CN106905434A - A kind of recombination fusion protein comprising hoof bat hepatitis B core protein and its preparation method and application - Google Patents
A kind of recombination fusion protein comprising hoof bat hepatitis B core protein and its preparation method and application Download PDFInfo
- Publication number
- CN106905434A CN106905434A CN201710111683.1A CN201710111683A CN106905434A CN 106905434 A CN106905434 A CN 106905434A CN 201710111683 A CN201710111683 A CN 201710111683A CN 106905434 A CN106905434 A CN 106905434A
- Authority
- CN
- China
- Prior art keywords
- protein
- fusion protein
- recombination fusion
- core
- amino acid
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/385—Haptens or antigens, bound to carriers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5258—Virus-like particles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6075—Viral proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10123—Virus like particles [VLP]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
- C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
- C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The invention provides a kind of recombination fusion protein comprising hoof bat hepatitis B core protein, the recombination fusion protein is made up of hoof bat hepatitis B core protein and the foreign protein fragments being inserted between the amino acid of the hoof bat hepatitis B core protein, and the amino acid sequence of the hoof bat hepatitis B core protein is as shown in SEQ ID NO.1.The present invention is first by the use of hoof bat hepatitis B core protein as foreign epitope in delivery carrier, by optimization design, recombination fusion protein is expressed in Hansenula yeast and forms Hybrid virus like particles, the Hybrid virus like particles are used as vaccine composition, on the premise of vaccine safety is ensured, can stimulate and produce stronger immune response, the infection for preventing RSV, with important science and application value.
Description
Technical field
The present invention relates to biomedicine field, more particularly to a kind of restructuring fusion comprising hoof bat hepatitis B core protein
Albumen and its preparation method and application.
Background technology
Respiratory Syncytial Virus(RSV) (Respiratory syncytial virus (RSV)) is to cause baby children in world wide
The main pathogens of youngster's ALRI.More than 90% infant at least experiences a rsv infection, 6 in two years after birth
Easily there is severe infections and death in month following baby, the elderly and immune deficiency crowd.For the disease that rsv infection causes, mesh
Preceding the only effective marketed drug is exactly palivizumab (Palivizumab).RSV belongs to Paramyxoviridae, and pneumonitis virus belongs to,
It is non-sections sub-thread strand RNA togavirus.Full length viral genome 15.2kb, comprising 10 encoding genes (be followed successively by NS1,
NS2, N, P, M, SH, G, F, M2, L), encode 11 kinds of albumen altogether, including 3 kinds of nucleocapsid proteins (N, P, L), 3 kinds of transmembrane proteins (F,
G, SH), 3 kinds of stromatins (M, M2-1, M2-2) and 2 kinds of non-structural proteins (NS1, NS2).Wherein two kinds of glycoprotein of G, F again quilt
Referred to as attachment proteins and fusion protein, respectively mediate retroviral and cell stick and merge, have for virus infection host cell
Play an important role, both albumen are also the main target antigen of current vaccine development.Can be by RSV according to G-protein antigentic specificity
It is divided into G-protein amino acid sequence between two hypotypes of A and B, amphitypy and only has 53% homology, and F protein then has same compared with homoamino acid
Source property, up to 90%, and two rsv infections of hypotype of A and B can simultaneously be prevented as vaccine antigen using F protein.
F protein is made up of two subunits of F1, F2, is I type glycoprotein, exists with trimeric form.There are two kinds of shapes in F protein
State：Before the fusion of meta-stable with relatively it is stable merge after state.Have now been found that F protein there are 6 neutralizing antibody epitopes：I、II、
IV, V and VI and fusion pro-antigen siteFor antigen siteI, II, IV have the report that corresponding neutralization monoclonal antibody is developed,
Wherein antigen site II is exactly RSV preventions and therapeutic monoclonal antibody handkerchief profit pearl (Palivizumab) and Mo Weizhu (Motavizumab)
Binding site, positioned at the 255-278 amino acids of F1 subunits, be the conformation type table of " α spirals-ring-α spirals " secondary structure
Position, during conformational state keeps constant before and after F protein fusion, therefore handkerchief profit pearl is respectively provided with for the RSV before fusion and after fusion
And activity.
The sixties in last century occurs in that serious ERD shows through the RSV vaccines (FI-RSV) of formalin-inactivated in clinical test
As the infant after being immunized does not play prevention effect not only when infecting RSV again, lung inflammation reaction is aggravated, finally made on the contrary
Into 2 Infant and child deaths.Therefore how to evade ERD phenomenons in the research and development of RSV vaccines and improve the security and validity of vaccine
As overriding concern problem.Existing more RSV candidate vaccines include attenuated live vaccine, restructuring live-virus vaccine, restructuring at present
Subunit's (G/F albumen) vaccine and nucleic acid (G/F) vaccine etc. are all in preclinical and clinical research.With Novavax companies,
Medimmune companies and the research and development institution that GSK companies are representative face using RSV F proteins as the target antigen of vaccine, and entrance
Bed conceptual phase, clinical data has obvious effect in being displayed in adult for control and prevention rsv infection.
Challenge test is carried out although F protein has good immunogenicity, after the immune cotton mouse of F protein after purification once to go out
ERD phenomenons are now crossed, F protein is disclosed and is still suffered from a certain degree of security risk.Meanwhile, the vaccine for not only being prepared by F protein may
ERD phenomenons occur, other immunogen proteins equally have similar risk.Therefore, a kind of being safely and effectively directed to is found to exhale
The vaccine for inhaling road syncytial virus is particularly important.
The content of the invention
It is weak and hidden in the presence of safety that the technical problem to be solved in the present invention is directed to vaccine immune response of the prior art
A kind of problem of trouble, there is provided recombination fusion protein comprising hoof bat hepatitis B core protein and its preparation method and application.
In order to solve the above-mentioned technical problem, the technical scheme of present invention offer is：
A kind of recombination fusion protein comprising hoof bat hepatitis B core protein, the recombination fusion protein is by hoof bat hepatitis
Viral core protein and the foreign protein fragments composition being inserted between the amino acid of the hoof bat hepatitis B core protein,
The amino acid sequence of the hoof bat hepatitis B core protein is as shown in SEQ ID NO.1.
