CN110201183A - It is a kind of can simultaneously delivery of antigens and immunopotentiator nanometer formulation - Google Patents

It is a kind of can simultaneously delivery of antigens and immunopotentiator nanometer formulation Download PDF

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CN110201183A
CN110201183A CN201910421369.2A CN201910421369A CN110201183A CN 110201183 A CN110201183 A CN 110201183A CN 201910421369 A CN201910421369 A CN 201910421369A CN 110201183 A CN110201183 A CN 110201183A
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gly
glu
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汤书兵
周伟
袁伟明
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Guangzhou Women and Childrens Medical Center
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Guangzhou Women and Childrens Medical Center
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Priority to CN201910421369.2A priority Critical patent/CN110201183A/en
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Priority to PCT/CN2020/091636 priority patent/WO2020233685A1/en
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    • 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
    • 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
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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

Abstract

The invention discloses it is a kind of can delivery of antigens and immunopotentiator simultaneously nanometer formulation, the nanometer formulation can efficiently delivery of antigens and immunopotentiator simultaneously, and there is good specificity.The nanometer formulation is merged with the C-terminal of connector gbl-intein by the N-terminal of ferritin and forms recombinant protein gbl-inteincSelf-chambering is made into 24 aggressiveness and constitutes nano particle after-ferrtin；The N-terminal of the C-terminal fusion intein of foreign protein forms recombinant protein, and the N-terminal specific recognition of intein simultaneously cuts off the ferritin for being exposed to the nano grain surface；Foreign protein and ferritin covalent cross-linking and obtain；Wherein, the foreign protein is immunopotentiator or antigen or both combination.

Description

It is a kind of can simultaneously delivery of antigens and immunopotentiator nanometer formulation
Technical field
The present invention relates to nanosecond medical science field, especially it is a kind of can delivery of antigens and immunopotentiator simultaneously nanometer system Agent.
Background technique
Vaccine is to prevent and control most effective, the most economical medical intervention means of communicable disease.Attenuation or inactivation cause of disease Immense success is obtained as traditional classical vaccine, the help mankind exterminate smallpox and infantile paralysis.However, traditional vaccine is deposited It can not prevent in security risk and effectively the virus with high frequency catastrophe characteristics, such as HIV, influenza virus.
Subunit vaccine because it is easy to purify, safely and effectively, stable in physicochemical property the advantages that due to pay close attention to by vaccine scholar. Subunit vaccine immunogenicity is poor, and induction immune response is weaker and the duration is short, limits its application clinically.A new generation Vaccine improves the range and dynamics of body response immune response using nano-delivery system and immunopotentiator.
Nano particle can be designed to multifunctional carrier, while delivery of antigens and Immune-enhancing effect by the means of genetic engineering Agent.The method Antigen and Immune-enhancing effect agent molecule of Gene Fusion are high-efficient, easy to operate, can be widely used in grinding for vaccine Hair.However the antigen of load may seriously affect nano particle assembling, cause recombinant protein to form precipitating, need a series of multiple Property-assembles again can just re-form nano particle.In addition, the method for Gene Fusion is difficult to antigen molecule and immunopotentiator point Son is integrated on the same nano particle simultaneously.
Chemical coupling can modify nano particle to convenient, polymorphism, make its multifunction, and realization is passed altogether on nano particle Send immunopotentiator and antigen.But this method efficiency is lower, poor specificity, side reaction may be with side effect.These disadvantages Largely limit the application of nanometer formulation.
Summary of the invention
The present invention is intended to provide one kind can efficiently delivery of antigens and immunopotentiator simultaneously, and being received with good specificity Metric system agent.
In order to achieve the above technical purposes, technical solution provided by the invention is such that
It is a kind of can simultaneously delivery of antigens and immunopotentiator nanometer formulation, by ferritin N-terminal and connector gbl- The C-terminal of intein merges to form recombinant protein gbl-inteincSelf-chambering is made into 24 aggressiveness and constitutes nano particle after-ferrtin；Outside The N-terminal of the C-terminal fusion intein of source protein forms recombinant protein, and the N-terminal specific recognition of intein and cutting off is exposed to described The ferritin of nano grain surface；Foreign protein and ferritin covalent cross-linking and obtain；
Wherein, the foreign protein is immunopotentiator or antigen or both combination.
Wherein, the immunopotentiator is flagellin or avidin monomer analog rhizavidin；Institute The antigen stated is the proteantigen of single polypeptide epitope or the proteantigen of different polypeptide epitopes.
Wherein, when the foreign protein is that both immunopotentiator and antigen combine, molar ratio is 1~3:5.
Wherein, the ferritin is the heavy chain ferritin of source of people or the heavy chain ferritin or other species of pyrococcus furiosus The ferritin in source.
Compared with prior art, the present invention having the advantage that
Nanometer formulation of the invention can efficiently show antigen protein, immunopotentiator, polypeptide epitope in nano grain surface And antibody, for enhanced preventative subunit vaccine and personalized polypeptide epitope tumor vaccine research and development and antibody coupling drug Field.
The present invention, which solves load protein vs protein self-assembly nanoparticles stable, to be influenced, and it is efficient to realize nanometer formulation Ground while delivery of antigens and immunopotentiator, while and there is good specificity.
Detailed description of the invention
Fig. 1 is the schematic diagram that nano-carrier module constructs principle.
Fig. 2 is to introduce connector gbl-inteinCThe analysis chart of nanoparticles stable is not influenced；
Fig. 2 a is the electrophoretogram of three kinds of recombinant proteins；Fig. 2 b is gb1-inteinC- HFT and gb1-inteinC- PFT is through sulphur Electrophoretogram behind sour ammonia-sinking shallow lake；Fig. 2 c is gb1-inteinCThe electrophoretogram of-HFT after purification；Fig. 2 d is gel-filtration purified gb1- inteinCThe UV of-HFT gel schemes；Fig. 2 e is the gb1-intein of TEM detection purifyingCThe electron microscope of-HFT；Fig. 2 f is TEM detection The gb1-intein of purifyingCThe electron microscope of-PFT.
Fig. 3 is that GFP albumen and strep2 are efficiently covalently coupled to the analysis chart after HFT；
Fig. 3 a is the electrophoretogram of Coomassie Brilliant Blue (CB assay) testing goal product GFP-HFT content；Fig. 3 b is to make With the electrophoretogram of Western-blotting detection (WB assay) GFP and HFT coupling efficiency；Fig. 3 c is coomassie brilliant blue inspection Survey the electrophoretogram of purpose product strep2-HFT content；Fig. 3 d be using Western-blotting analyze strep2 polypeptide with The electrophoretogram of HFT coupling efficiency.
Fig. 4 is to load GFP albumen and strep2 polypeptide by way of self cleavage not influencing nanoparticle structure stability Analysis chart；
Fig. 4 a is the dying method with coomassie brilliant blue electrophoretic analysis figure of gel-filtration purified GFP-HFT；Fig. 4 b is gel filtration Purify the UV curve graph of GFP-HFT；Fig. 4 c is the electron microscope of the GFP-HFT of TEM detection purifying；Fig. 4 d is gel-filtration purified The electrophoretic analysis figure of strep2-HFT；Fig. 4 e is the UV curve graph of gel-filtration purified strep2-HFT；Fig. 4 f is that TEM detection is pure The electron microscope of the strep2-HFT of change.
Fig. 5 is that nano particle provided by the invention and foreign protein are prepared not through intein mediating proteins editing technique montage With nano-device schematic diagram.
Fig. 6 be nano particle can high-efficient carrier polypeptide epitope and flagellin analysis chart；
Fig. 6 a is the gel-filtration purified electrophoretic analysis figure of nano vaccine SP70-HFT；Fig. 6 b is gel-filtration purified nanometer The UV curve graph of vaccine SP70-HFT；Fig. 6 c is the transmission electron microscope picture of nano vaccine SP70-HFT；Fig. 6 d is SP70 and CBLB total Deliver the gel-filtration purified electrophoretic analysis figure of nanometer formulation SP70-CBLB-HFT；Fig. 6 e is that gel-filtration purified total delivering is received The UV curve graph of metric system agent SP70-CBLB-HFT；Fig. 6 f is the transmission electron microscope picture for delivering nanometer formulation SP70-CBLB-HFT altogether； Fig. 6 g is the gel-filtration purified electrophoretic analysis figure of nanometer adjuvant CBLB-HFT；Fig. 6 h is gel-filtration purified nanometer adjuvant The UV curve graph of CBLB-HFT；Fig. 6 i is the transmission electron microscope picture of nanometer adjuvant CBLB-HFT.
Fig. 7 be modularization nano-carrier whether can high-efficient carrier CpG analysis chart；
Fig. 7 a is the gel-filtration purified electrophoretic analysis figure of rhizavidin-HFT；Fig. 7 b is rhizavidin-HFT gel The UV curve graph of Purification by filtration；Fig. 7 c is the transmission electron microscope picture of rhizavidin-HFT；Fig. 7 d is SP70-rhizavidin-HFT Gel-filtration purified electrophoretic analysis figure；Fig. 7 e is the gel-filtration purified UV curve graph of SP70-rhizavidin-HFT；Fig. 7 f For the transmission electron microscope picture of SP70-rhizavidin-HFT；Fig. 7 g is the two-dimentional class mean chart of SP70-rhizavidin-HFT；Figure 7h is purpose albumen rhizavidin-HFT combination biotinylation CpG electrophoretic analysis figure.
