not add or add the 2-10% hyclone, add the heparin of 10 μ g/mL).
3. Chinese hamster ovary celI: the storehouse cell is criticized in the Chinese hamster ovary celI work of getting stably express RS VLP or NDV/RSV VLP, by 20L cell factory or the 30L cell fermentation jar amplification culture that adds microcarrier during to suitable cell density, collecting cell is cultivated RS VLP or NDV/RSV VLP vaccinogen liquid with DMEM cell culture fluid (do not add or add the 2-10% hyclone, add the heparin of 10 μ g/mL).
4. Hep-2 cell: the Hep-2 cell work of getting stably express RS VLP or NDV/RSV VLP is criticized the storehouse cell, by 20L cell factory or the 30L cell fermentation jar amplification culture that adds microcarrier during to suitable cell density, collecting cell is cultivated RS VLP or NDV/RSV VLP vaccinogen liquid with DMEM cell culture fluid (do not add or add the 2-10% hyclone, add the heparin of 10 μ g/mL).
5. mdck cell: the storehouse cell is criticized in the mdck cell work of getting stably express RS VLP or NDV/RSV VLP, by 20L cell factory or the 30L cell fermentation jar amplification culture that adds microcarrier during to suitable cell density, collecting cell is cultivated RS VLP or NDV/RSV VLP vaccinogen liquid with DMEM cell culture fluid (do not add or add the 2-10% hyclone, add the heparin of 10 μ g/mL).
The purification of embodiment 6:RS VLP or NDV/RSV VLP vaccine
1. collect the RS VLP or the NDV/RSV VLP cell culture fluid of large-scale production, 4 ℃ of centrifugal 10min of 5000rmp remove cell and impurity; 2. get 4 ℃ of centrifugal 2h of supernatant 100000g, precipitation suspends with a small amount of TNE buffer (25mM Tris-HCl pH 7.4,150mM NaCl, 5mM EDTA).3. suspension is carried out sucrose gradient centrifugation, sucrose adopts TNE buffer preparation, and system adopts 20% sucrose 3.5mL; 65% sucrose 0.5mL.4 ℃ of centrifugal 2h of 100000g; 4. collect the intermediate layer, and use the refractometer density measurement; 5. the PBS balance Sepharose6FFTM medium with pH6.5-7.5 carries out gel chromatography, collects void volume stream and wears the peak.Foreign protein is removed and can be reached more than 95%; 6. adopt the DEAE-SepharoseFFTM medium to carry out ion-exchange chromatography, balance liquid is pH6.5-7.5, contains the PBS of 0.05-0.15M sodium chloride that eluent is the PBS that contains 0.2-0.5M sodium chloride, pH6.5-7.5.Foreign protein is removed and can be reached more than 99%, and the VLP stock solution behind the purification is vaccinogen liquid.
The calibrating of embodiment 7:RS VLP or NDV/RSV VLP vaccinogen liquid
The outward appearance of RS VLP and NDV/RSV VLP stock solution is a supernatant liquid; PH value is 7.0-7.2; Negative by the blood plate dientification of bacteria result that mixes; Negative by semifluid and broth bouillon cultivation carrying out mycoplasma qualification result; Show that the purpose band is clear, do not have other assorted bands by PAGE-SDS electrophoresis detection result; Adopt Reed﹠amp; The neutralization of Muench method calculating antiserum is tired and all is higher than 1: 2800.
