CN106986943A - A kind of recombination fusion protein comprising arctic ground squirrel hepatitis B core protein and its preparation method and application - Google Patents

A kind of recombination fusion protein comprising arctic ground squirrel hepatitis B core protein and its preparation method and application Download PDF

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CN106986943A
CN106986943A CN201710112206.7A CN201710112206A CN106986943A CN 106986943 A CN106986943 A CN 106986943A CN 201710112206 A CN201710112206 A CN 201710112206A CN 106986943 A CN106986943 A CN 106986943A
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fusion protein
protein
recombination fusion
ground squirrel
recombination
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CN106986943B (en
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李启明
梁宇
邵帅
靳玉琴
陈实
张靖
侯俊伟
唐芳
张学峰
杜丽芳
唐玉龙
栗子谦
王擎擎
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China National Pharmaceutical Biotechnology Research Institute Co Ltd
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BEIJING BIOLOGICAL PRODUCTS RESEARCH INSTITUTE Co Ltd
China National Pharmaceutical Biotechnology Research Institute Co Ltd
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Abstract

The invention provides a kind of recombination fusion protein for including arctic ground squirrel hepatitis B core protein, the recombination fusion protein is made up of arctic ground squirrel hepatitis B core protein and the foreign protein fragments being inserted between the amino acid of the arctic ground squirrel hepatitis B core protein.The present invention is first by the use of arctic ground squirrel hepatitis B core protein as foreign epitope in delivery carrier, pass through 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, it can stimulate and produce stronger immune response, available for prevention RSV infection, with important science and application value.

Description

A kind of recombination fusion protein comprising arctic ground squirrel hepatitis B core protein and its Preparation method and application
Technical field
The present invention relates to biomedicine field, more particularly to a kind of weight for including arctic ground squirrel hepatitis B core protein Group fusion protein 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 undergoes a rsv infection, 6 in two years after birth Severe infections and death easily occur for month following baby, the elderly and immune deficiency crowd.For disease caused by rsv infection, mesh Preceding the only effective marketed drug is exactly palivizumab (Palivizumab).RSV belongs to Paramyxoviridae, and pneumonitis virus belongs to, It is non-section 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 the rsv infection of two hypotypes of A and B can be prevented using F protein as vaccine antigen simultaneously.
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 site, for antigen site, I, II, IV have the report that corresponding neutralization monoclonal antibody is developed, Wherein antigen site II is exactly RSV preventions and the sharp pearl (Palivizumab) of therapeutic monoclonal antibody handkerchief 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 the sharp pearl of handkerchief is respectively provided with for the RSV before fusion and after fusion And activity.
The sixties in last century, the RSV vaccines (FI-RSV) through formalin-inactivated occurred in that serious ERD shows 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 GSK companies for representative research and development institution using RSV F proteins as the target antigen of vaccine, and entrance is faced Bed conceptual phase, clinical data is shown in adult for controlling and preventing rsv infection to have obvious effect.
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 still suffers from a certain degree of security risk.Meanwhile, the vaccine not only prepared by F protein may ERD phenomenons occur, other immunogen proteins equally have similar risk.Therefore, find a kind of safely and effectively for exhaling The vaccine for inhaling road syncytial virus is particularly important.
The content of the invention
The technical problem to be solved in the present invention is weak for some vaccine immune responses of the prior art, and there is peace The problem of full hidden danger, there is provided a kind of recombination fusion protein comprising arctic ground squirrel hepatitis B core protein and its preparation side Method and application.
In order to solve the above-mentioned technical problem, the technical scheme that provides of the present invention is：
A kind of recombination fusion protein for including arctic ground squirrel hepatitis B core protein, the recombination fusion protein is by north Polar region squirrel hepatitis B core protein and it is inserted between the amino acid of the arctic ground squirrel hepatitis B core protein Foreign protein fragments are constituted.