Fig. 8 is by model analysis difference vaccine dosage of SP70 epitope to the impact analysis figure of immune effect；
Fig. 8 a-c is that elisa assay different modes deliver CpG adjuvant and CBLB induces IgG titre；Fig. 8 d cell in vitro The antibody neutralization titer of titrimetry difference vaccine preparation induction；Fig. 8 e is the immune response of delivering nanometer formulation induction altogether.
Fig. 9 be albumen editor shearing technique can on HFT/PFT carrier high-efficient carrier HA antigenic analysis figure；
Fig. 9 a is recombination HA-HFT expression analysis electrophoretogram；Fig. 9 b is gel-filtration purified based on albumen editor shearing The electrophoretic analysis figure of the HA-HFT of technology preparation；Fig. 9 c is the UV curve graph of gel-filtration purified HA-HFT；Fig. 9 d is HA- The transmission electron microscope picture of HFT；Fig. 9 e is the electrophoretic analysis of the gel-filtration purified HA-PFT based on the preparation of albumen editor's shearing technique Figure；Fig. 9 f is the UV curve graph of gel-filtration purified HA-PFT；Fig. 9 g is the transmission electron microscope picture of HA-PFT.
Figure 10 is that nanometer adjuvant CpG-HFT enhances HA specific humoral immune response and regulates and controls the analysis chart of IgG type.
Figure 10 a is that elisa assay announcement nano-carrier and nanometer adjuvant can enhance HA specific humoral immune response；Figure 10b-d be elisa assay disclose vaccine induced antibody can specific recognition H1N1 (P), H3N2 and H7H9 strain, and nanometer adjuvant It is higher than nano vaccine itself with the antibody recognition capability of nano vaccine mix preparation induction.
Specific embodiment
With reference to embodiment, claim of the invention is described in further detail, but do not constituted pair Any restrictions of the invention, any limited times modification made in the claims in the present invention protection scope, still of the invention In claims.
Embodiment 1
Step 1: by the heavy chain ferritin (human heavy chain of human ferrtin, write a Chinese character in simplified form HFT) of source of people N-terminal merge to form recombinant protein gbl-intein with the C-terminal of connector gbl-inteincSelf-chambering is made into 24 aggressiveness after-ferrtin Constitute the nano particle that diameter is 18nm.
The N-terminal of the C-terminal fusion intein of step 2:SP70 forms recombinant protein, and the N-terminal specific recognition of intein is simultaneously cut Except the ferritin for being exposed to the nano grain surface；30 μM of foreign proteins and 30 μM of ferritin covalent cross-linkings generate nanometer system Agent.
Embodiment 2
Step 1: by heavy chain ferritin (the pyrococcus furiosus heavy chain of of pyrococcus furiosus Human ferrtin, writes a Chinese character in simplified form PFT) N-terminal merge to form recombinant protein gbl-intein with the C-terminal of connector gbl-inteinc- Self-chambering is made into 24 aggressiveness and constitutes the nano particle that diameter is 18nm after ferrtin.
Step 2: the N-terminal of the C-terminal fusion intein of flagellin forms recombinant protein, the N-terminal specific recognition of intein And cut off the ferritin for being exposed to the nano grain surface；60 μM of foreign proteins and 30 μM of ferritin covalent cross-linkings, generation are received Metric system agent.
Embodiment 3
Step 1: being merged by the N-terminal of the heavy chain ferritin of source of people with the C-terminal of connector gbl-intein and form recombinant protein gbl-inteincSelf-chambering is made into 24 aggressiveness and constitutes the nano particle that diameter is 18nm after-ferrtin.
Step 2: the C-terminal in the CpG of the amount of commaterial and area, influenza virus hemagglutinin stem is merged to the N-terminal of intein simultaneously Recombinant protein is formed, the N-terminal specific recognition of intein simultaneously cuts off the ferritin for being exposed to the nano grain surface；45 μM outer Source protein and 30 μM of ferritin covalent cross-linkings generate nanometer formulation.
Following protein involved in experiment and embodiment of the invention, polypeptide gene chemical synthesis reveal port biology section in Shanghai Skill Co., Ltd, primer are synthesized in Nanjing Genscript Biotechnology Co., Ltd..
Wherein, HFT is the heavy chain ferritin (human heavy chain of human ferrtin) of source of people, and PFT is The heavy chain ferritin (pyrococcus furiosus heavy chain of human ferrtin) of pyrococcus furiosus.
GFP-intein in the present inventionN-gb1、strep2-gp41-1-inteinN-gb1、SP70-gp41-1-inteinN- gb1、rhizavidin-gp41-1-intN-gb1、CBLB-gp41-1-inteinN、SP70-CBLB-gp41-1-inteinN、 H1HA10-gp41-1-inteinN- gb1 is inteinNRelevant " cargo " fusion protein.The gene of " cargo " fusion protein is logical Overlap PCR method amplifying target genes are crossed, the 5 ' primers and 3 ' primers used are respectively provided with NcoI and XhoI restriction enzyme site. Fusion is inserted into the linearisation pET28a carrier of identical digestion after digestion, and conversion to e. coli jm109 carries out recombination gram Grand building.gb1-gp41-1-inteinC-HFT、gb1-gp41-1-inteinC- PFT is inteinCRelevant " carrier " fusion Albumen.The gene of " carrier " fusion protein is also through overlap PCR method amplifying target genes, 5 ' primers and 3 ' primers difference With EcoRI and XhoI restriction enzyme site.After segment insertion pET28a linearized vector after digestion, e. coli jm109 is converted. Above-mentioned recombination extracts plasmid and converts to e. coli bl21 (DE3) plysS progress protein expression after being sequenced correctly.It chooses Monoclonal bacterial strain is taken, the card for being seeded to 20ml is received in the dual anti-LB culture medium of chloramphenicol, and 37 DEG C of shaking tables are incubated overnight.Second day 1:25 The card for being seeded to 500ml receives the dual anti-LB culture medium of chloramphenicol, 37 DEG C, 250rpm cultivate to OD be 0.6-0.8 when, be added 0.2mM IPTG is induced.25 DEG C, 250rpm induction 5h after, 4000rpm, 4 DEG C, centrifugation in 30 minutes receipts bacterium.Supernatant is abandoned, bacterium can It is frozen for next step purifying or -80 DEG C spare.
What the gene that the present invention recombinates primer and " cargo " fusion protein that " carrier " antigen-4 fusion protein gene uses used draws See Table 1 for details for object:
Table 1:
Note: primer used in the present invention is used to prepare the nano-device of different function.
Antigen or adjuvated protein purifying of the present invention: all albumen are all made of Ni2+The method of affinity chromatography carries out Purifying.Different recombinant proteins, because of its property difference, the purification process of different solubility, use slightly has difference.GFP- inteinN-gb1、strep2-inteinN-gb1、SP70-inteinN- gb1, H1HA10-inteinN-gb1 and Rhizavidin-inteinN-gb1 is soluble protein, and bacterial lysate supernatant can be directly added into Ni2+Carry out parent in resin And chromatographic purifying.30ml NT buffer cracks the supernatant of bacterium after being resuspended, 30 points are mixed on 4 DEG C of shaking tables with the resin of 1ml Clock.Then, non-specific binding albumen is flowed out in a manner of gravity with liquid.Then, it is added containing 10mM, 20mM, 40mM imidazoles 20ml NT buffer washs resin, removes non-specific binding albumen.Then, the NT buffer for 500mM imidazoles being added is washed It is de-.Finally, eluted product 4 DEG C of dialysed overnights in the NT buffer of 1L.CBLB-inteinNAnd SP70-CBLB-inteinNGreatly Part forms precipitating, and after the NT buffer of precipitating urea containing 2M is resuspended, bacterial lysate supernatant soluble protein is purified Method purifying.CBLB-inteinNAnd SP70-CBLB-inteinNIt is most of to form inclusion body precipitating, the NT of the urea containing 2M can be used After buffer is resuspended, the method that supernatant soluble protein is purified after re-suspension liquid centrifugation is purified.
The building of 1 nano-carrier modular platform of experimental example
1. intein (inteinC) and HFT progress Gene Fusion, obtain recombinant protein inteinC- ferrtin is (in Fig. 2 a Labeled as 1).
2. intein (intein) introduces streptococcal protein G B1 structural domain label (gb1) and constructs connector gb1-inteinC (A is shown as in Fig. 1).The N-terminal and connector gb1-intein of heavy chain ferritin (C is shown as in ferrti, Fig. 1)CCarry out Gene Fusion Constitute recombinant protein gb1-inteinC-ferrtin.Recombinant protein gb1-intein is constructed with HFT and PFT respectivelyC- HFT (figure Label is in 2a-2b) and gb1-inteinC- PFT (label is in Fig. 2 a-2b).In each figure of the application such as Fig. 2, this hair The abscissa of all UV profile of gel filtration represents elution volume in bright, and ordinate represents ultraviolet light Absorbance change at A280nm.