Embodiment 8:RS VLP or the preparation of NDV/RSV VLP vaccine dosage form
1. injection type preparation: purification RS VLP or NDV/RSV VLP are not added adjuvant, and preparation 10ug/0.5ml, 20ug/1.0ml do not have the adjuvant injection type; Purification RS VLP or NDV/RSV VLP albumen are added aluminium hydroxide or aluminum phosphate configuration 7.5ug (albumen)/0.5mg (aluminum salt)/0.5ml, 15 μ g (albumen)/1.0mg (aluminum salt)/0.5ml have the Adjuvanted vaccines dosage form; Purification RS VLP or NDV/RSV VLP albumen are added SP01 adjuvant (2% Squalene, 0.1% polyoxyethylene castor oil, 0.1% polyethers), SP02 adjuvant (2% Squalene, 0.1% polyoxyethylene castor oil, 0.1% polyethers, 10ug bacterial flagellum), SP03 adjuvant (2% Squalene, 0.1% polyoxyethylene castor oil, 0.1% polyethers, 10ug P3BSK4) mixing oil-in-water sample, and preparing 5.0ug/0.5ml, 10ug/1.0ml respectively has adjuvant injection respiratory syncytial virus sample particle vaccines dosage form.Above vaccinate solution is packed into and is inhaled the top bottle, put 2-8 ℃ standby.
2. nasal spray type preparation: purification RS VLP or NDV/RSV VLP are not added adjuvant absorption, and preparation 7.5ug μ g/0.2ml, 15ug/0.2ml do not have adjuvant spray nose vaccine dosage form; Purification RS VLP or NDV/RSV VLP albumen are added equal-volume Quaterisation chitosan hydrogel mucosa adjuvant and transmission system, and preparing 5.0 μ g/0.2ml, 10 μ g/0.2ml has adjuvant spray nose vaccine dosage form; Purification RS VLP or NDV/RSV VLP albumen are added equal-volume Quaterisation chitosan hydrogel and 10 μ g bacterial flagellums or 10 μ g P3BSK4 mucosa adjuvant and transmission systems, and preparing 4.0 μ g/0.2ml, 8 μ g/0.2ml has adjuvant spray nasal respiration road syncytial virus sample particle vaccines dosage form.The above spray nose vaccine solution quantitative nose sprayer of packing into, put 2-8 ℃ standby.
3. sublingual lozenge dosage form preparation: purification RS VLP or NDV/RSV VLP 10 μ g, 20 μ g are added 0.5% gelatin, 5% sucrose, 0.2% vitamin C, 0.1% oryzanol and 0.1% defatted milk powder respectively, transfer PH7.2 with phosphate buffer, the tabletting lyophilization is prepared 10 μ g/ sheets, 20 μ g/ sheets do not have adjuvant sublingual lozenge dosage form; Purification RS VLP or NDV/RSV VLP albumen 7.5 μ g, 15 μ g are added 0.5% gelatin, 5% sucrose, 0.2% vitamin C, 0.1% oryzanol and 0.1% defatted milk powder respectively with 15 μ gP3BSK4 or Flagellin, transfer PH7.2 with phosphate buffer, the tabletting lyophilization is prepared 7.5 μ g/ sheets, 15 μ g/ sheets have adjuvant respiratory syncytial virus sample particle vaccines sublingual lozenge dosage form; Above sublingual lozenge vaccine pack into the sealing plate in, put 2-8 ℃ standby.
4. transdermal patch dosage form preparation: purification RS VLP or NDV/RSV VLP 75 μ g, 100 μ g are added lipoid C respectively, dispose 75 μ g/ pasters, 100 μ g/ pasters do not have adjuvant transdermal patch dosage form; Purification RS VLP or NDV/RSV VLP albumen 50 μ g, 100 μ g are added oleic acid penetrating agent and LT adjuvant respectively, dispose 50 μ g/ pasters, 100 μ g/ pasters have adjuvant sublingual lozenge transdermal patch dosage form.Put 2-8 ℃ standby.
5. transdermal micro needle dosage form preparation: purification RS VLP or NDV/RSV VLP are not added adjuvant, prepare 5 μ g/0.5ml, 10 μ g/ micropin agent do not have the adjuvant dosage form; Purification RS VLP or NDV/RSV VLP albumen are added SP01 adjuvant (2% Squalene, 0.1% polyoxyethylene castor oil, 0.1% polyethers) or SP02 adjuvant (2% Squalene, 0.1% polyoxyethylene castor oil, 0.1% polyethers, 10 μ g bacterial flagellums) or SP03 adjuvant (2% Squalene, 0.1% polyoxyethylene castor oil, 0.1% polyethers, 10ug P3BSK4) mixing oil-in-water sample, prepare 3.0 μ g/ micropin agent respectively, 5 μ g/ agent have adjuvant sublingual lozenge micropin agent dosage form.Put 2-8 ℃ standby.