Heretofore described arctic ground squirrel hapatitis virus (Arctic Ground Squirrel Hepatitis Virus, AGSHV) belong to Hepadnaviridae, its host is arctic ground squirrel, grey squirrel, California ground squirrel and trip Mouse etc., it is nearer with human hepatitis B virus, groundhog hepatitis virus and ground squirrel hapatitis virus relation on phylogenetic tree, and AGSHV core proteins are similar with human hepatitis B virus core antigen (HBc) structure, can be by 240 or 180 AGSHV core proteins Independently it is assembled into virus-like particle (T=4 or T=3).The present inventor is on the basis of abundant reference has been fruitful, creatively It is carrier to have invented based on AGSHV, includes the recombination fusion protein of foreign epitope, by optimization design, in Hansenula yeast Expression forms Hybrid virus like particles (Chimeric VLP, cVLP), and the cVLP is ensureing vaccine safety as vaccine composition On the premise of, it can stimulate and produce stronger immune response, the infection for preventing RSV, with important science and application value.
In the present invention, the amino acid sequence of the arctic ground squirrel hepatitis B core protein is with wild-type protein Sequence (UniProtKB/Swiss-Prot:Q64897.1) it is reference sequences, the carrier sequence as shown in SEQ ID NO.1.
Above-mentioned foreign protein fragments can be arbitrary target protein fragments, include but is 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 peptide fragments with immunologic competence.
Preferably, the foreign protein fragments are inserted in the 78th of the arctic ground squirrel hepatitis B core protein Between position and the 79th amino acids, the foreign protein fragments pass through amino acid linking arm and the arctic ground squirrel hepatitis Malicious core protein connection.
In the present invention, inventor is had found when foreign protein fragments are inserted in arctic ground squirrel hepatitis B core protein The 78th between the 79th amino acids, and by appropriate amino acid linking arm connection after, its foreign protein fragments can The surface of virus-like particle is farthest presented in, the immunologic competence of the fragment can be improved.
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, no infectivity after structural proteins appropriate location insertion foreign epitope, can repeat high density Be illustrated in VLP surfaces.Therefore, the characteristics of vaccine has safe and efficient is prepared using Hybrid virus like particles technology.
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 arctic ground squirrel hepatitis B core protein And the restructuring for the foreign protein fragments composition being inserted between the amino acid of the arctic ground squirrel hepatitis B core protein Fusion protein is expressed in Hansenula yeast and forms Hybrid virus like particles.
It is highly preferred that in yet another embodiment of the present invention, it is described by arctic ground squirrel hepatitis B core protein And the 78th of the arctic ground squirrel hepatitis B core protein is inserted between the 79th amino acids and by amino acid The recombination fusion protein for the foreign protein fragments composition that linking arm is connected with the arctic ground squirrel hepatitis B core protein Expression forms Hybrid virus like particles in Hansenula yeast.
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 For 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 arctic ground squirrel hapatitis virus core 78th foreign protein fragments between the 79th amino acids of heart protein are 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 GILE amino acid linking arm of N-terminal and the L amino acid linking arm of C-terminal with it is described The 78th of arctic ground squirrel 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 Former epitope II amino acid sequence 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.
Preferably, above-mentioned by arctic ground squirrel hepatitis B core protein and respiratory syncytial virus f-protein epitope The recombination fusion protein of composition is assembled into virus-like particle.
It is highly preferred that above-mentioned by arctic ground squirrel hepatitis B core protein and respiratory syncytial virus f-protein epitope The recombination fusion protein of composition 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 obtained recombination fusion protein gene order replace S gene sequences in carrier PUC25-SU Row, obtain PUC25-AGRU recombinant plasmids；
Step 3) by step 2) in obtained recombinant plasmid transformed enter induced expression in Hansenula yeast, it is extracted, after purification Obtain the recombination fusion protein.