Above-mentioned three kinds of recombinant protein bacteria lysis are taken to carry out Tricine-SDS-PAGE electrophoresis, as a result as shown in Figure 2 a, In, control:pET28a carrier blank control bacterial lysate, 1:inteinC- ferrtin, 2:gb1-inteinC- HFT, 3: gb1-inteinC- PFT, all: bacterial lysate, up: supernatant.From Fig. 2 a as it can be seen that from 35kDa electrophoresis bacterial lysate have it is bright Aobvious band, but almost invisible protein band in the supernatant of corresponding position, show recombinant protein inteinC- ferrtin formation is forgiven Body.At 35kDa-40kDa electrophoresis band clearly, recombinant protein gb1-inteinC- HFT and gb1-inteinC- PFT expression quantity Significantly increase, and soluble high.By result as it can be seen that the table of heavy chain ferritin in the cell can be greatlyd improve by introducing gb1 Its solubility is reached and improved, scale is advantageously formed and prepares, therefore recombinant protein gb1-inteinC- ferrtin nano particle tool There is the feasibility for being used to prepare nano-carrier platform.
Bacterium is cultivated with the LB that 500ml is resuspended in the 50mM Tris, pH 7.5 of 30ml, 150mM Nacl (NT buffer), Then ultrasonication.After ultrasound, 12000rpm, it is centrifuged for 4 DEG C, 30 minutes, supernatant is transferred in the centrifuge tube of 50ml.It is added 12000rpm, the ammonium sulfate solids of 0.15g/ml are centrifuged for 4 DEG C, 30 minutes after 4 DEG C of shaking tables mix 15 minutes.Supernatant is abandoned, is obtained Ammonium sulfate precipitation product (label is in Fig. 2 b-2c).Ammonium sulfate precipitation product is resuspended with the NT buffer of 10ml, 4 DEG C of dialysed overnights in the NT buff of 1L.BCA measures gb1-inteinC- HFT and gb1-inteinC- PFT concentration is respectively 10mg/ml and 5mg/ml.After ammonium sulfate precipitation product is resuspended with 50mM pH8.0Tris and 150mM Nacl (NT buffer), It is utilized on the Shan Pu Biotechnology Co., Ltd ClearFirst-3000 type protein purification system of Shanghai Superpose6Increase gel filtration isolates and purifies, and carries out Tricine-SDS-PAGE and carry out electrophoresis observation purity, pure Change result and see Fig. 2 b-2d, wherein Fig. 2 b is gb1-inteinC- HFT and gb1-inteinCElectricity of-the PFT after ammonium sulfate precipitation Swimming figure, wherein up: supernatant, pellet: ammonium sulfate precipitation product；Fig. 2 c is gb1-inteinCThe electrophoresis of-HFT after purification Figure, wherein pellet: product, Purification of gb1-intein is resuspended in ammonium sulfate enrichment precipitatingC-HFT by gel Filtration: gel filtration method purifies gb1-inteinC- HFT, gel filtration of gb1-inteinC- HFT: Gel-filtration purified gb1-inteinC- HFT, 9-17: it is in charge of and collects gel-filtration purified gb1-inteinCThe volume of-HFT sample Number；Fig. 2 d is gel-filtration purified gb1-inteinCThe UV of-HFT gel schemes, and Detection wavelength 280nm, arrow is referred to gb1-inteinCThe distributing position of-HFT.
Pass through the recombination egg of transmission electron microscope (transmission electron microscope, TEM) observation after purification White gb1-inteinC- HFT and gb1-inteinC- PFT, transmission electron microscope amplification factor are 110k, length of the scale 20nm.As a result It is detailed in Fig. 2 e and 2f, wherein Fig. 2 e is the gb1-intein of TEM detection purifyingCThe electron microscope (negative staining sample preparation) of-HFT；Fig. 2 f is The gb1-intein of TEM detection purifyingCThe electron microscope of-PFT；Fig. 2 e and Fig. 2 f show gb1-inteinC- HFT and gb1- inteinC- PFT exists with form of nanoparticles, and scale used in transmission electron microscope observing negative staining sample represents in the present invention 20nm, arrows are nano particle.
Two kinds of recombinant proteins exist in the form of the spherical nanoparticle of 10-20nm, it is seen that recombinant protein gb1- inteinC- ferrtin forms nano particle by molecular self-assembling.After measured, recombinant protein gb1-inteinC- HFT and gb1- inteinCIt is 18nm nano particle that-PFT can be self-assembled into diameter with 24 monomers.
2 nano-carrier modular platform of experimental example can be with the efficient covalent coupling of foreign protein
The recombinant protein gb1-intein constructed with embodiment 1CFor-HFT nano particle, respectively with foreign protein GFP Albumen and strep2 Polypeptide tags (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) carry out the montage of intein mediating proteins and repair Decorations, observe the stability of the nano particle.
The C-terminal of 1.GFP albumen and strep2 Polypeptide tags introduces connector intein by Gene Fusion respectivelyN- gb1, point It Gou Cheng not GFP-inteinN- gb1 and strep2-inteinN-gb1。
2. taking 2 μ l concentration is the recombinant protein gb1-intein of 10mg/mlC- HFT mother liquor is added in DTT, and at room temperature 1 In hour, the GFP-intein that concentration is 3mg/ml is gradually increased in DTTNThe strep2- of-gb1 albumen mother liquor or 5mg/ml inteinN- gb1 albumen mother liquor is incremented by addition, and is observed purpose with 3 μ l, 6 μ l, 9 μ l, 12 μ l, 15 μ l, 18 μ l, 21 μ l, 24 μ l The amount of product GFP-HFT or strep2-HFT.Reaction condition: reaction system total volume is 30 μ l, 2mM DTT, room temperature 1 hour. Then Coomassie Brilliant Blue detection (CB assay) and western blotting detection (WB assay) are carried out respectively
3. testing result
As shown in Fig. 3 a and 3c, as the amount of GFP albumen and strep2 label gradually increases, purpose product can be promoted The amount of GFP-HFT (Fig. 3 a) and strep2-HFT (Fig. 3 c) increase.Fixed gb1-inteinCThe amount of-HFT increases GFP- inteinN- gb1 or strep2-inteinN- gb1 reaction density, reaction substrate gb1-inteinC- HFT and GFP-inteinN- Gb1 or strep2-inteinNUp to utmostly, intein self cleavage efficiency is close for reaction when the molar ratio of-gb1 is close to 1:1 Highest is not sheared gb1-inteinC- HFT almost noresidue.After the reaction was completed, anti-with GFP tag antibody, strep2 label Body and HFT antibody detect the variation of each reaction substrate and purpose product in reaction system respectively.Western-blotting detection Statistics indicate that trans cleavage efficiency is very high, react thoroughly (Fig. 3 b and 3d).As it can be seen that GFP and strep2 can be by efficient, special Ground is covalently cross-linking on HFT albumen.
After reaction, gained purpose product is in Shanghai Shan Pu Biotechnology Co., Ltd ClearFirst-3000 type egg Carry out gel filtration using 6 increase of superpose on white purification system to be purified.As a result it is detailed in Fig. 4, wherein figure 4a is the dying method with coomassie brilliant blue electrophoretic analysis figure of gel-filtration purified GFP-HFT；Fig. 4 b is gel-filtration purified GFP-HFT UV curve graph；Fig. 4 c is the electron microscope (negative staining sample preparation) of the GFP-HFT of TEM detection purifying；Fig. 4 d is gel-filtration purified The electrophoretic analysis figure (coomassie brilliant blue staining) of strep2-HFT；Fig. 4 e is the UV curve of gel-filtration purified strep2-HFT Figure；Fig. 4 f is the electron microscope (negative staining sample preparation) of the strep2-HFT of TEM detection purifying；Before: before intein self cleavage, After: after intein self cleavage, 9-20 in Fig. 4 a8-19 and 4d: it is in charge of and collects gel-filtration purified purpose product sample Number.
Such as Fig. 4 a, shown in 4b, molecular weight very close Protein G FP-HFT and GFP-inteinN- gb1 can be well It separates, and Protein G FP-HFT elution site ratio GFP-inteinN- gb1 is early, and arrow meaning is the peak of GFP-HFT in Fig. 4 b, shows Show that Protein G FP-HFT exists in the form of macromolecule polyalcohol.Fig. 4 d, shown in 4e, the very close albumen of molecular weight Strep2-HFT and strep2-inteinN- gb1 can also be separated well, and albumen strep2-HFT elution site ratio strep2-inteinN- gb1 is early, and arrow meaning is the peak of GFP-HFT in Fig. 4 e, shows albumen strep2-HFT with macromolecular The form of polymer exists.The purpose product GFP-HFT and strep2-HFT that isolate and purify are passed through into transmission electron microscope observing, GFP- There is (Fig. 4 c, 4f) in the form of nano particle in HFT and strep2-HFT.To sum up, albumen or polypeptide can be sheared efficiently to HFT Nano particle, and nanoparticles stable is not influenced, nano-carrier modular platform can be with the efficient covalent coupling of foreign protein.
The polymorphism of 3 nano-carrier modular platform of experimental example
Using the SP70 neutrality epitope of human enterovirus 71 (enterovirus 71) as antigen model, inquire into different The influence of drug delivery system and adjuvant to antigen immune response.Nanometer is modified with allowing polymorphism using nano-carrier building block technique The characteristics of grain, it is prepared for the nano particle of different function, to study influence of the different vaccine dosages to antigen immunogenicity, established Nanometer adjuvant, vaccine-adjuvant deliver nanometer formulation and nano vaccine altogether, are detailed in Fig. 5.