Figure ISA00000386805200011
Figure ISA00000386805200031
Figure ISA00000386805200061
Figure ISA00000386805200071
Figure ISA00000386805200081
Figure ISA00000386805200091
Figure ISA00000386805200101
Figure ISA00000386805200111
Figure ISA00000386805200121
Figure ISA00000386805200131
Figure ISA00000386805200141

Claims (34)

1. a RS VLP vaccine is characterized in that this vaccine contains the VLP of syncytial virus M, F, four kinds of structural protein compositions of NP, G.
2. RS VLP vaccine as claimed in claim 1 is characterized in that this vaccine relates to following nucleotide sequence: the 1. proteic nucleotide sequence of RSVM; 2. the proteic nucleotide sequence of RSV NP; 3. the proteic nucleotide sequence of RSV G; 4. the proteic nucleotide sequence of RSV F; Wherein, described G protein nucleic acid sequence comprises from RSV A hypotype Ga and RSV B hypotype Gb.
3. RS VLP vaccine as claimed in claim 1 is characterized in that this vaccine relates to following aminoacid sequence: the 1. proteic aminoacid sequence of RSV M; 2. the proteic aminoacid sequence of RSV NP; 3. the proteic aminoacid sequence of RSV G; 4. the proteic aminoacid sequence of RSV F; Wherein, described G protein nucleic acid sequence comprises from RSV A hypotype Ga and RSV B hypotype Gb.
4. as the arbitrary described RS VLP vaccine of claim 1-3, it is characterized in that this vaccine contains respiratory syncytial virus sample granule (RS VLP), and carry following surface protein: the 1. G albumen of RSV; 2. the F albumen of RSV; Wherein, described RSV G albumen comprises from the Ga of RSV A hypotype with from the Gb of B hypotype.
5. as the arbitrary RS VLP vaccine of stating of claim 1-3, it is characterized in that this vaccine comprises one and carries RSV and the proteic recombinant eukaryon expression vector of NDV primary structure, chick fibroblast, mammalian cell, insect cell; Wherein, described carrier for expression of eukaryon is selected from: 1. pHWD2000; 2. pOPI3CAT; 3. pCAGGS; 4. pcDNA6/TR; 5. pCMV-HA; Described chick fibroblast is selected from: 1. UMNSAH/DF-1 cell; 2. separate 9-10 fowl in age fibroblast; Described mammalian cell is selected from: 1. VERO cell; 2. 293 cells; 3. COS cell; 4. human diploid cell; 5.; Bhk cell; 6. Chinese hamster ovary celI; 7. mdck cell; 8. Hep-G2 cell; Described insect cell is selected from: 1. Tn5 cell; 2. SF9 Cell cell.
6. RS VLP vaccine as claimed in claim 5, it is characterized in that wherein said pHWD2000 carrier is to be obtained by the transformation of pHW2000 carrier, comprise the pCMV promoter of removing pHW2000 carrier upstream, and replace with chicken β-actin protein promoter, insert the hCMV-IE enhancer in chicken β-actin protein promoter word upstream simultaneously, improved pCWD2000 can be in eukaryotic system efficiently expressing exogenous gene, pCWD2000 has less molecular weight simultaneously, can carry bigger exogenous genetic fragment.
7. as the arbitrary described RS VLP vaccine of claim 1-6, it is characterized in that this vaccine does not contain adjuvant, or contain in the following adjuvant one or more: 1. aluminum salt: aluminium hydroxide or aluminum phosphate; 2. SP01 adjuvant: form the oil-in-water adjuvant by 2% Squalene, 0.1% polyoxyethylene castor oil, 0.1% polyethers; 3. SP02 adjuvant: in the SP01 adjuvant, add a certain amount of (20~60ug) recombinant bacteria flagellins; P3BSK4 (N-palmitoyl-S-[2,3-bis (palmitoyloxy)-(2RS)-propul]-[R]-propyl-[R]-cysteinyl-[S]-seryl-[S]-(lysyl) 3-lysine); 5. hydrogel adjuvant: quaternary ammoniated chitosan, 6. rLT: reorganization thermolability enterotoxin.