Specifically, above-mentioned preparation method can further comprise the steps：
Step A) gene optimization and synthesis
By the arctic territory pine containing amino acid linking arm and respiratory syncytial virus f-protein fragment gene sequence simultaneously Murine hepatitis virus core protein gene sequence, optimizes and closes according to the superiority-inferiority and tRNA abundance of Hansenula yeast codon Into；
Step B) recombinant plasmid structure
By step A) in obtained gene order replace S gene orders in carrier PUC25-SU, obtain PUC25-AGRU Recombinant plasmid；
Step C) recombination fusion protein expression and purifying
By step B) in obtained PUC25-AGRU recombinant plasmids, carry out after the digestion of EcoR I and Hind III, at linearisation Reason, electricity is transformed into NVSI-H.P-105 (△ URA3 △ LEU2) Hansenula yeast bacterium, is obtained recombinant, is sieved by ELISA method Choosing obtains the high expression quantity positive strain of recombination fusion protein, and fermentation inducement expression harvests thalline, and high pressure is crushed, centrifuging and taking supernatant, Gel filtration chromatography, produces 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 arctic ground squirrel hepatitis B core protein and breathing Recombination fusion protein the answering in Respiratory Syncytial Virus(RSV) preventative vaccine is prepared of road syncytial virus f-protein epitope composition With.
Another aspect of the present invention there is provided a kind of vaccine, and the vaccine is above-mentioned by arctic territory comprising effective dose The recombination fusion protein that squirrel hepatitis B core protein is constituted with respiratory syncytial virus f-protein epitope, and adjuvant.
Wherein, described is preferably to contain 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 particulates, 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, such as intracutaneous (i.d.), intraperitoneal (i.p.), Intramuscular (i.m.), it is 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 arctic ground squirrel hepatitis Obtained after the recombination fusion protein immune body that malicious core protein is constituted with respiratory syncytial virus f-protein epitope.
Another aspect of the present invention there is provided a kind of bacterial strain for including above-mentioned recombination fusion protein.
Beneficial effects of the present invention are：
1) present invention is first by the use of arctic ground squirrel hepatitis B core protein as foreign epitope in delivery carrier, Ke Yiyou Effect ground makes foreign epitope repeat highdensity distribution on VLP surfaces, and do not influence VLP's in VLP surface display foreign epitopes Autonomous assembling.
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 common by arctic ground squirrel hepatitis B core protein and respiratory syncytial virus f-protein epitope The recombination fusion protein of composition, can combine the sharp pearl monoclonal antibody of handkerchief, after immune mouse, can produce special for RSV cause of diseases Property neutralizing antibody, for research and development RSV vaccines provide feasibility.
4) it is carrier based on AGSHV 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 being protected as vaccine composition On the premise of demonstrate,proving vaccine safety, it can stimulate and produce stronger immune response, the infection for preventing RSV, with important section Learn and application value.
Brief description of the drawings
Fig. 1 is PUC25-AGRU construction of recombinant plasmid schematic diagrames；
Fig. 2 is PUC25-AGRU recombinant plasmid digestion qualification result figures, and wherein Lane1 is the digestion of PUC25-AGRU plasmids As a result；
Fig. 3 is the high expression quantity positive yeast bacterial strain PCR qualification result figures of the albumen screened, and wherein Lane1 is bacterial strain PCR Electrophoresis result；
The recombination fusion protein component qualification result figure that Fig. 4 designs for the present invention, wherein,
A:Whole cell is analyzed after induced expression；
Lane1 is not induce thalline；
Lane2 is thalline after induction；
B:Recombination fusion protein SDS-PAGE is analyzed after purification；
Lane3 is destination protein electrophoresis result after purification；
C:The Western-blot analyses of recombination fusion protein；
Lane4 is the immunoblot results of purpose albumen；
Fig. 5 is the combination result figure of recombination fusion protein and the sharp pearl antibody of handkerchief；
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 horizontal result figure of the specific neutralizing antibody titers of cause of disease produced after fusion protein immunization mouse；
Sequence explanation
SEQ ID NO.1 are the amino acid sequence of arctic ground squirrel hepatitis B core protein in the present invention；
SEQ ID NO.2 are the amino acid sequence of the recombination fusion protein of the present 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 the recombination fusion protein of the present invention.