It is that adjuvant prepares corresponding preparations respectively with CpG or flagellin (flagellin).Flagellin specifically uses CBLB.CBLB is a truncate of flagellin, has its adjuvant function.
Nano particle can the analysis chart of high-efficient carrier polypeptide epitope and flagellin be detailed in Fig. 6: where Fig. 6 a is nanometer epidemic disease The gel-filtration purified electrophoretic analysis figure of seedling SP70-HFT；Fig. 6 b is the UV curve of gel-filtration purified nano vaccine SP70-HFT Figure；Fig. 6 c is the transmission electron microscope picture of nano vaccine SP70-HFT；Fig. 6 d is that SP70 and CBLB deliver nanometer formulation SP70- altogether The gel-filtration purified electrophoretic analysis figure of CBLB-HFT；Fig. 6 e is gel-filtration purified delivering nanometer formulation SP70-CBLB- altogether The UV curve graph of HFT；Fig. 6 f is the transmission electron microscope picture (negative staining sample preparation) for delivering nanometer formulation SP70-CBLB-HFT altogether；Fig. 6 g is The gel-filtration purified electrophoretic analysis figure of nanometer adjuvant CBLB-HFT；Fig. 6 h is gel-filtration purified nanometer adjuvant CBLB-HFT's UV curve graph；Fig. 6 i is the transmission electron microscope picture (negative staining sample preparation) of nanometer adjuvant CBLB-HFT；Before: before intein self cleavage, After: after intein shearing, the 8-19 in Fig. 6 a, 6d and 6g: it is in charge of the volume for collecting gel-filtration purified purpose product sample Number.
1. nano vaccine: 1. intein (intein) introduces streptococcal protein G B1 structural domain label (gb1) and constructs connector gb1-inteinC(A is shown as in Fig. 1).The N-terminal and connector gb1-intein of source of people heavy chain ferritin HFTCCarry out Gene Fusion structure At recombinant protein gb1-inteinC-HFT.24 recombinant protein gb1-inteinC- HFT molecular self-assembling is at 24 aggressiveness nanometers Grain.2. the C-terminal of SP70 introduces inteinN- gb1 constitutes Protein S P70-inteinN-gb1.In 2mM DTT solution, in molar ratio Nano particle and Protein S P70-intein are added for 1:1N- gb1 obtains SP70-HFT nano particle through protein splice, as receives Rice vaccine delivery system.
2. nanometer adjuvant:
The C-terminal of 2.1CBLB introduces inteinNConstitute PROTEIN C BLB-inteinN, in 2mM DTT solution, by HFT with CBLB molar ratio 1:1 adds nano particle and CBLB-inteinN, CBLB-HFT is obtained through protein splicing, and be self-assembly of CBLB-HFT nano particle.
2.2CpG cannot be directly covalently attached with HFT, but biotinylated CpG can pass through the height of avidin-biotin Close combination, is loaded to nano grain surface.Biotinylated CpG can be combined by the height parent of avidin-biotin to be made With being loaded to nano grain surface.The C-terminal of avidin rhizavidin introduces inteinN- gb1 constitutes albumen rhizavidin-inteinN- gb1 adds nano particle and rhizavidin-intein in 2mM DTT solutionN- gb1, warp Protein splicing obtains rhizavidin-HFT, and is self-assembly of rhizavidin-HFT nano particle.
Modularization nano-carrier whether can the analysis chart of high-efficient carrier CpG be detailed in Fig. 7；Wherein, Fig. 7 a is rhizavidin- The gel-filtration purified electrophoretic analysis figure of HFT；Fig. 7 b is the gel-filtration purified UV curve graph of rhizavidin-HFT；Fig. 7 c is The transmission electron microscope picture (negative staining sample preparation) of rhizavidin-HFT；Fig. 7 d is the gel-filtration purified of SP70-rhizavidin-HFT Electrophoretic analysis figure；Fig. 7 e is the gel-filtration purified UV curve graph of SP70-rhizavidin-HFT；Fig. 7 f is SP70- The transmission electron microscope picture (negative staining sample preparation) of rhizavidin-HFT；Fig. 7 g is the two-dimentional class mean chart of SP70-rhizavidin-HFT； Fig. 7 h is purpose albumen rhizavidin-HFT combination biotinylation CpG electrophoretic analysis figure；Before: before intein self cleavage, After: after intein shearing, the 8-19 in Fig. 7 a, 7d and 7g is numbered: being in charge of and is collected gel-filtration purified purpose product sample Number.
As shown in figs 7 a-b: rhizavidin can efficiently be coupled to HFT, and purpose product can be purified with gel filtration. There is (Fig. 7 c) in the form of spherical nanoparticle through transmission electron microscope observing in gel-filtration purified rhizavidin-HFT.And Rhizavidin and SP70 is coupled with the molar ratio of 1:5, is not also influenced nanoparticle structure, is detailed in Fig. 7 d-f.Such as Fig. 7 g institute Show, two-dimentional class average (2D-class average) analysis, white circle indicates destination protein self-assembly at spherical nano-scale Particle, the white speck of discrete distribution is rhizavidin albumen (electron density is bigger than polypeptide) in circle, and speck is average The rhizavidin-intein of number and initial reactionN- gb1 and SP70-inteinNThe concentration of-gb1 is consistent.As shown in Fig. 7 h, After purpose product rhizavidin-HFT is in conjunction with enough biotinylated CpG, protein band migration discloses CpG by success Load.
Therefore, biotinylated CpG can be covalently attached with nano particle by the following method.As shown in figure 5,1. intein (intein) it introduces streptococcal protein G B1 structural domain label (gb1) and constructs connector gb1-inteinC(A is shown as in Fig. 1).Source of people The N-terminal and connector gb1-intein of heavy chain ferritin HFTCIt carries out Gene Fusion and constitutes recombinant protein gb1-inteinC-HFT。24 A recombinant protein gb1-inteinC- HFT molecular self-assembling is at 24 aggressiveness nano particles.2. the C-terminal of rhizavidin introduces inteinN- gb1 constitutes albumen rhizavidin-inteinN-gb1.It is in molar ratio 1:1:1 addition in 2mM DTT solution Nano particle, biotinylated CpG and albumen rhizavidin-inteinN- gb1, through protein splice and avidin and life Object element CpG high affine interaction obtains CpG-HFT nano particle, as nanometer adjuvant drug delivery system.
3. vaccine-adjuvant delivers nanometer formulation altogether
3.1 CBLB are 1:1 at the molar ratio of Protein S P70-CBLB, CBLB and SP70 with SP70 Gene Fusion.SP70- The C-terminal of the CBLB of CBLB introduces inteinNConstitute Protein S P70-CBLB-inteinN, in 2mM DTT solution, by HFT with SP70-CBLB molar ratio 1:1 adds nano particle and SP70-CBLB-inteinN, SP70-CBLB- is obtained through protein splicing HFT, and it is self-assembly of SP70-CBLB-HFT nano particle.
Purpose product is isolated and purified as seen in figures 6a-b for Tricine-SDS-PAGE and UV tracing analysis gel filtration The result of SP70-HFT.As the result is shown: this method can purify purer purpose product；Before and after are respectively indicated are as follows: are cut certainly Front and back sample is cut, digital 8-19 represents gel-filtration purified purpose product and collects sample number into spectrum (1ml/ pipe).Fig. 6 c:TEM analysis Purpose product SP70-HFT exists with form of nanoparticles, arrow indicative purpose product.For the purpose of Fig. 6 d-f and Fig. 6 g-i difference The purifying of product SP70-CBLB-HFT and CBLB-HFT gel filtration and quality testing.Fig. 6 a, 6d and 6g, which are shown, not to be sheared gb1-inteinC- HFT band is almost invisible, illustrates that the self cleavage of nano particle is high-efficient.Fig. 6 c, 6f and 6i show three kinds Purpose product exists with form of nanoparticles, shows that nano particle provided by the invention can be by self cleavage and antigen, adjuvant Covalent cross-linking, to assign nano particle different functions.
3.2 as shown in figure 5,1. intein (intein) introduces streptococcal protein G B1 structural domain label (gb1) building connector gb1-inteinC(A is shown as in Fig. 1).The N-terminal and connector gb1-intein of source of people heavy chain ferritin HFTCCarry out Gene Fusion structure At recombinant protein gb1-inteinC-HFT.24 recombinant protein gb1-inteinC- HFT molecular self-assembling is at 24 aggressiveness nanometers Grain.2. the C-terminal of SP70 introduces inteinN- gb1 constitutes Protein S P70-inteinN-gb1.In 2mM DTT solution, in molar ratio Nano particle and Protein S P70-intein are added for 1:1N- gb1 obtains Protein S P70-HFT through protein splice.rhizavidin C-terminal introduce inteinN- gb1 constitutes albumen rhizavidin-inteinN-gb1.In 2mM DTT solution, nanometer is added Grain, biotinylated CpG, albumen rhizavidin-inteinN- gb1 and Protein S P70-inteinN- gb1 is obtained through protein splice To SP70-CpG-HFT nano particle, as nanometer delivers drug delivery system altogether.Wherein, albumen rhizavidin-inteinN-gb1 With Protein S P70-inteinNThe molar ratio of-gb1 is 1:5, nano particle and albumen rhizavidin-inteinN- gb1 and albumen SP70-inteinNThe molar ratio of the mixture of-gb1 is 6:1:5, biotinylated CpG and albumen rhizavidin-inteinN- The molar ratio of gb1 is 1:1.