8. as the arbitrary described RS VLP vaccine of claim 1-6, it is characterized in that this vaccine makes the dosage form that various clinical is suitable for: injection type, nasal spray type, sublingual lozenge, transdermal patch or micropin dosage form.
9. as the arbitrary described RS VLP vaccine of claim 1-6, it is following several to it is characterized in that this vaccine comprises: RS VLP vaccine, NDV-HNF/RSV-GF VLP vaccine, NDV-NH/RSV-G VLP vaccine, NDV-F/RSV-F VLP vaccine, NDV-NH/RSV-G+NDV-F/RSV-F VLP vaccine.
10. as the preparation method of the arbitrary described RS VLP vaccine of claim 1-6, it is characterized in that this method comprises the steps:
1. make up the pHWD2000 carrier for expression of eukaryon; 2. the recombinant eukaryon expression vector that contains coding RSV M protein nucleic acid sequence; 3. the recombinant eukaryon expression vector that contains the proteic nucleotide sequence of coding RSV NP; 4. the recombinant eukaryon expression vector that contains the proteic nucleotide sequence of coding recombinant RSV incorporate G; 5. the recombinant eukaryon expression vector that contains the proteic nucleotide sequence of coding recombinant RSV incorporate F; 6. a kind of cell line that is used to express is provided, after transfection, can discharges RS VLP; 7. be provided for the method for purification RS VLP, method comprises sucrose gradient centrifugation purification process and column chromatography purification method.
11. the preparation method of RS VLP vaccine as claimed in claim 10, the method that it is characterized in that wherein making up the recombinant eukaryon expression vector that contains recombinant RSV incorporate G and RSV F protein nucleic acid sequence comprises the steps: 1. to pass through bioinformatics, analyzes NDV F and the proteic intracellular region of HN and strides film district nucleotide sequence; 2. by bioinformatics, analyze RSV F and the proteic extracellular region nucleotide sequence of G; 3. by the synthetic method of gene synthetic reorganization HN/G and F/F protein nucleic acid sequence; 4. on this method basis, utilize enzyme action and the means that are connected to make up and contain coding reorganization NDV NH/RSV G and the proteic carrier for expression of eukaryon of NDV F/RSV F.
12. the preparation method of RS VLP vaccine as claimed in claim 10, it is characterized in that the method that wherein makes up recombinant expression carrier comprises the steps: coding RSV M albumen, F albumen, NP albumen, the proteic dna segment of G are inserted in the pHWD2000 carrier, constitute reorganization pHWD2000-M, pHWD2000-F, pHWD2000-NP, pHWD2000-G expression vector; In another scheme, comprise with coding NDV M albumen, NP albumen, F albumen, HN albumen, reorganization F/F albumen, and the proteic dna segment of reorganization NH/G be inserted in the pHWD2000 carrier, constitute reorganization pHWD2000-M, pHWD2000-NP, pHWD2000-F, pHWD2000-HN, pHWD2000-F/F, pHWD2000-NH/G expression vector; Described reorganization NH is NDV-NH/RSV-G, i.e. pHWD2000-NH/G; Described reorganization F is NDV-F/RSV-F, i.e. pHWD2000-F/F.
13. the preparation method of RS VLP vaccine as claimed in claim 10 is characterized in that institute's phalangeal cell is in the wherein said expression host cell system: chick fibroblast or mammalian cell, chick fibroblast has: 1. UMNSAH/DF-1 cell; 2. separate 9-10 fowl in age fibroblast; Mammalian cell is selected from: 1. VERO cell; 2. 293 cells; 3. COS cell; 4. human diploid cell; 5. bhk cell; 6. Chinese hamster ovary celI; 7. mdck cell; 8. Hep-G2 cell; 9. Tn5 cell; 10. SF9Cell cell.