Heretofore described hoof bat hepatitis viruse (Roundleaf bat hepatitis B virus, RBHV) belongs to
Hepadnaviridae, its host is bat, hoof bat etc., on phylogenetic tree with human hepatitis B virus, WCHV and
Ground squirrel hapatitis virus relation is nearer, and RBHV core proteins are similar with human hepatitis B virus core antigen (HBc) structure, can be by
240 or 180 RBHV core proteins are independently assembled into virus-like particle (T=4 or T=3).The present inventor is fully using for reference
On the basis of productive, it is carrier creatively to have invented based on RBHV, includes the recombination fusion protein of foreign epitope, is passed through
Optimization design, expression forms Hybrid virus like particles (Chimeric VLP, cVLP) in Hansenula yeast, and the cVLP is used as vaccine
Composition, on the premise of vaccine safety is ensured, can stimulate and produce stronger immune response, and the infection for preventing RSV has
Important science and application value.
In the present invention, the amino acid sequence of the hoof bat hepatitis B core protein and wild type hoof bat hepatitis viruse core
The amino acid sequence of heart protein is different, and in order to improve expressing quantity and protein stability, the present inventor creatively cuts out open country
Raw type protein reference sequence (NCBI Reference Sequence:YP_009045994.1 28 amino acid of N-terminal), from
And form the carrier sequence as shown in SEQ ID NO.1.
Above-mentioned foreign protein fragments can be arbitrary target protein fragments, including but not limited to, with immunogenicity
Protein fragments, such as B cell or φt cell receptor, the epitope with neutralization activity, protein tag (GFP or EGFP
Deng) and other there is the peptide fragment of immunologic competence.
Preferably, the foreign protein fragments are inserted in the 78th and of the hoof bat hepatitis B core protein
Between 79 amino acids, the foreign protein fragments are connected by amino acid linking arm with the hoof bat hepatitis B core protein
Connect.
In the present invention, inventor has found to be inserted in the 78th of hoof bat hepatitis B core protein when foreign protein fragments
Between position and the 79th amino acids, and after appropriate amino acid linking arm is connected, its foreign protein fragments can maximum journey
The surface for being presented in virus-like particle of degree, can improve the immunologic competence of the fragment.
Virus-like particle (virus-like particles, VLPs) is by the egg of virus structural protein being made by manufacturers or users
White particle, deleted virus nucleic acid, without infectivity, after structural proteins appropriate location insertion foreign epitope, can repeat high density
Be illustrated in VLP surfaces.Therefore, the characteristics of preparing vaccine using Hybrid virus like particles technology and have safe and efficient.
Preferably, in an embodiment of the invention, the recombination fusion protein is assembled into virus-like particle.
It is highly preferred that in yet another embodiment of the present invention, the recombination fusion protein is formed in expression system
Hybrid virus like particles (cVLPs).
It is highly preferred that in yet another embodiment of the present invention, it is described by hoof bat hepatitis B core protein and insertion
The recombination fusion protein of the foreign protein fragments composition between the amino acid of the hoof bat hepatitis B core protein is in the Chinese
Expression forms Hybrid virus like particles in inferior yeast.
It is highly preferred that in yet another embodiment of the present invention, it is described by hoof bat hepatitis B core protein and insertion
The 78th of the hoof bat hepatitis B core protein and the 79th amino acids between and by amino acid linking arm with it is described
The recombination fusion protein of the foreign protein fragments composition of hoof bat hepatitis B core protein connection expresses shape in Hansenula yeast
Into Hybrid virus like particles.
Another aspect of the present invention there is provided a kind of above-mentioned recombination fusion protein in antigen presentation carrier is prepared
Using.
Another aspect of the present invention there is provided a kind of application of above-mentioned recombination fusion protein in vaccine is prepared.
Preferably, the vaccine is Respiratory Syncytial Virus(RSV) preventative vaccine.
Preferably, above-mentioned recombination fusion protein is expressed in Hansenula yeast forms Hybrid virus like particles, it is described chimeric
Virus-like particle as the vaccine a part.
Preferably, in an embodiment of the invention, the foreign protein fragments in above-mentioned recombination fusion protein
It is the protein fragments with immunogenicity.
It is highly preferred that the foreign protein fragments are respiratory syncytial virus f-protein fragment.
Preferably, in an embodiment of the invention, it is above-mentioned to be inserted in the hoof bat hepatitis B core protein
The 78th and the foreign protein fragments between the 79th amino acids be the protein fragments with immunogenicity.
It is highly preferred that the foreign protein fragments are respiratory syncytial virus f-protein fragment, the respiratory tract is closed
Cellular virus fusion protein fragment by the L amino acid linking arm of the GILE amino acid linking arm of N-terminal and C-terminal with it is described
The 78th of hoof bat hepatitis B core protein and the connection of the 79th amino acids.
In the present invention, the respiratory syncytial virus f-protein fragment can be any coding in RSV genomes
The albumen of gene code, the encoding gene includes but is not limited to NS1, NS2, N, P, M, SH, G, F, M2 or L gene, or its combination.
Preferably, in an embodiment of the invention, the respiratory syncytial virus f-protein fragment is RSV
F protein.
It is highly preferred that the respiratory syncytial virus f-protein fragment is the epitope II in RSV F proteins.This resists
The amino acid sequence of former epitope II is as shown in SEQ ID NO.3.
Preferably, in an embodiment of the invention, the amino acid sequence such as SEQ of the recombination fusion protein
Shown in ID NO.2.
It is made up of hoof bat hepatitis B core protein and respiratory syncytial virus f-protein epitope preferably, above-mentioned
Recombination fusion protein is assembled into virus-like particle.
It is made up of hoof bat hepatitis B core protein and respiratory syncytial virus f-protein epitope it is highly preferred that above-mentioned
Recombination fusion protein is expressed in Hansenula yeast and forms Hybrid virus like particles.
Another aspect of the present invention there is provided a kind of preparation method of above-mentioned recombination fusion protein, including following step
Suddenly：
Step 1) gene order of recombination fusion protein is entered according to the superiority-inferiority and tRNA abundance of Hansenula yeast codon
Row optimization and synthesis, the recombination fusion protein gene order after being optimized；
Step 2) with step 1) in the S gene sequences replaced in carrier PUC25-SU of the recombination fusion protein gene order that obtains
Row, obtain PUC25-RBRU recombinant plasmids；
Step 3) by step 2) in the recombinant plasmid transformed that obtains enter induced expression in Hansenula yeast, it is extracted, after purification
Obtain the recombination fusion protein.