The immune effect of the different immune formulation of experimental example 4
1. establishing mouse test group: SP70-CpG-HFT group using molar ratio be 1:1 0.4 μ g/ biotinylation CpG and After 10 μ g/ SP70-rhizavidin-HFT ice baths mix 30 minutes, mouse is immunized.SP70-HFT+CpG-HFT group use is received Rice vaccine SP70-HFT and nanometer adjuvant CpG-HFT co-immunization mouse, dosage are respectively 10 μ g/ and 1.6 μ g/. Nano vaccine SP70-HFT group: mouse is immunized in SP70-HFT, and dosage is respectively 10 μ g/.SP70-HFT+CpG group: SP70-HFT and 0.4 μ g/ biotinylation CpG co-immunization mouse.Each group mouse 5,3 inoculations, inoculation is spaced 2 every time Week.Immunoassay is carried out in third time posterior orbit blood sampling in immune 2 weeks.
Table 2:
Note: SP70-HFT: nano vaccine；SP70-HFT+CpG: nano vaccine and free CpG intermixture；SP70-CpG- HFT:SP70 and CpG deliver nanometer formulation altogether；SP70-HFT+CpG-HFT: nano vaccine and nanometer adjuvant combination formulations.
After different dosage forms vaccinated mice, be inoculated with 3 times, every minor tick 2 weeks, the immune 2 weeks posterior orbits of third time take blood into Row elisa assay.
As shown at 8a, elisa assay different modes delivering CpG adjuvant induces IgG titre.0.4 free μ g/ of low dosage is only CpG is without obvious adjuvant effect, and the power-assisted effect of nanometer adjuvant CpG-HFT is obvious, and SP70-CpG-HFT delivers nanometer formulation altogether Adjuvant effect is best.As it can be seen that 0.4 μ g/ of low dosage only dissociates, CpG adjuvant effect is unobvious.But delivered with nano particle It is significant that nanometer adjuvant CpG-HFT and antigen-adjuvant deliver nano particle SP70-CpG-HFT adjuvant effect altogether, and delivers nanometer altogether Preparation induces B cell immune response most efficient.This is because delivering nanometer formulation utmostly guarantees that antigen and adjuvant position altogether To the same antigen presenting cell (antigen presenting cell, abbreviation APC), the power-assisted effect of adjuvant is given full play to. Using the IgG antibody titre (1:1000) of ELISA detection different subtype, IgG Subtype the results show that deliver nanometer formulation altogether The IgG titre of Th1 and Th2 type is remarkably reinforced in SP70-CpG-HFT simultaneously, is shown in Table 1, wherein the IgG1 accounting example highest of Th2 type.
2. mouse test group (every group of 5 mouse are inoculated with 3 times, every minor tick 2 weeks) is established according to the 1st of this experimental example the point, Respectively SP70-CBLB-HFT (10 μ g/ are only) group and SP70-HFT (10 μ g/ are only)+CBLB-HFT (10 μ g/ are only) group, then Take blood to carry out elisa assay (the 3rd immune eye socket after two weeks takes blood).As shown in Figure 8 b, the antibody of SP70-CBLB-HFT induction Titre is lower than SP70-HFT+CBLB-HFT.
3.CpG is better than flagellin as adjuvant, to the power-assisted effect of B cell immune response；Compare the CpG that delivers altogether and Flagellin, discovery CpG induction of antibodies titre are higher；It is detailed in Fig. 8 c.
4. the serum of the 1st~2 point of each test group mouse of this experimental example is taken respectively, with 2 times for gradient series diluted blood (50 μ l) clearly, the EV71 G082 (50 μ l) with 100 TCID50 is in 37 DEG C of CO2After placing 1h in incubator, it is added 15000 Human embryo human rhabdomyosarcoma cells (human embryo rhabdomyosarcoma cells, RD cells).After infection 3 days CPE is observed, neutralize antibody titers are counted.As shown in figure 8d, antibody neutralization titer analysis is found, SP70-CpG-HFT, SP70- The antibody titer of HFT+CpG-HFT, SP70-CBLB-HFT and SP70-HFT+CBLB-HFT are all remarkably higher than SP70-HFT, In with SP70-CpG-HFT induce antibody titer highest.
To sum up, Fig. 8 is detailed in by impact analysis figure of the model analysis difference vaccine dosage to immune effect of SP70 epitope: its In, Fig. 8 a-c is that elisa assay different modes deliver CpG adjuvant and CBLB induces IgG titre；Fig. 8 a is different modes delivering Influence of the CpG to SP70IgG antibody titer, compared with nano vaccine SP70-HFT, the free CpG (SP70- of nano vaccine mixing HFT+CpG) without obvious adjuvant effect.But nano vaccine and nanometer adjuvant CpG-HFT mix preparation and SP70 and CpG are passed altogether Nanometer formulation SP70-CpG-HFT is sent to be remarkably improved Specific antibody titre.And delivering nanometer formulation effect compares nano vaccine altogether It is higher with the antibody titer of nanometer adjuvant mix preparation induction.Fig. 8 b is disclosed when CBLB is adjuvant, and delivering nanometer formulation induces altogether Specific antibody titers are higher than nano vaccine SP70-HFT and nanometer adjuvant CBLB-HFT mix preparation.Fig. 8 c shows the adjuvant of CpG Effect is higher than CBLB.The antibody neutralization titer of Fig. 8 d cell in vitro titrimetry difference vaccine preparation induction.The neutralization of antibody is imitated Valence is the results show that the antibody neutralising capacity highest of delivering nanometer formulation induction, nano vaccine and nanometer adjuvant mix preparation pierce altogether Swash the antibody neutralising capacity generated to take second place, obviously higher than nano vaccine itself.And CpG is the antibody dilution factor of adjuvant induction Better than CBLB adjuvant.Fig. 8 e: the analysis of lethal protection in vivo, which discloses delivering nanometer formulation altogether, can provide immunoprotection the most efficient (75%), the protecting effect that nano vaccine and nanometer adjuvant provide takes second place (50%), is both higher than nano vaccine (25%).Exempt from Epidemic disease analysis is the results show that the immune response of delivering nanometer formulation induction is most effective altogether, and nanometer adjuvant and nano vaccine intermixture are also There is significant adjuvant effect.
The stability of 5 nano-carrier modular platform of experimental example
Area, influenza virus hemagglutinin stem is the important target of universal influenza vaccines, wherein Bacillus coli expression The antibody of H1HA10 tripolymer induction can neutralize the strain of different subtype.H1HA10 is carried out using existing Gene Fusion method (being abbreviated as HA) merges with ferrtin obtains recombinant protein HA-HFT.
As a result be detailed in Fig. 9: i.e. albumen editor shearing technique can on HFT/PFT carrier high-efficient carrier HA antigen；Wherein, scheme 9a is recombination HA-HFT expression analysis electrophoretogram；In figure, control:pET28a carrier blank control bacterial lysate；All: Bacterial lysate；Up: bacterial lysate supernatant；HA-HFT:HA (H1HA10 is abbreviated as HA) merges to be formed with HFT direct gene Recombinant protein.Fig. 9 b is the electrophoretic analysis figure of the gel-filtration purified HA-HFT based on the preparation of albumen editor's shearing technique.Fig. 9 c For the UV curve graph of gel-filtration purified HA-HFT.Fig. 9 d is the transmission electron microscope picture (negative staining sample preparation) of HA-HFT.Fig. 9 e is solidifying The electrophoretic analysis figure for the HA-PFT that glue Purification by filtration is prepared based on albumen editor's shearing technique.Fig. 9 f is gel-filtration purified The UV curve graph of HA-PFT.Fig. 9 g is the transmission electron microscope picture (negative staining sample preparation) of HA-PFT.Before: before intein self cleavage, After: after intein shearing, the 8-19 in Fig. 9 b and 9e is numbered: being in charge of and is collected gel-filtration purified purpose product sample It numbers (1ml/ pipe).
As illustrated in fig. 9, recombinant protein HA-HFT is not contained in the supernatant of bacterial lysate, it is seen that recombinant protein forms packet Contain body precipitating.
HA is covalently coupled to by HFT using method of the invention and forms HA-HFT, albumen editor is based on as shown in Fig. 9 b-d The HA-HFT of shearing preparation exists with form of nanoparticles.
HA is covalently coupled to by PFT using method of the invention and forms HA-PFT, albumen editor is based on as shown in Fig. 9 e-g The HA-PFT of shearing preparation equally exists with form of nanoparticles, that is to say, that PFT can also load HA.
Since H1HA10 needs to maintain natural trimeric configuration, CpG-HFT pairs of nanometer adjuvant is only studied in the present invention The influence of nano vaccine HA-HFT/PFT immunogenicity.