14. the preparation method of RS VLP vaccine as claimed in claim 10 is characterized in that wherein cell culture processes comprises the steps: that adding concentration in cell culture medium is the glycosaminoglycans of 2-100 μ g/mL; Wherein, the indication glycosaminoglycans is heparin sulfate, chondroitin sulfate B, sulfated hyaluronic acid or heparin.
15. the preparation method of RS VLP vaccine as claimed in claim 10 is characterized in that wherein said purification process comprises the steps: sucrose gradient centrifugation, sucrose concentration is 30%, 45% and 60%; Or the column chromatography purification method, with the Sepharose6FFTM molecular sieve and carry out DEAE-SepharoseFFTM ion exchange.
16. the preparation method of RS VLP vaccine as claimed in claim 15 is characterized in that the step of wherein said sucrose gradient centrifugation is: 1. collect the RS VLP cell culture fluid of large-scale production, 4 ℃ of centrifugal 10min of 5000rmp remove cell and impurity; 2. get 4 ℃ of centrifugal 2h of supernatant 100000g, precipitation suspends with TNE buffer, and TNE buffer consists of 25mM Tris-HCl pH 7.4,150mM NaCl, 5mM EDTA; 3. suspension is carried out sucrose gradient centrifugation, sucrose adopts TNE buffer preparation, and system adopts 30% sucrose 1mL; 45% sucrose 1mL; 60% sucrose 4mL; 4 ℃ of centrifugal 2h of 100000g; 4. collect the intermediate layer, and use the refractometer density measurement; 5. the PBS balance Sepharose6FFTM medium with pH6.5-7.5 carries out gel chromatography, collects void volume stream and wears the peak; Foreign protein is removed and can be reached more than 95%; 6. adopt the DEAE-SepharoseFFTM medium to carry out ion-exchange chromatography, balance liquid is pH6.5-7.5, contains the PBS of 0.05-0.15M sodium chloride that eluent is the PBS that contains 0.2-0.5M sodium chloride, pH6.5-7.5; Foreign protein is removed and can be reached more than 99%, and the VLP stock solution behind the purification is vaccinogen liquid.
17. NDV/RSV VLP vaccine, it is characterized in that this vaccine contains Avian pneumo-encephalitis virus M, F, four kinds of structural protein of NP, NH, and the VLP of two kinds of surface proteins compositions of NDV HN/RSV G fusion rotein of the NDV F/RSV F fusion rotein of Avian pneumo-encephalitis virus F albumen and syncystial virus F composition, newcastle disease virus HN albumen and syncytial virus G albumen composition.
18. NDV/RSV VLP vaccine as claimed in claim 17 is characterized in that this vaccine relates to following nucleotide sequence: the 1. proteic nucleotide sequence of NDV M; 2. the proteic nucleotide sequence of NDV NP; 3. the proteic nucleotide sequence of NDV HN; 4. the proteic nucleotide sequence of NDVF; 5. NDV HN/RSV G fusion rotein nucleotide sequence; 6. NDV F/RSV F fusion rotein nucleotide sequence; Wherein, described G protein nucleic acid sequence comprises from RSV A hypotype Ga and RSV B hypotype Gb.
19. NDV/RSV VLP vaccine as claimed in claim 17 is characterized in that this vaccine relates to following aminoacid sequence: the 1. proteic aminoacid sequence of NDV M; 2. the proteic aminoacid sequence of NDV NP; 3. the proteic aminoacid sequence of NDV HN; 4. the proteic aminoacid sequence of NDV F; 5. the aminoacid sequence of NDV HN/RSV G fusion rotein; 6. the aminoacid sequence of NDV F/RSVF fusion rotein; Wherein, described G protein nucleic acid sequence comprises from RSV A hypotype Ga and RSV B hypotype Gb.