Specifically, above-mentioned preparation method can be further comprising the steps：
Step A) gene optimization with synthesis
By the hoof bat hepatitis containing amino acid linking arm and respiratory syncytial virus f-protein fragment gene sequence simultaneously
Viral core protein gene order, optimizes and synthesis according to the superiority-inferiority and tRNA abundance of Hansenula yeast codon；
Step B) recombinant plasmid structure
By step A) in the gene order that obtains replace S gene orders in carrier PUC25-SU, obtain PUC25-RBRU
Recombinant plasmid；
Step C) recombination fusion protein expression and purifying
By step B) in the PUC25-RBRU recombinant plasmids that obtain, after carrying out EcoR I and the digestions of Hind III, at linearisation
Reason, electricity is transformed into NVSI-H.P-105 (△ URA3 △ LEU2) Hansenula yeast bacterium, obtains recombinant, is sieved by ELISA method
Choosing obtains recombination fusion protein expression quantity positive strain high, and fermentation inducement expression harvests thalline, and high pressure is crushed, centrifuging and taking supernatant,
Gel filtration chromatography, obtains final product recombination fusion protein.
Wherein, step A) in involved gene be amino acid sequence Design and optimization according to fusion protein and synthesize.
Preferably, its nucleotide sequence is as shown in SEQ ID NO.4.
Another aspect of the present invention there is provided a kind of above-mentioned by hoof bat hepatitis B core protein and respiratory syncystial
Application of the recombination fusion protein of virus amalgamation protein epitope composition in Respiratory Syncytial Virus(RSV) preventative vaccine is prepared.
Another aspect of the present invention there is provided a kind of vaccine, and the vaccine is above-mentioned by hoof bat liver comprising effective dose
The recombination fusion protein that scorching viral core protein is constituted with respiratory syncytial virus f-protein epitope, and adjuvant.
Wherein, described being preferably contains low-solubility aluminium component, more preferably aluminium hydroxide.It is of course also possible to using such as
MF59, aluminum phosphate, calcium phosphate, cell factor (such as IL-2, IL-12, GM-CSF), saponin(e (such as QS21), MDP derivatives,
CpG ODN, LPS, MPL, polyphosphazene (polyphosphazenes), emulsion (such as Freund (Freund ' s), SAF), fat
Plastid, lipopeptid, virion (virosome), Iscoms, cochleates, PLG particulate, poloxamer (poloxamer)
Grain, virus-like particle, heat labile enterotoxin (LT), cholera toxin (CT), Mutant toxins (such as LTK63 and LTR72) are micro-
The adjuvant such as grain and/or polymerized liposome.
Vaccine of the present invention can be used by any suitable means, for example intracutaneous (i.d.), intraperitoneal (i.p.),
Intramuscular (i.m.), intranasal, orally, subcutaneous (s.c.) etc. and in any suitable delivery apparatus (O ' Hagan etc., Nature
Reviews, Drug Discovery2 (9), (2003), 727-735) in use.Preferably, vaccine of the present invention is in skin
Interior, subcutaneous or intramuscular is used.
Above-mentioned vaccine can be made into any appropriate formulation, including but not limited to freeze-dried, liquid agent, spray.
Another aspect of the present invention there is provided a kind of antibody, and the antibody is by above-mentioned by hoof bat hepatitis virus core
Obtained after the recombination fusion protein immune body that albumen is constituted with respiratory syncytial virus f-protein epitope.
Another aspect of the present invention there is provided a kind of bacterial strain comprising above-mentioned recombination fusion protein.
Beneficial effects of the present invention are：
1) present invention is in first delivery carrier by the use of hoof bat hepatitis B core protein as foreign epitope, can effectively be existed
VLP surface display foreign epitopes, make foreign epitope repeat highdensity distribution on VLP surfaces, and do not influence the autonomous dress of VLP
Match somebody with somebody.
2) recombination fusion protein of the present invention is expressed using expressed by Hansenula yeast system, and expressing quantity is high, with one
Determine the protein translation post-processing modification of degree, and recombination fusion protein can be automatically assembled into cVLP, expression product in yeast body
Easy purification.
3) present invention is collectively constituted by hoof bat hepatitis B core protein and respiratory syncytial virus f-protein epitope
Recombination fusion protein, can combine handkerchief profit pearl monoclonal antibody, after immune mouse, can produce and be neutralized for RSV cause of diseases specificity
Antibody, feasibility is provided for research and development RSV vaccines.
4) based on RBHV it is carrier in the present invention, includes the recombination fusion protein of foreign epitope, by optimization design,
Expression forms Hybrid virus like particles (Chimeric VLP, cVLP) in Hansenula yeast, and the cVLP is ensureing as vaccine composition
On the premise of vaccine safety, can stimulate and produce stronger immune response, the infection for preventing RSV, with important science
And application value.
Brief description of the drawings
Fig. 1 is PUC25-RBRU construction of recombinant plasmid schematic diagrames；
Fig. 2 is PUC25-RBRU recombinant plasmid digestion qualification result figures, and wherein Lane1 is PUC25-RBRU plasmid enzyme restrictions electricity
Swimming result；
Fig. 3 is the albumen for screening expression quantity positive yeast bacterial strain PCR qualification result figures high, and wherein Lane1 is bacterial strain PCR
Electrophoresis result；
Fig. 4 is the recombination fusion protein component qualification result figure of present invention design, wherein,
Left figure:Whole cell analysis after induced expression；
Lane1 is not induce thalline；
Lane2 is thalline after induction；
Middle figure:Recombination fusion protein SDS-PAGE analyses after purification；
Lane3 is destination protein electrophoresis result after purification；
Right figure:The Western-blot analyses of recombination fusion protein；
Lane5 is purpose protein immunoblot result；
Fig. 5 is the combination result figure of recombination fusion protein and handkerchief profit pearl antibody；
Fig. 6 is recombination fusion protein cVLP dynamic light scattering result figures；
Fig. 7 is the cVLP transmission electron microscope pictures of recombination fusion protein after phosphotungstic acid negative staining；
Fig. 8 is to recombinate the cause of disease specificity horizontal result figure of NAT produced after fusion protein immunization mouse；
Sequence explanation
SEQ ID NO.1 are the amino acid sequence of hoof bat hepatitis B core protein in the present invention；
SEQ ID NO.2 are the amino acid sequence of recombination fusion protein of the invention；
SEQ ID NO.3 are the amino acid sequence of epitope in recombination fusion protein of the invention；
SEQ ID NO.4 are the nucleotide sequence of recombination fusion protein of the invention.