6 CpG-HFT of experimental example enhancing is directed to the Th1 type IgG response of HA
It establishes mouse test group: every group mouse 5, being immunized three times, every time inoculation interval 2 weeks, posterior orbit blood sampling in 3 weeks is immunized For immunoassay.HA group uses H1HA10-inteinNImmune mouse, 10 μ g/ of dosage is only.HA-HFT+CpG-HFT group Using nano vaccine HA-HFT and nanometer adjuvant CpG-HFT co-immunization mouse, dosage is respectively 10 μ g/ and 1.6 μ g/ Only.Nano vaccine HA-HFT group: mouse is immunized in HA-HFT, and dosage is respectively 10 μ g/.Nano vaccine HA-PFT group: HA- Mouse is immunized in PFT, and dosage is respectively 10 μ g/.HA-PFT+CpG-HFT group is helped using nano vaccine HA-PFT and nanometer Agent CpG-HFT co-immunization mouse, dosage are respectively 10 μ g/ and 1.6 μ g/.
After different dosage forms vaccinated mice, the 3rd time immune 2 weeks posterior orbits take blood to carry out elisa assay.Use nano particle HA is delivered, improves 10 times than HA induction of antibodies titre.CpG-HFT's is used in combination, and specific antibody gradient improves 10 times again. It is higher than HFT by carrier induction of antibodies titre of PFT.PFT is low with the ferrtin similarity of mouse, may have certain adjuvant Effect.
Nanometer adjuvant CpG-HFT enhancing HA specific humoral immune response simultaneously regulates and controls the analysis chart of IgG type and is detailed in Figure 10； Wherein, Figure 10 a is that elisa assay announcement nano-carrier and nanometer adjuvant can enhance HA specific humoral immune response.With HA Antigen is compared, nano vaccine HA-HFT can by HA specific IgG antibodies titre improve 10 times or so, nanometer adjuvant CpG-HFT with Nano vaccine mix preparation can further improve Specific antibody titre (10 times or so).In addition, PFT is higher than for carrier effect HFT, it may be possible to it is low with ferritin ferritin homology in mouse by PFT, there is certain adjuvant effect to cause.Source of people HFT with Mouse ferritin very high homology (amino acid sequence 99% is identical), HFT albumen itself is without adjuvant effect.Figure 10 b-dELISA analysis Disclose, vaccine induced antibody can specific recognition H1N1 (P), H3N2 and H7H9 strain, and nanometer adjuvant is mixed with nano vaccine The antibody recognition capability of preparation induction is higher than nano vaccine itself.(primary antibody is the serum for being inoculated with 3 dose vaccine mouse, secondary antibody It is the mouse secondary antibody of horseradish peroxidase HRP label).
Figure 10 a shows that nano particle delivering HA significantly improves the B cell immune response of body response HA, more anti-than HA induction Body titre improves 10 times, is used in combination with nanometer adjuvant CpG-HFT, specific antibody gradient improves 10 times again, further mentions High response is horizontal.It is higher than HFT carrier using the antibody titer that PFT is induced as carrier, prompts the ferrtin similarity of PFT and mouse Low, PFT carrier may have the function of certain adjuvant.Figure 10 b-d shows that HA specificity associated antibodies can recognize different subtype Influenza strain, identification hypotype cover: (the writing a Chinese character in simplified form P) of H1NI, H3N2 and H7N9 (Anhui strain, write a Chinese character in simplified form Ahpri).Elisa assay IgG hypotype (1:105Dilution inoculation different dosage forms vaccine mouse serum) discovery, CpG-HFT mainly enhance Th2 type IgG2 and IgG3 immune response has very strong adjuvant effect to the IgG of Th1 type, is shown in Table 3.With nanoscale building blocks load C pG preparation nanometer assistant Agent is used in combination with the nano vaccine of load HA, significantly increases HA specific antibody immune response, and induction of antibodies identifies different Asias The Influenza virus strain ability of type also enhances.
Table 3:
Note: HA:H1HA10-inteinN；HA-HFT:HFT nano particle delivers H1HA10；HA-HFT+CpG-HFT:HA- HFT and CpG-HFT combination formulations；HA-PFT:PFT nano particle delivers H1HA10；HA-PFT+CpG-HFT:HA-PFT and CpG- HFT combination formulations (remarks: since gb1 specifically combines all types of IgG, in detection HA Specific antibody titre When use H1HA10-inteinNAlbumen.When shearing using albumen editor, the solubility of albumen can be significantly improved based on gb1, Therefore H1HA10-intein is usedN-gb1)。
In conclusion nano-carrier modular platform of the invention can efficiently show antigen protein in nano grain surface, exempt from Epidemic disease reinforcing agent, polypeptide epitope, for enhanced preventative subunit vaccine and personalized polypeptide epitope tumor vaccine research and development etc. Field.Solve load protein vs protein self-assembly nanoparticles stable influence and nano particle be difficult to total delivering adjuvant and The problem of antigen molecule.
Sequence table
<110>Guangzhou Women and Children's Medical Center
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Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ser
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ser Val
85 90 95
Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser Ile Gln
100 105 110
Asp Glu Ile Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser Asn Gln
115 120 125
Thr Gln Phe Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile Asp Leu Gln
145 150 155 160
Lys Ile Asp Val Lys Ser Leu Gly Leu Asp Gly Phe Asn Val Asn Ser
165 170 175
Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser Met Gly Thr
180 185 190
Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr Ala Asn Pro
195 200 205
Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys Val Asp Ala Val Arg Ser
210 215 220
Ser Leu Gly Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr Asn Leu
225 230 235 240
Gly Asn Thr Val Thr Asn Leu Asn Ser Ala Arg Ser Arg Ile Glu Asp
245 250 255
Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln Ile Leu
260 265 270
Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala Asn Gln Val Pro Gln
275 280 285
Asn Val Leu Ser Leu Leu Arg
290 295
<210> 9
<211> 172
<212> PRT
<213> H1HA10
<400> 9
Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser
1 5 10 15
Val Asn Leu Leu Glu Asp Ser His Gly Ser Ala Asn Ser Ser Leu Pro
20 25 30
Tyr Gln Asn Thr His Pro Thr Thr Asn Gly Glu Ser Pro Lys Tyr Val
35 40 45
Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn Gly Ser Ala
50 55 60
Gly Ser Ala Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn
65 70 75 80
Thr Val Ile Glu Lys Met Asn Ile Gln Asp Thr Ala Thr Gly Lys Glu
85 90 95
Phe Asn Lys Asp Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp
100 105 110
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
115 120 125
Leu Glu Asn Glu Arg Thr Leu Asp Ala His Asp Ser Gln Gly Thr Gly
130 135 140
Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg
145 150 155 160
Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu
165 170
<210> 10
<211> 182
<212> PRT
<213> HFT
<400> 10
Thr Thr Ala Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp Ser
1 5 10 15
Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser Tyr
20 25 30
Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu
35 40 45
Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu
50 55 60
His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile
65 70 75 80
Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly
85 90 95
Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn Gln
100 105 110
Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His
115 120 125
Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala
130 135 140
Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala
145 150 155 160
Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly
165 170 175
Asp Ser Asp Asn Glu Ser
180
<210> 11
<211> 173
<212> PRT
<213> PFT
<400> 11
Leu Ser Glu Arg Met Leu Lys Ala Leu Asn Asp Gln Leu Asn Arg Glu
1 5 10 15
Leu Tyr Ser Ala Tyr Leu Tyr Phe Ala Met Ala Ala Tyr Phe Glu Asp
20 25 30
Leu Gly Leu Glu Gly Phe Ala Asn Trp Met Lys Ala Gln Ala Glu Glu
35 40 45
Glu Ile Gly His Ala Pro Arg Phe Tyr Asn Tyr Ile Tyr Asp Arg Asn
50 55 60
Gly Arg Val Glu Leu Asp Glu Ile Pro Lys Pro Pro Lys Glu Trp Glu
65 70 75 80
Ser Pro Leu Lys Ala Phe Glu Ala Ala Tyr Glu His Glu Lys Phe Ile
85 90 95
Ser Lys Ser Ile Tyr Glu Leu Ala Ala Leu Ala Glu Glu Glu Lys Asp
100 105 110
Tyr Ser Thr Arg Ala Phe Leu Glu Trp Phe Ile Asn Glu Gln Val Glu
115 120 125
Glu Glu Ala Ser Val Lys Lys Ile Leu Asp Lys Leu Lys Phe Ala Lys
130 135 140
Asp Ser Pro Gln Ile Leu Phe Met Leu Asp Lys Glu Leu Ser Ala Arg
145 150 155 160
Ala Pro Lys Leu Pro Gly Leu Leu Met Gln Gly Gly Glu
165 170
<210> 12
<211> 405
<212> PRT
<213> strep2-GFP-gp41-1-inteinN-gb1-6xHis