20., it is characterized in that this vaccine includes a kind of Avian pneumo-encephalitis virus core, and carry following surface protein: 1. NDV HN/RSV G fusion rotein as the arbitrary described NDV/RSV VLP vaccine of claim 17-19; 2. NDV F/RSV F fusion rotein; Wherein, described RSV G albumen comprises from the Ga of RSV A hypotype with from the Gb of B hypotype.
21., it is characterized in that this vaccine comprises one and carries RSV and the proteic recombinant eukaryon expression vector of NDV primary structure, chick fibroblast, mammalian cell, insect cell as the arbitrary described NDV/RSV VLP vaccine of claim 17-19; Wherein, described carrier for expression of eukaryon is selected from: 1. pHWD2000; 2. pOPI3CAT; 3. pCAGGS; 4. pcDNA6/TR; 5. pCMV-HA; Described chick fibroblast is selected from: 1. UMNSAH/DF-1 cell; 2. separate 9-10 fowl in age fibroblast; Described mammalian cell is selected from: 1. VERO cell; 2. 293 cells; 3. COS cell; 4. human diploid cell; 5.; Bhk cell; 6. Chinese hamster ovary celI; 7. mdck cell; 8. Hep-G2 cell; Described insect cell is selected from: 1. Tn5 cell; 2. SF9 Cell cell.
22. NDV/RSV VLP vaccine as claimed in claim 21, pHWD2000 carrier described in it is characterized in that altogether is to be obtained by the transformation of pHW2000 carrier, comprise the pCMV promoter of removing pHW2000 carrier upstream, and replace with chicken β-actin protein promoter, insert the hCMV-IE enhancer in chicken β-actin protein promoter word upstream simultaneously, improved pCWD2000 can be in eukaryotic system efficiently expressing exogenous gene, pCWD2000 has less molecular weight simultaneously, can carry bigger exogenous genetic fragment.
23., it is characterized in that this vaccine does not contain adjuvant, or contain in the following adjuvant one or more: 1. aluminum salt: aluminium hydroxide or aluminum phosphate as the arbitrary described NDV/RSV VLP vaccine of claim 17-22; 2. SP01 adjuvant: form the oil-in-water adjuvant by 2% Squalene, 0.1% polyoxyethylene castor oil, 0.1% polyethers; 3. SP02 adjuvant: in the SP01 adjuvant, add a certain amount of (20~60ug) recombinant bacteria flagellins;
P3BSK4 (N-palmitoyl-S-[2,3-bis (palmitoyloxy)-(2RS)-propul]-[R]-propyl-[R]-cysteinyl-[S]-seryl-[S]-(lysyl) 3-lysine); 5. hydrogel adjuvant: quaternary ammoniated chitosan, 6. rLT: reorganization thermolability enterotoxin.
24., it is characterized in that this vaccine makes the dosage form that various clinical is suitable for: injection type, nasal spray type, sublingual lozenge, transdermal patch or micropin dosage form as the arbitrary described NDV/RSV VLP vaccine of claim 17-22.
25. as the arbitrary described NDV/RSV VLP vaccine of claim 17-22, it is following several to it is characterized in that this vaccine comprises: RS VLP vaccine, NDV-HNF/RSV-GF VLP vaccine, NDV-NH/RSV-G VLP vaccine, NDV-F/RSV-F VLP vaccine, NDV-NH/RSV-G+NDV-F/RSV-F VLP vaccine.