Specific embodiment
The invention discloses a kind of recombination fusion protein comprising hoof bat hepatitis B core protein and preparation method thereof and
Using those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.It is important to note that institute
There is similar replacement and change apparent to those skilled in the art, they are considered as being included in the present invention,
And related personnel can substantially be modified on the basis of present invention, spirit and scope is not departed to content described herein
Or suitably change with combining and to realize and apply the technology of the present invention.
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology
The implication that personnel are generally understood that.Also, cell culture used herein, molecular genetics, nucleic acid chemistry, immunological experiment
Room operating procedure is widely used conventional steps in corresponding field.
In order that those skilled in the art more fully understands technical scheme, with reference to specific embodiment pair
The present invention is described in further detail.The experimental technique of unreceipted actual conditions in preferred embodiment, generally according to conventional strip
Part, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brookers etc. are write, and Huang Peitang etc. is translated, Science Press, 2002)
Described in condition, or according to the condition proposed by manufacturer and operating process.
Experiment material：
EcoR I, the restriction enzymes of Hind III and PCR reaction reagents are all from TaKaRa companies；
RSVsF albumen comes from Sino Biological Inc., is insect cell recombination expression product, freezes
Agent；
Handkerchief profit pearl monoclonal antibody comes from Medimmune companies；
Goat anti human IgG-HRP comes from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge；
Aluminum hydroxide adjuvant comes from SERVA Electrophoresis GmbH companies；
BALB.c female mices come from Beijing Vital River Experimental Animals Technology Co., Ltd.；
PUC25-SU expression plasmid of yeast comes from Beijing Institute of Biological Products Co., Ltd.；
NVSI-H.P-105 (△ URA3 △ LEU2) Hansenula yeast strain comes from Beijing Biological Product Inst.'s Limited Liability
Company；
RSV Long plants (ATCC VR26) comes from American Type Culture Collecti (ATCC)；
All relevant gene sequencing and primer synthesis commission Beijing promise match genome research center have in following embodiments
Limit company completes；
It is all in following embodiments to entrust Shanghai Jierui Biology Engineering Co., Ltd about gene chemical synthesis and genetic manipulation
Complete.
Percentage in following culture medium prescriptions is mass volume ratio：
MD culture mediums：1.34% without amino acid yeast nitrogen, 2% glucose；
MM culture mediums：1.34% without amino acid yeast nitrogen, 0.8% absolute methanol；
SM-leu culture mediums：1.34% without amino acid yeast nitrogen, 2% glucose, 0.01% leucine；
MM-leu culture mediums：1.34% without amino acid yeast nitrogen, 0.8% absolute methanol, 0.01% leucine；
YPD culture mediums：1% yeast extract, 2% peptone, 2% glucose；
Solid medium be aforesaid liquid culture medium in add 1.5% agar, autoclave sterilization.
Embodiment 1：The structure of recombination fusion protein expression plasmid of yeast and identification
1st, the design of recombination fusion protein
With hoof bat hepatitis B core protein amino sequence (NCBI Reference Sequence:YP_
009045994.1) it is reference sequences, cuts out carrier of the 28 amino acid formation of N-terminal of reference sequences as shown in SEQ ID NO.1
Sequence, the 78th in SEQ ID NO.1 sequences is inserted respiratory syncytial virus f-protein and the 79th amino acids between
(Genebank:ACO83301.1) 254-277 amino acids epitope (shown in SEQ ID NO.3), the epitope is handkerchief profit pearl
The binding site of antibody, is in series by " GILE " and " L " amino acid linking arm, forms the ammonia as shown in SEQ ID NO.2
Base acid sequence.
2nd, gene optimization and synthesis
Recombination fusion protein amino acid sequence according to SEQ ID NO.2, according to the quality of Hansenula yeast codon
Property and tRNA abundance carry out the optimization of gene order, form the coding gene sequence as shown in SEQ ID NO.4, the sequence is entered
Row gene chemical synthesis.
3rd, the structure of expression plasmid
According to the plasmid construction schematic diagram shown in Fig. 1, genes of interest is replaced into expression vector using gene recombination technology
S genes in PUC25-SU expression plasmid of yeast, formation includes recombination fusion protein gene order (as shown in SEQ ID NO.4)
Expression plasmid of yeast PUC25-RBRU, the plasmid with 25S rDNA on Hansenula yeast genome be homologous recombination integrate arm, with
URA3 genes are marker gene, and efficiently starting destination protein with MOX promoters expresses.
4th, the digestion identification of PUC25-RBRU expression plasmids and gene sequencing
EcoR I and the identification of the double digestions of Hind III, 37 DEG C, digestion 1 hour, enzyme are carried out to PUC25-RBRU expression plasmid of yeast
Cut system as shown in table 1, the product after digestion is carried out into 1% agarose gel electrophoresis detection, as shown in Figure 2, it is seen that the plasmid
Can digestion go out two genetic fragments, respectively may be about the large fragment of 4kb and the small fragment of 2kb, large fragment is and includes restructuring and melt
The Yeast expression frame of hop protein genes of interest, gene sequencing is carried out by the plasmid, as a result shows, nothing consistent with expected results
The change of genes of interest.
The digestion system of table 1
Embodiment 2：The screening and identification of height expression positive yeast bacterial strain
1st, convert
NVSI-H.P-105 (△ URA3 △ LEU2) Hansenula yeast bacterium is incubated in YPD fluid nutrient mediums, cell density
(OD600) when reaching 1.0, the preparation of competent yeast is carried out, by by the PUC25-RBRU after EcoR I and the double digestions of Hind III
Large fragment gene is transformed into NVSI-H.P-105 saccharomycete by way of electricity conversion in plasmid, most transformed bacteria solution coating at last
In on SM-leu solid mediums, 37 DEG C are cultivated 3-5 days, obtain conversion recombinant.