<400> 12
Met Gly Trp Ser His Pro Gln Phe Glu Lys Val Ser Lys Gly Glu Glu
1 5 10 15
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
20 25 30
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
35 40 45
Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro
50 55 60
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys
65 70 75 80
Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
85 90 95
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
100 105 110
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
115 120 125
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly
130 135 140
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val
145 150 155 160
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys
165 170 175
Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr
180 185 190
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
195 200 205
His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys
210 215 220
Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr
225 230 235 240
Leu Gly Met Asp Glu Leu Tyr Lys Thr Arg Ser Gly Tyr Cys Leu Asp
245 250 255
Leu Lys Thr Gln Val Gln Thr Pro Gln Gly Met Lys Glu Ile Ser Asn
260 265 270
Ile Gln Val Gly Asp Leu Val Leu Ser Asn Thr Gly Tyr Asn Glu Val
275 280 285
Leu Asn Val Phe Pro Lys Ser Lys Lys Lys Ser Tyr Lys Ile Thr Leu
290 295 300
Glu Asp Gly Lys Glu Ile Ile Cys Ser Glu Glu His Leu Phe Pro Thr
305 310 315 320
Gln Thr Gly Glu Met Asn Ile Ser Gly Gly Leu Lys Glu Gly Met Cys
325 330 335
Leu Tyr Val Lys Glu Gln Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu
340 345 350
Lys Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys
355 360 365
Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr
370 375 380
Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Leu Glu His
385 390 395 400
His His His His His
405
<210> 13
<211> 172
<212> PRT
<213> strep2-gp41-1-inteinN-gb1-6xHis
<400> 13
Met Gly Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Thr
1 5 10 15
Arg Ser Gly Tyr Cys Leu Asp Leu Lys Thr Gln Val Gln Thr Pro Gln
20 25 30
Gly Met Lys Glu Ile Ser Asn Ile Gln Val Gly Asp Leu Val Leu Ser
35 40 45
Asn Thr Gly Tyr Asn Glu Val Leu Asn Val Phe Pro Lys Ser Lys Lys
50 55 60
Lys Ser Tyr Lys Ile Thr Leu Glu Asp Gly Lys Glu Ile Ile Cys Ser
65 70 75 80
Glu Glu His Leu Phe Pro Thr Gln Thr Gly Glu Met Asn Ile Ser Gly
85 90 95
Gly Leu Lys Glu Gly Met Cys Leu Tyr Val Lys Glu Gln Tyr Lys Leu
100 105 110
Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val
115 120 125
Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn
130 135 140
Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr
145 150 155 160
Val Thr Glu Lys Leu Glu His His His His His His
165 170
<210> 14
<211> 185
<212> PRT
<213> SP70-gp41-1-inteinN-gb1-strep2-6xHis
<400> 14
Met Gly Tyr Pro Thr Phe Gly Glu His Lys Gln Glu Lys Asp Leu Glu
1 5 10 15
Tyr Gly Thr Arg Ser Gly Tyr Cys Leu Asp Leu Lys Thr Gln Val Gln
20 25 30
Thr Pro Gln Gly Met Lys Glu Ile Ser Asn Ile Gln Val Gly Asp Leu
35 40 45
Val Leu Ser Asn Thr Gly Tyr Asn Glu Val Leu Asn Val Phe Pro Lys
50 55 60
Ser Lys Lys Lys Ser Tyr Lys Ile Thr Leu Glu Asp Gly Lys Glu Ile
65 70 75 80
Ile Cys Ser Glu Glu His Leu Phe Pro Thr Gln Thr Gly Glu Met Asn
85 90 95
Ile Ser Gly Gly Leu Lys Glu Gly Met Cys Leu Tyr Val Lys Glu Gln
100 105 110
Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr
115 120 125
Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala
130 135 140
Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys
145 150 155 160
Thr Phe Thr Val Thr Glu Lys Trp Ser His Pro Gln Phe Glu Lys Gly
165 170 175
Ser Leu Glu His His His His His His
180 185
<210> 15
<211> 317
<212> PRT
<213> rhizavidin-gp41-1-intN-gb1-6xHis
<400> 15
Met Gly Phe Asp Ala Ser Asn Phe Lys Asp Phe Ser Ser Ile Ala Ser
1 5 10 15
Ala Ser Ser Ser Trp Gln Asn Gln His Gly Ser Thr Met Ile Ile Gln
20 25 30
Val Asp Ser Phe Gly Asn Val Ser Gly Gln Tyr Val Asn Arg Ala Glu
35 40 45
Gly Thr Gly Cys Gln Asn Ser Pro Tyr Pro Leu Thr Gly Arg Val Asn
50 55 60
Gly Thr Phe Ile Asp Phe Ser Val Lys Trp Asn Asn Ser Thr Glu Asn
65 70 75 80
Cys Asn Ser Asn Thr Gln Trp Thr Gly Tyr Ala Gln Val Asn Gly Asn
85 90 95
Asn Thr Glu Ile Val Thr Arg Trp Asn Leu Lys Tyr Glu Gly Gly Ser
100 105 110
Gly Pro Ala Ile Trp Gln Gly Gln Asp Thr Phe Gln Tyr Val Pro Thr
115 120 125
Thr Glu Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala
130 135 140
Ala Lys Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Thr Arg
145 150 155 160
Ser Gly Tyr Cys Leu Asp Leu Lys Thr Gln Val Gln Thr Pro Gln Gly
165 170 175
Met Lys Glu Ile Ser Asn Ile Gln Val Gly Asp Leu Val Leu Ser Asn
180 185 190
Thr Gly Tyr Asn Glu Val Leu Asn Val Phe Pro Lys Ser Lys Lys Lys
195 200 205
Ser Tyr Lys Ile Thr Leu Glu Asp Gly Lys Glu Ile Ile Cys Ser Glu
210 215 220
Glu His Leu Phe Pro Thr Gln Thr Gly Glu Met Asn Ile Ser Gly Gly
225 230 235 240
Leu Lys Glu Gly Met Cys Leu Tyr Val Lys Glu Gln Tyr Lys Leu Ile
245 250 255
Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val Asp
260 265 270
Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly
275 280 285
Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val
290 295 300
Thr Glu Lys Gly Ser Leu Glu His His His His His His
305 310 315
<210> 16
<211> 408
<212> PRT
<213> CBLB-gp41-1-inteinN-strep2-6xHis
<400> 16
Met Gly Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln
1 5 10 15
Asn Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg
20 25 30
Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly
35 40 45
Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln
50 55 60
Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu
65 70 75 80
Gly Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu
85 90 95
Ser Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser
100 105 110
Ile Gln Asp Glu Ile Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser
115 120 125
Asn Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln
130 135 140
Met Lys Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile Asp
145 150 155 160
Leu Gln Lys Ile Asp Val Lys Ser Leu Gly Leu Asp Gly Phe Asn Val
165 170 175
Asn Ser Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser Met
180 185 190
Gly Thr Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr Ala
195 200 205
Asn Pro Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys Val Asp Ala Val
210 215 220
Arg Ser Ser Leu Gly Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr
225 230 235 240
Asn Leu Gly Asn Thr Val Thr Asn Leu Asn Ser Ala Arg Ser Arg Ile
245 250 255
Glu Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln
260 265 270
Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala Asn Gln Val
275 280 285
Pro Gln Asn Val Leu Ser Leu Leu Arg Thr Arg Ser Gly Tyr Cys Leu
290 295 300
Asp Leu Lys Thr Gln Val Gln Thr Pro Gln Gly Met Lys Glu Ile Ser
305 310 315 320
Asn Ile Gln Val Gly Asp Leu Val Leu Ser Asn Thr Gly Tyr Asn Glu
325 330 335
Val Leu Asn Val Phe Pro Lys Ser Lys Lys Lys Ser Tyr Lys Ile Thr
340 345 350
Leu Glu Asp Gly Lys Glu Ile Ile Cys Ser Glu Glu His Leu Phe Pro
355 360 365
Thr Gln Thr Gly Glu Met Asn Ile Ser Gly Gly Leu Lys Glu Gly Met
370 375 380
Cys Leu Tyr Val Lys Glu Trp Ser His Pro Gln Phe Glu Lys Gly Ser
385 390 395 400
Leu Glu His His His His His His
405
<210> 17
<211> 424
<212> PRT
<213> SP70-CBLB-gp41-1-inteinN-strep2-6xHis
<400> 17
Met Gly Tyr Pro Thr Phe Gly Glu His Lys Gln Glu Lys Asp Leu Glu
1 5 10 15
Tyr Gly Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln
20 25 30
Asn Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg
35 40 45
Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly
50 55 60
Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln
65 70 75 80
Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu
85 90 95
Gly Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu
100 105 110
Ser Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser
115 120 125
Ile Gln Asp Glu Ile Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser
130 135 140
Asn Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln
145 150 155 160
Met Lys Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile Asp
165 170 175
Leu Gln Lys Ile Asp Val Lys Ser Leu Gly Leu Asp Gly Phe Asn Val
180 185 190
Asn Ser Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser Met
195 200 205
Gly Thr Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr Ala
210 215 220
Asn Pro Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys Val Asp Ala Val
225 230 235 240
Arg Ser Ser Leu Gly Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr
245 250 255
Asn Leu Gly Asn Thr Val Thr Asn Leu Asn Ser Ala Arg Ser Arg Ile
260 265 270
Glu Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln
275 280 285
Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala Asn Gln Val
290 295 300
Pro Gln Asn Val Leu Ser Leu Leu Arg Thr Arg Ser Gly Tyr Cys Leu
305 310 315 320