26., it is characterized in that this method comprises the steps: as the preparation method of the arbitrary described NDV/RSV VLP vaccine of claim 17-22
To contain the recombinant eukaryon expression vector and the common cell line that is used to express that transforms of the recombinant eukaryon expression vector of the nucleotide sequence that contains NDV-HN/RSV-G fusion rotein, NDV F/RSV F fusion rotein of the nucleotide sequence of coding NDV M, NDV NP, form the NDV-NHF/RSV-GF VLP that has the NDV core and carry RSV surface antigen G, F;
Maybe will contain the recombinant eukaryon expression vector and the common cell line that is used to express that transforms of the recombinant eukaryon expression vector of the nucleotide sequence that contains the NDV-HN/RSV-G fusion rotein of coding NDV M, NDV NP, the proteic nucleotide sequence of NDV F, form the NDV-NH/RSV-G VLP that has the NDV core and carry RSV surface antigen G;
Maybe will contain the recombinant eukaryon expression vector and the common cell line that is used to express that transforms of the recombinant eukaryon expression vector of the nucleotide sequence that contains NDV F/RSV F fusion rotein of coding NDV M, NDV NP, the proteic nucleotide sequence of NDV HN, form the NDV-F/RSV-F VLP that has the NDV core and carry RSV surface antigen F;
Wherein the described NDV-HNF/RSV-GF VLP of this method, NDV-NH/RSV-G VLP, NDV-F/RSV-F VLP and NDV-NH/RSV-G+NDV-F/RSV-F VLP all can excite the dual immunne response of body fluid and cell; Wherein said RSV G albumen comprises from RSV A hypotype Ga with from B hypotype Gb.
27. the preparation method of NDV/RSV VLP vaccine as claimed in claim 26 is characterized in that wherein this method is described and with NDV is that the VLP (NDV-HNF/RSV-GF VLP, NDV-NH/RSV-G VLP, NDV-F/RSV-F VLP) of core comprises: 1. NDV M albumen; 2. NDV NP albumen; 3. NDV NH albumen; 4. NDV F albumen; 5. the proteic fusion rotein NDV-F/RSV-F of NDV F albumen born of the same parents and RSV F; 6. NDV HN albumen and the proteic fusion rotein NDV of RSV G HN/RSVG.
28., it is characterized in that wherein this method comprises the steps: as the preparation method of the arbitrary described NDV/RSV VLP vaccine of claim 17-22
1. make up the pHWD2000 carrier for expression of eukaryon; 2. the recombinant eukaryon expression vector that contains coding NDV M protein nucleic acid sequence; 3. the recombinant eukaryon expression vector that contains the proteic nucleotide sequence of coding NDV NP; 4. the recombinant eukaryon expression vector that contains the proteic nucleotide sequence of coding reorganization NDV NH; 5. the recombinant eukaryon expression vector that contains the proteic nucleotide sequence of coding reorganization NDV F; 6. the recombinant eukaryon expression vector that contains the nucleotide sequence of coding reorganization NDV HN/RSV G fusion rotein; 7. the recombinant eukaryon expression vector that contains the nucleotide sequence of coding reorganization NDV F/RSV F fusion rotein; 8. a kind of cell line that is used to express is provided, after transfection, can high efficiency discharges NDV/RSV VLP; 9. be provided for the method for purification NDV/RSV VLP, method comprises sucrose gradient centrifugation purification process and column chromatography purification method.
29. the preparation method of NDV/RSV VLP vaccine as claimed in claim 28, the method that it is characterized in that wherein making up the recombinant eukaryon expression vector that contains reorganization NDV NH and NDV F protein nucleic acid sequence comprises the steps: 1. to pass through bioinformatics, analyzes NDV F and the proteic intracellular region of HN and strides film district nucleotide sequence; 2. by bioinformatics, analyze RSV F and the proteic extracellular region nucleotide sequence of G; 3. by the synthetic method of gene synthetic reorganization HN/G and F/F protein nucleic acid sequence; 4. on this method basis, utilize enzyme action and the means that are connected to make up and contain coding reorganization NDV NH/RSV G and the proteic carrier for expression of eukaryon of NDV F/RSV F.