2nd, ELISA screenings
The monoclonal bacterium colony of picking SM-leu cultured on solid medium carries out bacterium in 2ml SM-leu fluid nutrient mediums
Body culture, 37 DEG C, 250rpm shaken cultivations 24 hours.Take 200 μ l bacterium solutions and transfer and continue in 4ml SM-leu fluid nutrient mediums
Culture, after cell density (OD600) reaches more than 10,3000rpm is harvested by centrifugation thalline, and is resuspended in 4ml MM-leu cultures
Thalline culture is carried out in base, this stage added 1% absolute methanol every 6 hours, induce the expression of destination protein, induction 24 is small
When；Thalline is harvested by centrifugation and 200 μ l yeast thalline disruption buffer (20mM PB, pH value 7.2) and 200mg beades is added, it is high
The vibration of frequency low temperature is broken, using coating buffer (Na2CO3-NaHCO3Solution, pH value 9.6) albumen supernatant is diluted and 500 times and is wrapped
By on ELISA Plate, 100 μ l/ holes, 4 DEG C are coated with 8 hours；The PBS containing 1%BSA is added, 100 μ l/ holes, 37 DEG C of closings 3 are small
When；Palivizumab is diluted to 1 μ g/ml, 100 μ l/ holes, 37 DEG C, 1 hour；Goat anti human IgG-HRP is diluted 10000 times,
100 μ l/ holes, 37 DEG C, 1 hour；50 μ l nitrite ions A and 50 μ l nitrite ion B, color development at room temperature 10 minutes are added to add 50 μ l to terminate
Liquid C；OD values are read in the case where wavelength is 450nm and 630nm wavelength, selection OD value highest bacterial strains are used as recombination fusion protein table high
Up to yeast strain.Because the yeast strain for screening is only supplemented with URA3 genes, it is only capable of in the culture medium for being supplemented with leucine
Growth, therefore LEU2 genetic transformation is entered in the saccharomycete for screening, the bacterial strain is grown in MD basal mediums.
3rd, strain genes of interest identification
Recombination fusion protein expression yeast strain genome high is extracted, genes of interest is carried out using the primer shown in table 2
PCR is expanded, and PCR primer is carried out 1% agarose gel electrophoresis inspection by PCR reaction systems and reaction condition as shown in table 3, table 4
Survey, as a result as shown in figure 3, it is amplifiable go out expected size DNA fragmentation, PCR primer is carried out into gene sequencing, genes of interest
Without change.
The strain idenfication primer information table of table 2
The PCR reaction systems of table 3
The PCR reaction conditions of table 4
Embodiment 3：The preparation and identification of cVLP
1st, yeast fermentation and bacterial cell disruption
The MD Liquid Cultures that the recombination fusion protein for obtaining expression barms high is inoculated in 10ml will be screened in embodiment 2
Shaken cultivation 24 hours in base, then Amplification Culture 24 hours in 100mlMD fluid nutrient mediums of transferring, prepare fermentation seed, connect
Kind in carrying out saccharomycetes to make fermentation culture in 5L fermentation tanks, and the induced expression of destination protein is carried out using methyl alcohol.After fermentation ends,
Using brine thalline 2 times, most thalline is resuspended in disruption buffer (20mM PB, 50mM NaCl, pH value 7.2) at last
In enter horizontal high voltage crush, centrifuging and taking albumen supernatant.Whole cell albumen before and after induction is carried out into 10%SDS-PAGE electrophoresis point
Analysis, as a result as shown in the left figure of Fig. 4, arrow is signified to be destination protein band.
2nd, destination protein purifying and Western-blot detections
Gel filtration chromatography, medium selection are carried out using AKTA chromatogram purifications instrument (GE AKTA explorer)
Sephacryl S500-HR, column volume is 900ml, and detailed process is as follows：
A, column equilibration：With 3 times of level pads of column volume (50mM PB+0.2MNacl, pH7.3) balance chromatographic column extremely
280nm absorption values, less than 0.5mAU, the absorption value of detector are zeroed without obvious change.
B, loading：During the thick pure liquid of 100ml albumen pumped into chromatographic column according to 5ml/min pump speeds, after end of the sample, balance is slow
Fliud flushing continues to flow through chromatographic column.
C, receipts sample：Liquid to be buffered gradually rises to 280nm absorption values during 1/3 column volume, is collected according to 5ml/ pipes are automatic, and
SDS-PAGE electrophoretic analysis is carried out to the sample after collection.
D, post are reclaimed and recycled：0.2M sodium hydroxide solutions process chromatographic column, then using equilibration buffer
Chromatographic column, continues the chromatographic purifying of next albumen.
Destination protein after purification is carried out into 10%SDS-PAGE electrophoretic analysis result as shown in the middle figure of Fig. 4, arrow institute
Refer to and be destination protein band, molecular size range is about 25kD, it is basically identical with expected size.Simultaneously by destination protein after purification
Western-blot detections are carried out, conventionally by after in destination protein transferring film to pvdf membrane, is entered using handkerchief profit pearl antibody
Row detection, as a result as shown in the right figure of Fig. 4, arrow is signified to be destination protein strip-like developing pipe.
3 and the binding tests of handkerchief profit pearl antibody
CVLP after purification is carried out into 2 times of gradient dilutions from 1 μ g/ml startings, and is coated on ELISA Plate, while with
RSVsF albumen is positive control, and the handkerchief profit pearl AC of addition is 1 μ g/ml, to verify cVLP albumen with handkerchief profit pearl antibody
Combination degree, as a result as shown in figure 5, cVLP can be combined effectively with handkerchief profit pearl antibody.
4th, the dynamic light scattering and transmission electron microscope observing of cVLP
Albumen after purification is carried out into dynamic light scattering (Malvern, NANO-8S90) analysis, as a result as shown in fig. 6, knot
Fruit display VLP sizes homogeneity is good, and hydration particle diameter is about 26nm.Destination protein is dripped on the plating charcoal copper mesh film of 300 mesh, is inhaled
Attached 5 minutes, phosphotungstic acid negative staining 1 minute, transmission electron microscope (HITACHI, JEM-1400) observed the particle shape of sample, as a result as schemed
Shown in 7, it is seen that VLP sizes are between 20-30nm, and size is homogeneous, and form is good.
Embodiment 4：The immunological effect research of recombination fusion protein of the present invention
SPF grades of 6-8 week old BALB/c female mices 32 are taken, 4 groups of A, B, C and D, every group 8 is randomly divided into.Press respectively
Following design immunity inoculation：The aluminum hydroxide adjuvant of recombination fusion protein (cVLP) and 250 μ g that A groups inject 0.5 μ g is mixed
Compound；B groups inject the RSVsF albumen of 0.5 μ g and the aluminum hydroxide adjuvant mixture of 250 μ g；C groups injecting normal saline and 250 μ
The aluminum hydroxide adjuvant of g, is control group.Take abdominal channels to be immunized, each group is immunized 3 times, be spaced 2 weeks.Each group mouse immune
It is daily afterwards to observe mouse state and changes of weight.Immune to terminate two weeks after, disconnected cone is taken a blood sample and separates serum.Using virus it is micro in
With RSV specificity NAT levels in test method detection serum, as a result as shown in figure 8, recombination fusion protein
(cVLP) after mixing with aluminum hydroxide adjuvant, the RSV specificity NATs of higher level can be produced, points out the present invention weight
Group fusion protein has can be used as the potentiality of RSV preventative vaccines.