Asp Leu Lys Thr Gln Val Gln Thr Pro Gln Gly Met Lys Glu Ile Ser
325 330 335
Asn Ile Gln Val Gly Asp Leu Val Leu Ser Asn Thr Gly Tyr Asn Glu
340 345 350
Val Leu Asn Val Phe Pro Lys Ser Lys Lys Lys Ser Tyr Lys Ile Thr
355 360 365
Leu Glu Asp Gly Lys Glu Ile Ile Cys Ser Glu Glu His Leu Phe Pro
370 375 380
Thr Gln Thr Gly Glu Met Asn Ile Ser Gly Gly Leu Lys Glu Gly Met
385 390 395 400
Cys Leu Tyr Val Lys Glu Trp Ser His Pro Gln Phe Glu Lys Gly Ser
405 410 415
Leu Glu His His His His His His
420
<210> 18
<211> 340
<212> PRT
<213> H1HA10-gp41-1-inteinN-gb1-6xHis
<400> 18
Met Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
1 5 10 15
Ser Val Asn Leu Leu Glu Asp Ser His Gly Ser Ala Asn Ser Ser Leu
20 25 30
Pro Tyr Gln Asn Thr His Pro Thr Thr Asn Gly Glu Ser Pro Lys Tyr
35 40 45
Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn Gly Ser
50 55 60
Ala Gly Ser Ala Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val
65 70 75 80
Asn Thr Val Ile Glu Lys Met Asn Ile Gln Asp Thr Ala Thr Gly Lys
85 90 95
Glu Phe Asn Lys Asp Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val
100 105 110
Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val
115 120 125
Leu Leu Glu Asn Glu Arg Thr Leu Asp Ala His Asp Ser Gln Gly Thr
130 135 140
Gly Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val
145 150 155 160
Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Gly
165 170 175
Ser Gly Gly Gly Ser Thr Arg Ser Gly Tyr Cys Leu Asp Leu Lys Thr
180 185 190
Gln Val Gln Thr Pro Gln Gly Met Lys Glu Ile Ser Asn Ile Gln Val
195 200 205
Gly Asp Leu Val Leu Ser Asn Thr Gly Tyr Asn Glu Val Leu Asn Val
210 215 220
Phe Pro Lys Ser Lys Lys Lys Ser Tyr Lys Ile Thr Leu Glu Asp Gly
225 230 235 240
Lys Glu Ile Ile Cys Ser Glu Glu His Leu Phe Pro Thr Gln Thr Gly
245 250 255
Glu Met Asn Ile Ser Gly Gly Leu Lys Glu Gly Met Cys Leu Tyr Val
260 265 270
Lys Glu Gln Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu
275 280 285
Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys
290 295 300
Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp
305 310 315 320
Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Gly Ser Leu Glu His His
325 330 335
His His His His
340
<210> 19
<211> 316
<212> PRT
<213> gb1-gp41-1-inteinC-HFT
<400> 19
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Glu Phe Gln Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys
35 40 45
Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val
50 55 60
Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr
65 70 75 80
Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Met Leu Lys Lys
85 90 95
Ile Leu Lys Ile Glu Glu Leu Asp Glu Arg Glu Leu Ile Asp Ile Glu
100 105 110
Val Ser Gly Asn His Leu Phe Tyr Ala Asn Asp Ile Leu Thr His Asn
115 120 125
Ser Ser Ser Ser Asp Val Thr Thr Ala Ser Thr Ser Gln Val Arg Gln
130 135 140
Asn Tyr His Gln Asp Ser Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu
145 150 155 160
Glu Leu Tyr Ala Ser Tyr Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp
165 170 175
Arg Asp Asp Val Ala Leu Lys Asn Phe Ala Lys Tyr Phe Leu His Gln
180 185 190
Ser His Glu Glu Arg Glu His Ala Glu Lys Leu Met Lys Leu Gln Asn
195 200 205
Gln Arg Gly Gly Arg Ile Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys
210 215 220
Asp Asp Trp Glu Ser Gly Leu Asn Ala Met Glu Cys Ala Leu His Leu
225 230 235 240
Glu Lys Asn Val Asn Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr
245 250 255
Asp Lys Asn Asp Pro His Leu Cys Asp Phe Ile Glu Thr His Tyr Leu
260 265 270
Asn Glu Gln Val Lys Ala Ile Lys Glu Leu Gly Asp His Val Thr Asn
275 280 285
Leu Arg Lys Met Gly Ala Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe
290 295 300
Asp Lys His Thr Leu Gly Asp Ser Asp Asn Glu Ser
305 310 315
<210> 20
<211> 307
<212> PRT
<213> gb1-gp41-1-inteinC-PFT
<400> 20
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Glu Phe Gln Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys
35 40 45
Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val
50 55 60
Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr
65 70 75 80
Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Met Leu Lys Lys
85 90 95
Ile Leu Lys Ile Glu Glu Leu Asp Glu Arg Glu Leu Ile Asp Ile Glu
100 105 110
Val Ser Gly Asn His Leu Phe Tyr Ala Asn Asp Ile Leu Thr His Asn
115 120 125
Ser Ser Ser Ser Asp Val Leu Ser Glu Arg Met Leu Lys Ala Leu Asn
130 135 140
Asp Gln Leu Asn Arg Glu Leu Tyr Ser Ala Tyr Leu Tyr Phe Ala Met
145 150 155 160
Ala Ala Tyr Phe Glu Asp Leu Gly Leu Glu Gly Phe Ala Asn Trp Met
165 170 175
Lys Ala Gln Ala Glu Glu Glu Ile Gly His Ala Pro Arg Phe Tyr Asn
180 185 190
Tyr Ile Tyr Asp Arg Asn Gly Arg Val Glu Leu Asp Glu Ile Pro Lys
195 200 205
Pro Pro Lys Glu Trp Glu Ser Pro Leu Lys Ala Phe Glu Ala Ala Tyr
210 215 220
Glu His Glu Lys Phe Ile Ser Lys Ser Ile Tyr Glu Leu Ala Ala Leu
225 230 235 240
Ala Glu Glu Glu Lys Asp Tyr Ser Thr Arg Ala Phe Leu Glu Trp Phe
245 250 255
Ile Asn Glu Gln Val Glu Glu Glu Ala Ser Val Lys Lys Ile Leu Asp
260 265 270
Lys Leu Lys Phe Ala Lys Asp Ser Pro Gln Ile Leu Phe Met Leu Asp
275 280 285
Lys Glu Leu Ser Ala Arg Ala Pro Lys Leu Pro Gly Leu Leu Met Gln
290 295 300
Gly Gly Glu
305

Claims (5)

1. one kind can simultaneously delivery of antigens and immunopotentiator nanometer formulation, which is characterized in that by the N-terminal and connector of ferritin The C-terminal of gbl-intein merges to form recombinant protein gbl-inteincSelf-chambering is made into 24 aggressiveness and constitutes nanometer after-ferrtin Grain；The N-terminal of the C-terminal fusion intein of foreign protein forms recombinant protein, and the N-terminal specific recognition of intein and cutting off is exposed to The ferritin of the nano grain surface；Foreign protein and ferritin covalent cross-linking and obtain；
Wherein, the foreign protein is immunopotentiator or antigen or both combination.
2. according to claim 1 can simultaneously delivery of antigens and immunopotentiator nanometer formulation, which is characterized in that it is described Immunopotentiator is flagellin or avidin monomer analog rhizavidin.
3. according to claim 1 can simultaneously delivery of antigens and immunopotentiator nanometer formulation, which is characterized in that it is described Antigen is the proteantigen of single polypeptide epitope or the proteantigen of different polypeptide epitopes.
4. according to claim 1 can simultaneously delivery of antigens and immunopotentiator nanometer formulation, which is characterized in that it is described When foreign protein is that both immunopotentiator and antigen combine, molar ratio is 1~3:5.
5. according to claim 1 can simultaneously delivery of antigens and immunopotentiator nanometer formulation, which is characterized in that it is described Ferritin is the ferritin of the heavy chain ferritin of source of people or the heavy chain ferritin of pyrococcus furiosus or other source of species.
CN201910421369.2A 2019-05-21 2019-05-21 It is a kind of can simultaneously delivery of antigens and immunopotentiator nanometer formulation Pending CN110201183A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020233685A1 (en) * 2019-05-21 2020-11-26 广州市妇女儿童医疗中心 Intein-mediated nano-carrier and application thereof, and nano preparation capable of simultaneously delivering antigen and immunopotentiator
WO2020233460A1 (en) * 2019-05-21 2020-11-26 广州市妇女儿童医疗中心 Intein-mediated nanocarrier module platform, construction method therefor and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1927401A (en) * 2005-09-07 2007-03-14 中国人民解放军军事医学科学院基础医学研究所 Self-assembly composite nano granule tumor vaccine and method for preparing same
CN104755502B (en) * 2012-10-12 2018-05-18 清华大学 The generation of polypeptide and purification process
CN108434450A (en) * 2018-02-06 2018-08-24 中国科学院生物物理研究所 Vaccine and preparation method thereof based on ferritin nano particle

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1927401A (en) * 2005-09-07 2007-03-14 中国人民解放军军事医学科学院基础医学研究所 Self-assembly composite nano granule tumor vaccine and method for preparing same
CN104755502B (en) * 2012-10-12 2018-05-18 清华大学 The generation of polypeptide and purification process
CN108434450A (en) * 2018-02-06 2018-08-24 中国科学院生物物理研究所 Vaccine and preparation method thereof based on ferritin nano particle

Cited By (2)

* Cited by examiner, † Cited by third party
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WO2020233685A1 (en) * 2019-05-21 2020-11-26 广州市妇女儿童医疗中心 Intein-mediated nano-carrier and application thereof, and nano preparation capable of simultaneously delivering antigen and immunopotentiator
WO2020233460A1 (en) * 2019-05-21 2020-11-26 广州市妇女儿童医疗中心 Intein-mediated nanocarrier module platform, construction method therefor and application thereof

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