30. the preparation method of NDV/RSV VLP vaccine as claimed in claim 28, it is characterized in that wherein making up the method for recombinant expression carrier, comprise the steps: coding RSV M albumen, F albumen, NP albumen, the proteic dna segment of G are inserted in the pHWD2000 carrier, constitute reorganization pHWD2000-M, pHWD2000-F, pHWD2000-NP, pHWD2000-G expression vector; In another scheme, comprise with coding NDV M albumen, NP albumen, F albumen, HN albumen, reorganization F/F albumen, and the proteic dna segment of reorganization NH/G be inserted in the pHWD2000 carrier, constitute reorganization pHWD2000-M, pHWD2000-NP, pHWD2000-F, pHWD2000-HN, pHWD2000-F/F, pHWD2000-NH/G expression vector; Described reorganization NH is NDV-NH/RSV-G, i.e. pHWD2000-NH/G; Described reorganization F is NDV-F/RSV-F, i.e. pHWD2000-F/F.
31. the preparation method of NDV/RSV VLP vaccine as claimed in claim 28 is characterized in that institute's phalangeal cell is in the wherein said expression host cell system: chick fibroblast or mammalian cell, chick fibroblast has: 1. UMNSAH/DF-1 cell; 2. separate 9-10 fowl in age fibroblast; Mammalian cell is selected from: 1. VERO cell; 2. 293 cells; 3. COS cell; 4. human diploid cell; 5. bhk cell; 6. Chinese hamster ovary celI; 7. mdck cell; 8. Hep-G2 cell; 9. Tn5 cell; 10. SF9Cell cell; Wherein, described NDV/RSV VLP expression host cell system comprises NDV-HNF/RSV-GF VLP, NDV-NH/RSV-G VLP, and NDV-F/RSV-F VLP.
32. the preparation method of NDV/RSV VLP vaccine as claimed in claim 28 is characterized in that wherein cell culture processes comprises the steps: that adding concentration in cell culture medium is the glycosaminoglycans of 2-100 μ g/mL; Wherein, the indication glycosaminoglycans is heparin sulfate, chondroitin sulfate B, sulfated hyaluronic acid or heparin.
33. the preparation method of NDV/RSV VLP vaccine as claimed in claim 28 is characterized in that wherein purification process comprises the steps: sucrose gradient centrifugation, sucrose concentration is 30%, 45% and 60%; Or the column chromatography purification method, with the Sepharose6FFTM molecular sieve and carry out DEAE-SepharoseFFTM ion exchange.
34. the preparation method of NDV/RSV VLP vaccine as claimed in claim 28 is characterized in that wherein the step of sucrose gradient centrifugation is: 1. collect the NDV/RSV VLP cell culture fluid of large-scale production, 4 ℃ of centrifugal 10min of 5000rmp remove cell and impurity; 2. get 4 ℃ of centrifugal 2h of supernatant 100000g, precipitation suspends with TNE buffer, and TNE buffer consists of 25mM Tris-HCl pH 7.4,150mM NaCl, 5mM EDTA; 3. suspension is carried out sucrose gradient centrifugation, sucrose adopts TNE buffer preparation, and system adopts 30% sucrose 1mL; 45% sucrose 1mL; 60% sucrose 4mL; 4 ℃ of centrifugal 2h of 100000g; 4. collect the intermediate layer, and use the refractometer density measurement; 5. the PBS balance Sepharose6FFTM medium with pH6.5-7.5 carries out gel chromatography, collects void volume stream and wears the peak; Foreign protein is removed and can be reached more than 95%; 6. adopt the DEAE-SepharoseFFTM medium to carry out ion-exchange chromatography, balance liquid is pH6.5-7.5, contains the PBS of 0.05-0.15M sodium chloride that eluent is the PBS that contains 0.2-0.5M sodium chloride, pH6.5-7.5; Foreign protein is removed and can be reached more than 99%, and the VLP stock solution behind the purification is vaccinogen liquid.
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CN103642761A (en) * 2013-12-10 2014-03-19 北京交通大学 Respiratory syncytial virus-like particles and preparation method and application thereof
CN104293741A (en) * 2014-10-10 2015-01-21 武汉大学 Respiratory syncytial virus virus-like particle vaccine as well as preparation method and application thereof
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CN114685676A (en) * 2020-12-28 2022-07-01 兰州生物制品研究所有限责任公司 Recombinant protein, expression method, purification method and application thereof
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