Comparative example：The comparative analysis of recombination fusion protein of the present invention and other types recombination fusion protein
According to the design of embodiment 1, separately design and carried to present with WCHV cAg (WHC)
The cVLP of body and be in the cVLP of delivery carrier, according to described in embodiment 1,2 and 3 with human hepatitis B virus core antigen (HBC)
Method is prepared respectively, finally gives WHC-cVLP and HBC-cVLP, basic herein according to the animal immune method in embodiment 4
On increase by two groups, every group 8, D groups inject the aluminum hydroxide adjuvant mixture of the WHC-cVLP and 250 μ g of 0.5 μ g；E groups are noted
The aluminum hydroxide adjuvant mixture of the HBC-cVLP and 250 μ g of 0.5 μ g is penetrated, takes abdominal channels to be immunized, each group is immunized 3 times,
Interval 2 weeks.Daily observation mouse state and changes of weight after each group mouse immune.Immune two weeks after, disconnected cone is taken a blood sample and separates blood
Clearly, using RSV specificity NAT levels in viral microneutralization test method detection serum.Result as shown in figure 8,
The RSV specificity NATs of display RBHV-cVLP are about the RSV specificity NAT of 8.5, WHC-cVLP about
It is that the RSV specificity NATs of 6.5, HBC-cVLP are about 3.0, RBHV-cVLP and are produced compared with WHC-cVLP and HBC-cVLP
Raw neutralizing antibody level is higher.Result above prompting RBHV-cVLP has a clear superiority in terms of external source Epitope presentation, this hair
Bright recombination fusion protein has can be used as the potentiality of RSV preventative vaccines.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Sequence table
<110>Co., Ltd of traditional Chinese medicines Zhong Sheng Bioteknologisk Institut；Beijing Institute of Biological Products Co., Ltd.
<120>A kind of recombination fusion protein comprising hoof bat hepatitis B core protein and its preparation method and application
<130> None
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 189
<212> PRT
<213> RBHV
<400> 1
Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Ser Gln Leu Ile
1 5 10 15
Ser Phe Leu Pro Glu Asp Phe Phe Pro Asn Leu Ala Glu Leu Val Glu
20 25 30
Thr Thr Thr Ala Leu Tyr Glu Glu Glu Leu Val Gly Lys Glu His Cys
35 40 45
Ser Pro His His Thr Ala Leu Arg Ser Leu Leu Asn Cys Trp Gly Glu
50 55 60
Thr Val Arg Leu Ile Thr Trp Val Arg Asn Ser Val Glu Gly Pro Leu
65 70 75 80
Ile Gln Asp Ala Ile Val Gln Gln Val Gln Ala Ser Val Gly Leu Arg
85 90 95
Met Arg Gln Leu Met Trp Phe His Leu Ser Cys Leu Thr Phe Gly Gln
100 105 110
Pro Thr Val Ile Glu Phe Leu Val Ser Phe Gly Thr Trp Ile Arg Thr
115 120 125
Pro Gln Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro
130 135 140
Glu His Thr Ile Val Arg Arg Arg Gly Gly Ser Arg Ala Thr Arg Ser
145 150 155 160
Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro
165 170 175
Arg Arg Arg Arg Ser Gln Ser Pro Ala Ser Ser Asn Cys
180 185
<210> 2
<211> 218
<212> PRT
<213>Recombination fusion protein
<400> 2
Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Ser Gln Leu Ile
1 5 10 15
Ser Phe Leu Pro Glu Asp Phe Phe Pro Asn Leu Ala Glu Leu Val Glu
20 25 30
Thr Thr Thr Ala Leu Tyr Glu Glu Glu Leu Val Gly Lys Glu His Cys
35 40 45
Ser Pro His His Thr Ala Leu Arg Ser Leu Leu Asn Cys Trp Gly Glu
50 55 60
Thr Val Arg Leu Ile Thr Trp Val Arg Asn Ser Val Glu Gly Gly Ile
65 70 75 80
Leu Glu Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr
85 90 95
Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Leu Pro Leu Ile Gln Asp
100 105 110
Ala Ile Val Gln Gln Val Gln Ala Ser Val Gly Leu Arg Met Arg Gln
115 120 125
Leu Met Trp Phe His Leu Ser Cys Leu Thr Phe Gly Gln Pro Thr Val
130 135 140
Ile Glu Phe Leu Val Ser Phe Gly Thr Trp Ile Arg Thr Pro Gln Ala
145 150 155 160
Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu His Thr
165 170 175
Ile Val Arg Arg Arg Gly Gly Ser Arg Ala Thr Arg Ser Pro Arg Arg
180 185 190
Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg
195 200 205
Arg Ser Gln Ser Pro Ala Ser Ser Asn Cys
210 215
<210> 3
<211> 24
<212> PRT
<213>Epitope
<400> 3
Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp
1 5 10 15
Gln Lys Lys Leu Met Ser Asn Asn
20
<210> 4
<211> 657
<212> DNA
<213> DNA
<400> 4
atggacatcg acccttacaa ggagttcggc gcctcgtcgc agctgatctc gttcctgcct 60
gaggacttct tccctaacct ggccgagctg gtggagacca ccaccgccct gtacgaggag 120
gagctggtgg gcaaggagca ctgctcgcct caccacacgg ccctgagatc gctgctgaac 180
tgctggggcg agacggtgag actgatcacg tgggtgagaa actcggtgga gggcggcatc 240
ctggagaact cggagctgct gtcgctgatc aacgacatgc ctatcaccaa cgaccagaag 300
aagctgatgt cgaacaacct gcctctgatc caggacgcca tcgtgcagca ggtgcaggcc 360
tcggtgggcc tgagaatgag acagctgatg tggttccacc tgtcgtgcct gaccttcggc 420
cagcctaccg tgatcgagtt cctggtgtcg ttcggcacct ggatcagaac ccctcaggcc 480
tacagacctc ctaacgcccc tatcctgtcg accctgcctg agcacaccat cgtgagaaga 540
agaggcggct cgagagccac cagatcgcct agaagaagaa ccccttcgcc tagaagaaga 600
agatcgcagt cgcctagaag aagaagatcg cagtcgcctg cctcgtcgaa ctgctaa 657
Claims (16)
1. a kind of recombination fusion protein comprising hoof bat hepatitis B core protein, it is characterised in that the recombination fusion protein
By hoof bat hepatitis B core protein and the foreign protein being inserted between the amino acid of the hoof bat hepatitis B core protein
Matter fragment is constituted, and the amino acid sequence of the hoof bat hepatitis B core protein is as shown in SEQ ID NO.1.