Embodiment
The invention discloses a kind of recombination fusion protein comprising arctic ground squirrel hepatitis B core protein and its preparation Methods and applications, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.Need what is particularly pointed out It is that all similar replacements and change are apparent to those skilled in the art, they are considered as being included in this Invent, and related personnel can substantially enter on the basis of present invention, spirit and scope are not departed to content described herein Row is changed or suitably changed with combining, 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 method 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：
The restriction enzyme of EcoRI, 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；
Long plants of RSV (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 arctic territory squirrel hepatitis B core protein amino sequence (UniProtKB/Swiss-Prot:Q64897.1) it is Reference sequences, form the carrier sequence as shown in SEQ ID NO.1, in the 78th and the 79th ammonia of SEQ ID NO.1 sequences Insertion respiratory syncytial virus f-protein (Genebank between base acid:ACO83301.1) 254-277 amino acids epitope (shown in SEQ ID NO.3), the epitope is the binding site of the sharp pearl antibody of handkerchief, passes through " GILE " and " L " amino acid linking arm It is in series, forms the amino acid sequence as shown in SEQ ID NO.2.
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 coding gene sequence as shown in SEQ ID NO.4, the sequence entered Row gene chemical synthesis.
3rd, the structure of expression plasmid
According to the plasmid construction schematic diagram shown in Fig. 1, target gene 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-AGRU, the plasmid integrates arm by homologous recombination of 25S rDNA on Hansenula yeast genome, with URA3 genes are marker gene, and destination protein expression is efficiently started with MOX promoters.
4th, the digestion identification of PUC25-AGRU expression plasmids and gene sequencing
EcoRI and the identification of the double digestions of Hind III, 37 DEG C, digestion 1 hour, enzyme are carried out to PUC25-AGRU expression plasmid of yeast Cut system as shown in table 1, the product after digestion is subjected to 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 4kb large fragment and 2kb small fragment, large fragment is to include restructuring to melt The Yeast expression frame of hop protein target gene, gene sequencing is carried out by the plasmid, as a result shows, nothing consistent with expected results The change of target gene.
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 the PUC25- after the double digestion of EcoR I and Hind III Large fragment gene is transformed into NVSI-H.P-105 saccharomycete by way of electricity conversion in AGRU plasmids, most transformed bacteria solution at last It is coated on SM-leu solid mediums, 37 DEG C are cultivated 3-5 days, obtains conversion recombinant.
2nd, ELISA is screened
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 to transfer in 4ml SM-leu fluid nutrient mediums to continue Culture, after cell density (OD600) reaches more than 10, thalline is harvested by centrifugation in 3000rpm, 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 buffers (20mM PB, pH value 7.2) and 200mg beades is added, it is high The vibration of frequency low temperature is broken, utilizes coating buffer (Na2CO3-NaHCO3Solution, pH value 9.6) albumen supernatant is diluted 500 times and 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 are added, color development at room temperature 10 minutes adds 50 μ l and terminated 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 the high table of recombination fusion protein Up to yeast strain.Because the yeast strain screened only supplements URA3 genes, it is only capable of in the culture medium of leucine is supplemented with Growth, therefore LEU2 genetic transformation is entered in the saccharomycete screened, the bacterial strain is grown in MD basal mediums.
3rd, strain target gene is identified
Recombination fusion protein height expression yeast strain genome is extracted, target gene 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 subjected to gene sequencing, target gene 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：CVLP preparation and identification
1st, yeast fermentation and bacterial cell disruption
Obtained recombination fusion protein height expression barms will be screened in embodiment 2 and is inoculated in 10ml MD Liquid Cultures Shaken cultivation 24 hours in base, then expansion culture 24 hours in 100mlMD fluid nutrient mediums of transferring, prepare fermentation seed, connect Plant in progress saccharomycetes to make fermentation culture in 5L fermentation tanks, and utilize the induced expression of methanol progress destination protein.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 subjected to 10%SDS-PAGE electrophoresis point Analysis, as a result as shown in Figure 4 A, arrow meaning are 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 level pad (50mM PB+0.2MNacl, pH7.3) the balance chromatographic column of 3 times of column volumes extremely 280nm absorption values, less than 0.5mAU, the absorption value of detector are zeroed without obvious change.
B, loading：The thick pure liquid of 100ml albumen is 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 handle chromatographic column, then utilize equilibration buffer Chromatographic column, continues the chromatographic purifying of next albumen.
Destination protein after purification is subjected to 10%SDS-PAGE electrophoretic analysis result as shown in Figure 4 B, arrow meaning is Destination protein band, molecular size range is about 25kD, basically identical with expected size.Destination protein after purification is carried out simultaneously Western-blot is detected, conventionally by after in destination protein transferring film to pvdf membrane, is examined using the sharp pearl antibody of handkerchief Survey, as a result as shown in Figure 4 C, arrow meaning is destination protein strip-like developing pipe.
3rd, with the binding tests of the sharp pearl antibody of handkerchief
CVLP after purification is subjected to 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 sharp pearl antibody concentration of handkerchief of addition is 1 μ g/ml, to verify the sharp pearl antibody of cVLP albumen and handkerchief Combination degree, as a result as shown in figure 5, cVLP can effectively with the sharp pearl antibody binding of handkerchief.
4th, cVLP dynamic light scattering and transmission electron microscope observing
Albumen after purification is subjected to dynamic light scattering (Malvern, NANO-8S90) analysis, as a result as shown in fig. 6, knot Fruit shows that VLP sizes homogeneity is good, and hydration particle diameter is about 29nm.Destination protein is dripped on the plating charcoal copper mesh film of 300 mesh, 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 0.5 μ g RSVsF albumen and 250 μ g aluminum hydroxide adjuvant mixture；C groups injecting normal saline and 250 μ G aluminum hydroxide adjuvant, 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 blood sampling simultaneously separates serum.Using virus it is micro in The specific neutralizing antibody titers levels of RSV in serum are detected with test method, as a result as shown in figure 8, recombination fusion protein (cVLP) after being mixed with aluminum hydroxide adjuvant, the specific neutralizing antibody titers of RSV of higher level can be produced, the present invention is pointed out weight Group fusion protein have can as RSV preventative vaccines potentiality.
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, it is in delivery carrier to separately design with groundhog hepatitis virus cAg (WHC) CVLP and with human hepatitis B virus core antigen (HBC) be in delivery carrier cVLP, according to the side described in embodiment 1,2 and 3 Method is prepared respectively, finally gives WHC-cVLP and HBC-cVLP.According to the animal immune method in embodiment 4, basis herein Upper two groups of increase, every group 8, D groups inject 0.5 μ g WHC-cVLP and 250 μ g aluminum hydroxide adjuvant mixture；E groups are injected 0.5 μ g HBC-cVLP and 250 μ g aluminum hydroxide adjuvant mixture, take abdominal channels to be immunized, and each group is immunized 3 times, Every 2 weeks.Daily observation mouse state and changes of weight after each group mouse immune.Immune two weeks after, disconnected cone blood sampling simultaneously separates serum, Utilize the specific neutralizing antibody titers levels of RSV in viral microneutralization test method detection serum.As a result as shown in figure 8, aobvious Show specific neutralizing antibody titers of RSV that the AGSHV-cVLP specific neutralizing antibody titers of RSV are about 9.5, WHC-cVLP about It is about that 3.0, AGSHV-cVLP is produced compared with WHC-cVLP and HBC-cVLP for 6.5, the HBC-cVLP specific neutralizing antibody titers of RSV Raw neutralizing antibody level is higher.Result above prompting AGSHV-cVLP has a clear superiority in terms of external source Epitope presentation, this Invention recombination fusion protein have can as RSV preventative vaccines potentiality.