2. recombination fusion protein according to claim 1, it is characterised in that the foreign protein fragments are inserted in described
And the 79th amino acids between, the foreign protein fragments are connected the 78th of hoof bat hepatitis B core protein by amino acid
Arm is connect to be connected with the hoof bat hepatitis B core protein.
3. recombination fusion protein according to claim 1 and 2, it is characterised in that the recombination fusion protein is assembled into disease
Malicious sample particle.
4. the recombination fusion protein as described in any one in a kind of 1-3 such as claim in antigen presentation carrier is prepared should
With.
5. application of the recombination fusion protein as described in any one in a kind of 1-3 such as claim in vaccine is prepared.
6. application according to claim 5, it is characterised in that the recombination fusion protein expresses formation in Hansenula yeast
Hybrid virus like particles, the Hybrid virus like particles as the vaccine a part.
7. recombination fusion protein according to claim 1 and 2, it is characterised in that the foreign protein fragments be with
The protein fragments of immunogenicity.
8. recombination fusion protein according to claim 7, it is characterised in that the foreign protein fragments are that respiratory tract is closed
Cellular virus fusion protein fragment.
9. recombination fusion protein according to claim 8, it is characterised in that the respiratory syncytial virus f-protein piece
Section is by the GILE amino acid linking arm of N-terminal and the L amino acid linking arm and the hoof bat hepatitis virus core of C-terminal
The 78th of albumen and the connection of the 79th amino acids.
10. the recombination fusion protein described in any one in claim 1-2,8-9, it is characterised in that the restructuring
The amino acid sequence of fusion protein is as shown in SEQ ID NO.2.
11. recombination fusion proteins according to claim 10, it is characterised in that the recombination fusion protein is assembled into virus
Sample particle.
A kind of preparation method of 12. recombination fusion proteins as described in any one in claim 1-3,7-11, its feature
It is to comprise the following steps：
Step 1) gene order of recombination fusion protein is carried out according to the superiority-inferiority and tRNA abundance of Hansenula yeast codon it is excellent
Change and synthesize, the recombination fusion protein gene order after being optimized；
Step 2) with step 1) in the S gene orders replaced in carrier PUC25-SU of the recombination fusion protein gene order that obtains,
Obtain PUC25-RBRU recombinant plasmids；
Step 3) by step 2) in the recombinant plasmid transformed that obtains enter induced expression in Hansenula yeast, it is extracted, obtain after purification
The recombination fusion protein.
The recombination fusion protein as described in any one in a kind of 13. 7-11 such as claim is pre- in preparation Respiratory Syncytial Virus(RSV)
Application in anti-property vaccine.
14. a kind of vaccines, it is characterised in that the vaccine is comprising effective dose such as any one institute in claim 7-11
The recombination fusion protein stated, and adjuvant.
15. a kind of antibody, it is characterised in that the antibody is merged as the restructuring as described in any one in claim 7-11
Obtained after protein immunization individuality.
A kind of 16. bacterial strains of the recombination fusion protein comprising as described in any one in claim 1-3,7-11.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710111683.1A CN106905434B (en) | 2017-02-28 | 2017-02-28 | Recombinant fusion protein containing hepialus hepatitis virus core protein and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710111683.1A CN106905434B (en) | 2017-02-28 | 2017-02-28 | Recombinant fusion protein containing hepialus hepatitis virus core protein and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106905434A true CN106905434A (en) | 2017-06-30 |
| CN106905434B CN106905434B (en) | 2021-01-26 |
Family
ID=59208582
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710111683.1A Active CN106905434B (en) | 2017-02-28 | 2017-02-28 | Recombinant fusion protein containing hepialus hepatitis virus core protein and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106905434B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108456686A (en) * | 2017-02-17 | 2018-08-28 | 厦门大学 | It is a kind of to be used to show peptide carrier of desired polypeptides and application thereof |
| CN111138550A (en) * | 2020-01-06 | 2020-05-12 | 国药中生生物技术研究院有限公司 | Recombinant respiratory syncytial virus chimeric vaccine and preparation method and application thereof |
| CN111166881A (en) * | 2020-01-06 | 2020-05-19 | 国药中生生物技术研究院有限公司 | Recombinant respiratory syncytial virus multi-epitope chimeric vaccine and preparation method and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1668331A (en) * | 2002-07-18 | 2005-09-14 | 赛托斯生物技术公司 | Hapten-carrier conjugates and uses thereof |
| CN1712529A (en) * | 2004-06-24 | 2005-12-28 | 中国人民解放军军事医学科学院野战输血研究所 | Chimeric virus particle vaccine carrier protein, its production and use |
| WO2012061815A3 (en) * | 2010-11-05 | 2013-03-14 | Novavax Inc. | RABIES GLYCOPROTEIN VIRUS-LIKE PARTICLES (VLPs) |
| CN103087933A (en) * | 2011-11-08 | 2013-05-08 | 北京生物制品研究所 | Recombinant polymorphic hansenula polymorpha and preparation method 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 |
| CN104745492A (en) * | 2015-04-21 | 2015-07-01 | 江苏赛锘威生物医药有限公司 | Hansenula polymorpha mutant and application of expression vector of hansenula polymorpha mutant to recombinant protein expression |
| WO2015143335A1 (en) * | 2014-03-20 | 2015-09-24 | The University Of North Carolina At Chapel Hill | Methods and compositions for chimeric coronavirus spike proteins |
| CN105497886A (en) * | 2015-12-23 | 2016-04-20 | 中国医学科学院医学生物学研究所 | Application of HBcAg (hepatitis B core antigen) virus-like particle serving as cancer therapeutic vaccine carrier |
-
2017
- 2017-02-28 CN CN201710111683.1A patent/CN106905434B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1668331A (en) * | 2002-07-18 | 2005-09-14 | 赛托斯生物技术公司 | Hapten-carrier conjugates and uses thereof |
| CN1712529A (en) * | 2004-06-24 | 2005-12-28 | 中国人民解放军军事医学科学院野战输血研究所 | Chimeric virus particle vaccine carrier protein, its production and use |
| WO2012061815A3 (en) * | 2010-11-05 | 2013-03-14 | Novavax Inc. | RABIES GLYCOPROTEIN VIRUS-LIKE PARTICLES (VLPs) |