Described 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 arctic ground squirrel hepatitis B core protein and its preparation method and application
<130> None
<160> 4
<170> PatentIn version 3.5
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Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gln Leu Leu
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Thr Ala Thr Ala Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys
35 40 45
Ser Pro His His Thr Ala Ile Arg Gln Ala Leu Val Cys Trp Glu Glu
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Leu Thr Arg Leu Ile Ala Trp Met Ser Ala Asn Ile Asn Ser Glu Glu
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Val Arg Arg Val Ile Val Ala His Val Asn Asp Thr Trp Gly Leu Lys
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Val Arg Gln Asn Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly Gln
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His Thr Val Gln Glu Phe Leu Val Ser Phe Gly Val Arg Ile Arg Thr
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Pro Ala Pro Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro
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Glu His Thr Val Ile Arg Arg Arg Gly Ser Ala Arg Val Val Arg Ser
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Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro
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<213>Recombination fusion protein
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Ser Pro His His Thr Ala Ile Arg Gln Ala Leu Val Cys Trp Glu Glu
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gagctgaccg gcagagagca ctgctcgcct caccacacgg ccatcagaca ggccctggtg 180
tgctgggagg agctgacgag actgatcgcc tggatgtcgg ccaacatcaa ctcgggcatc 240
ctggagaact cggagctgct gtcgctgatc aacgacatgc ctatcaccaa cgaccagaag 300
aagctgatgt cgaacaacct ggaggaggtg agaagagtga tcgtggccca cgtgaacgac 360
acctggggcc tgaaggtgag acagaacctg tggttccacc tgtcgtgcct gaccttcggc 420
cagcacaccg tgcaggagtt cctggtgtcg ttcggcgtga gaatcagaac ccctgcccct 480
tacagacctc ctaacgcccc tatcctgtcg accctgcctg agcacaccgt gatcagaaga 540
agaggctcgg ccagagtggt gagatcgcct agaagaagaa ccccttcgcc tagaagaaga 600
agatcgcagt cgcctagaag aagacctcag tcgcctgcct cgaactgcta a 651

Claims (16)

1. a kind of recombination fusion protein for including arctic ground squirrel hepatitis B core protein, it is characterised in that the restructuring is melted Hop protein is by arctic ground squirrel hepatitis B core protein and the ammonia for being inserted in the arctic ground squirrel hepatitis B core protein Foreign protein fragments composition between base acid.
2. recombination fusion protein according to claim 1, it is characterised in that the foreign protein fragments are inserted in described The 78th of arctic ground squirrel hepatitis B core protein is between the 79th amino acids, and the foreign protein fragments pass through ammonia Base acid linking arm is connected with the arctic ground squirrel hepatitis B core protein.
3. recombination fusion protein according to claim 1 or 2, it is characterised in that the recombination fusion protein is assembled into disease Malicious sample particle.
4. a kind of the answering in antigen presentation carrier is prepared of the recombination fusion protein as described in any one in 1-3 such as claim With.
5. application of the recombination fusion protein as described in any one in vaccine is prepared in a kind of 1-3 such as claim.
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 or 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 close for respiratory tract 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 passes through the GILE amino acid linking arm of N-terminal and the L amino acid linking arm of C-terminal and the arctic ground squirrel hepatitis The 78th of malicious core protein 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 protein according to claim 10, it is characterised in that the recombination fusion protein is assembled into virus Sample particle.
12. a kind of preparation method of recombination fusion protein as described in any one in claim 1-3,7-11, its feature It is, comprises the following steps：
Step 1) gene order of recombination fusion protein is excellent according to superiority-inferiority and tRNA the abundance progress of Hansenula yeast codon Change and synthesize, the recombination fusion protein gene order after being optimized；
Step 2) with step 1) in obtained recombination fusion protein gene order replace S gene orders in carrier PUC25-SU, Obtain PUC25-AGRU recombinant plasmids；
Step 3) by step 2) in obtained recombinant plasmid transformed enter induced expression in Hansenula yeast, it is extracted, obtain after purification The recombination fusion protein.
13. the recombination fusion protein as described in any one in a kind of 7-11 such as claim is pre- in preparation Respiratory Syncytial Virus(RSV) Application in anti-property vaccine.
14. a kind of vaccine, it is characterised in that the vaccine 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 individual.
16. a kind of bacterial strain of the recombination fusion protein comprising as described in any one in claim 1-3,7-11.
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