| CN103087933A (en) * | 2011-11-08 | 2013-05-08 | 北京生物制品研究所 | Recombinant polymorphic hansenula polymorpha and preparation method thereof |
| WO2015143335A1 (en) * | 2014-03-20 | 2015-09-24 | The University Of North Carolina At Chapel Hill | Methods and compositions for chimeric coronavirus spike proteins |
| 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 |
| CN104745492A (en) * | 2015-04-21 | 2015-07-01 | 江苏赛锘威生物医药有限公司 | Hansenula polymorpha mutant and application of expression vector of hansenula polymorpha mutant to recombinant protein expression |
| CN105497886A (en) * | 2015-12-23 | 2016-04-20 | 中国医学科学院医学生物学研究所 | Application of HBcAg (hepatitis B core antigen) virus-like particle serving as cancer therapeutic vaccine carrier |
Non-Patent Citations (3)
| Title |
|---|
| DREXLER等: "ACCESSION：YP_009045994，core protein [Roundleaf bat hepatitis B virus]", 《GENBANK》 * |
| JEANNE H.SCHICKLI等: "Palivizumab epitope-displaying virus-like particles protect rodents from RSV challenge", 《THE JOURNAL OF CLINICAL INVESTIGATION》 * |
| 国家药典委员会: "《中华人民共和国药典 三部 注释》", 31 March 2016, 复旦大学出版社 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108456686A (en) * | 2017-02-17 | 2018-08-28 | 厦门大学 | It is a kind of to be used to show peptide carrier of desired polypeptides and application thereof |
| EP3617317A4 (en) * | 2017-02-17 | 2021-01-13 | Xiamen University | Peptide carrier for displaying target polypeptide and use thereof |
| CN108456686B (en) * | 2017-02-17 | 2021-03-05 | 厦门大学 | Polypeptide carrier for displaying target polypeptide and application thereof |
| US10987418B2 (en) | 2017-02-17 | 2021-04-27 | Xiamen University | Polypeptide carrier for presenting target polypeptide and uses thereof |
| CN111138550A (en) * | 2020-01-06 | 2020-05-12 | 国药中生生物技术研究院有限公司 | Recombinant respiratory syncytial virus chimeric vaccine and preparation method and application thereof |
| CN111166881A (en) * | 2020-01-06 | 2020-05-19 | 国药中生生物技术研究院有限公司 | Recombinant respiratory syncytial virus multi-epitope chimeric vaccine and preparation method and application thereof |
| CN111138550B (en) * | 2020-01-06 | 2022-12-13 | 国药中生生物技术研究院有限公司 | Recombinant respiratory syncytial virus chimeric vaccine and preparation method and application thereof |
| CN111166881B (en) * | 2020-01-06 | 2023-03-24 | 国药中生生物技术研究院有限公司 | Recombinant respiratory syncytial virus multi-epitope chimeric vaccine and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106905434B (en) | 2021-01-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112076315B (en) | Nanometer antigen particle fused by novel coronavirus S protein and ferritin subunit, novel coronavirus vaccine, preparation method and application thereof | |
| CN107841507B (en) | Efficiently expressed porcine circovirus type 2 Cap-cell-penetrating peptide fusion protein gene and application thereof | |
| CN107427571A (en) | Novel multivalent vaccine based on nano particle | |
| CN111825768B (en) | Self-assembly ferritin-based nano antigen particle, influenza vaccine and preparation method | |
| CN113512096A (en) | Weever rhabdovirus recombinant G2 protein and application thereof | |
| CN106905434A (en) | A kind of recombination fusion protein comprising hoof bat hepatitis B core protein and its preparation method and application | |
| CN109303916A (en) | Coke dies GAP-associated protein GAP GSDMD and is preparing the application in ghost vaccine | |
| CN114891074A (en) | Seasonal influenza A universal virus-like particle and preparation method and application thereof | |
| CN113862284B (en) | Gene, virus-like particle, vaccine and preparation and application for encoding recombinant avian influenza virus HA protein | |
| CN109966483A (en) | A kind of more antigen universal influenza vaccines and its preparation method and application based on ferritin | |
| CN111454989B (en) | Chimeric gene type I encephalitis B virus-like particle vaccine and preparation method and application thereof | |
| CN111607605B (en) | Construction method of multivalent epitope and subunit vaccine | |
| CN110669142B (en) | RGD-fused porcine circovirus type 2 virus-like particle, mutant infectious clone, preparation method and application thereof | |
| CN103667319B (en) | The trivalent vaccine and its preparation method and purposes of anti-human papilloma virus (anti-HPV) | |
| CN107936123A (en) | A kind of transmissible gastro-enteritis virus fusion protein and its preparation method and application | |
| KR20180064158A (en) | Soluble Multi-Epitope Antigen of Foot-and-Mouth Disease Virus and Uses Thereof | |
| CN106986942A (en) | A kind of recombination fusion protein comprising bat hepatitis B core protein and its preparation method and application | |
| CN106986943A (en) | A kind of recombination fusion protein comprising arctic ground squirrel hepatitis B core protein and its preparation method and application | |
| CN111166881B (en) | Recombinant respiratory syncytial virus multi-epitope chimeric vaccine and preparation method and application thereof | |
| CN109136198A (en) | A kind of expression Chicken Infectious Anemia Virus VP1, VP2 genetic recombination bird pox virus live vector vaccine | |
| CN113827714A (en) | H7N9 subtype avian influenza virus-like particle vaccine preparation and application thereof | |
| CN112321718B (en) | Self-assembly ferritin-based nano antigen particle, peste des petits ruminants vaccine and preparation method and application thereof | |
| CN114196701A (en) | Bivalent recombinant Newcastle disease virus vector of SARS-COV-2, corresponding vaccine strain and preparation method | |
| CN112442130B (en) | Self-assembly ferritin-based nano antigen particle, rabies vaccine prepared from same and application of self-assembly ferritin-based nano antigen particle | |
| CN109517044B (en) | Porcine epidemic diarrhea virus genetic engineering antigen and antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20190730 Address after: 101111 No. 38 Jinghai Second Road, Daxing District, Beijing Economic and Technological Development Zone Applicant after: China National Pharmaceutical Biotechnology Research Institute Co., Ltd. Address before: 101111 No. 38 Jinghai Second Road, Daxing District, Beijing Economic and Technological Development Zone Applicant before: China National Pharmaceutical Biotechnology Research Institute Co., Ltd. Applicant before: Beijing Biological Products Research Institute Co., Ltd. |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |