WO2020233685A1 - Intein-mediated nano-carrier and application thereof, and nano preparation capable of simultaneously delivering antigen and immunopotentiator - Google Patents

Intein-mediated nano-carrier and application thereof, and nano preparation capable of simultaneously delivering antigen and immunopotentiator Download PDF

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WO2020233685A1
WO2020233685A1 PCT/CN2020/091636 CN2020091636W WO2020233685A1 WO 2020233685 A1 WO2020233685 A1 WO 2020233685A1 CN 2020091636 W CN2020091636 W CN 2020091636W WO 2020233685 A1 WO2020233685 A1 WO 2020233685A1
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halo
intein
int
protein
hft
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PCT/CN2020/091636
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French (fr)
Chinese (zh)
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汤书兵
周伟
袁伟明
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广州市妇女儿童医疗中心
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Priority claimed from CN201910421369.2A external-priority patent/CN110201183A/en
Priority claimed from CN201910421450.0A external-priority patent/CN110272498A/en
Application filed by 广州市妇女儿童医疗中心 filed Critical 广州市妇女儿童医疗中心
Publication of WO2020233685A1 publication Critical 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present disclosure relates to the field of nanocarriers and nanoformulations, in particular to intein-mediated nanocarriers, construction methods and applications thereof, and nanoformulations capable of simultaneously delivering antigens and immune enhancers.
  • the C-terminus of the protein is the main site for the insertion of the cargo molecule gene, which is used to load the cargo protein on the surface of the nanocarrier.
  • the C-terminus of the protein is in a "meta-stable state", and the smaller protein molecules of the C-terminus gene fusion are usually misassembly of the recombinant protein, which limits its application prospects.
  • Nano-delivery technology is becoming increasingly important in biomedical fields such as vaccine design, targeted drug delivery, and live imaging diagnosis.
  • studies using the properties of encapsulin protein to encapsulate the C-terminal protein with signal peptide and developing it as a cargo transport carrier have been studies using the properties of encapsulin protein to encapsulate the C-terminal protein with signal peptide and developing it as a cargo transport carrier.
  • this application is severely restricted by the internal space capacity of nanoparticles.
  • Another loading technology of Encapsulin nanocarriers is the site-directed mutation of amino acids on its surface to lysine or cysteine to covalently couple the load molecule.
  • chemical coupling methods have disadvantages such as low efficiency, poor specificity, and potential safety hazards in side reaction products, which limit the breadth of its application.
  • Vaccines are the most effective and economical means of medical intervention to prevent and control infectious diseases. Attenuated or inactivated pathogens have achieved great success as a traditional classic vaccine, helping humans eliminate smallpox and polio. However, traditional vaccines have potential safety hazards and cannot effectively prevent viruses with high-frequency mutation characteristics, such as HIV and influenza viruses.
  • subunit vaccines have attracted much attention from vaccinologists, but subunit vaccines have poor immunogenicity, weak inducing immune responses and short duration, which limits their clinical applications.
  • Nanoparticles can be designed as a multifunctional carrier by means of genetic engineering to deliver antigens and immune enhancers at the same time.
  • the loaded antigen may seriously affect the assembly of nanoparticles, leading to the formation of precipitates of recombinant proteins, and a series of renaturation-reassembly is required to re-form nanoparticles.
  • the method of gene fusion is difficult to integrate antigen molecules and immune enhancer molecules into the same nanoparticle at the same time.
  • the purpose of this disclosure includes the comprehensive use of genetic engineering technology and intein-mediated protein editing and shearing technology to perfectly solve the C-terminal metastability problem of encapsulin, and provide a universal nanocarrier that can be used in encapsulin Surface-specific and efficient delivery of different cargo molecules.
  • the present disclosure provides a nanocarrier mediated by intein.
  • the nanocarrier is constructed through the following steps:
  • the C-terminus of the encapsulin protein is introduced by gene fusion method into three tandem proteins int N , gb1 and halo to form a recombinant protein encapsulin-int N -gb1-halo, the nucleic acid sequence of which is shown in SEQ ID No. 8;
  • the intein intein is self-excised, forming by-products int N -gb1-halo and gb1-int C , and encapsulin is covalently coupled with cargo to produce the target product encapsulin-cargo.
  • nucleic acid sequence of the encapsulin protein is shown in SEQ ID No. 1
  • nucleic acid sequence of int N is shown in SEQ ID No. 2
  • nucleic acid sequence of gb1 is shown in SEQ ID No. 3.
  • the nucleic acid sequence of the halo is shown in SEQ ID No. 4, and the nucleic acid sequence of the int C is shown in SEQ ID No. 16.
  • the cargo is GFP or M44 or strep2.
  • nucleic acid sequence of the GFP is shown in SEQ ID No. 9.
  • nucleic acid sequence of M44 is shown in SEQ ID No. 10.
  • nucleic acid sequence of strep2 is shown in SEQ ID No. 11.
  • the above-mentioned intein-mediated nanocarrier, step 3) is encapsulin-int N -gb1-halo and gb1-int C- GFP according to the concentration ratio of 1:1-2, and both are at room temperature. After incubating for 2 hours, the target product encapsulin-GFP was separated by superose 6B increase molecular exclusion method.
  • the above-mentioned intein-mediated nanocarrier, step 3) is encapsulin-int N -gb1-halo and gb1-int C -strep2 in a concentration ratio of 1:1-3, and both are at room temperature. After incubating for 2 hours, the target product encapsulin-strep2 was separated by superose 6B increase molecular exclusion method.
  • the above-mentioned intein-mediated nanocarrier, step 2) is encapsulin-int N -gb1-halo and gb1-int C -M44 in a concentration ratio of 1:1-2, and both are at room temperature. After incubating for 2 hours, the target product encapsulin-M44 was separated by superose 6B increase molecular exclusion method.
  • the present disclosure also provides the application of the above-mentioned nanocarrier as the delivery cargo protein and inoculating mice to induce high-titer specific antibodies.
  • the above-mentioned nanocarrier is used as a cargo protein to be used to inoculate mice to induce high-titer specific antibodies.
  • the antibody purification method is:
  • the mixture is mixed with Promega halo TM beads, and the target product halo-cargo is covalently bound to halo TM beads. After centrifugation to discard the supernatant, the immunized serum is added. Affinity interaction is combined with halo TM -halo-cargo, other antibodies and proteins are separated after centrifugation and washing, and finally the target antibody is eluted with 200mM glycine pH2.8.
  • the above-mentioned nanocarrier is used to deliver cargo protein and inoculate mice to induce high-titer specific antibodies.
  • the method for preparing and purifying halo TM -halo-GFP is: combining halo-int N -gb1 and gb1 -int C -GFP was incubated for 4 hours at room temperature according to a concentration ratio of 1:1-2, 250 ⁇ L of the reaction solution was mixed with 50 ⁇ L of the balanced Promega halo TM room temperature shaker for half an hour and centrifuged, and the beads were washed with 500 ⁇ L of PBS.
  • the above-mentioned nanocarrier is used as a cargo protein to inoculate mice to induce high-titer specific antibodies.
  • the preparation method of halo TM -halo-M44 and the purification method of M44 related antibodies are: halo-int N- After incubating gb1 and gb1-int C- M44 at room temperature for 2-4 hours at a concentration ratio of 1:1-2, 250 ⁇ L of the reaction solution was mixed with 50 ⁇ L of the balanced Promega halo TM room temperature shaker for half an hour, and then centrifuged and used Wash the beads with 500 ⁇ L PBS to remove non-specific binding proteins.
  • the above-mentioned nanocarrier is used to deliver cargo protein, inoculate mice to induce high-titer specific antibodies, and the induced antibodies are used for WB and/or IF analysis.
  • the present disclosure also provides a nano-formulation capable of simultaneously delivering antigen and immune enhancer.
  • the nano-formulation is constructed by the following steps: 1) The N-terminal of ferritin is fused with the C-terminal of the linker gbl-intein.
  • the recombinant protein gbl-intein c- ferrtin self-assembles into twenty-four polymers to form nanoparticles;
  • the C-terminus of the foreign protein is fused with the N-terminus of the intein to form a recombinant protein, and the N-terminus of the intein specifically recognizes and excises the ferritin exposed on the surface of the nanoparticle;
  • the foreign protein is an immune enhancer or an antigen or a combination of the two.
  • nucleic acid sequence of the gbl is shown in SEQ ID No. 22, and the nucleic acid sequence of the intein c is shown in SEQ ID No. 19.
  • the immune enhancer is flagellin or rhizavidin, a monomer analog of avidin.
  • the antigen is a protein antigen of a single polypeptide epitope or a protein antigen of different polypeptide epitopes.
  • the exogenous protein is a combination of an immune enhancer and an antigen
  • the molar ratio is 1 to 3:5.
  • the ferritin is human-derived heavy-chain ferritin or Pyrococcus furiosus or ferritin derived from other species.
  • the technical solution provided by the present disclosure successfully introduces the intein-mediated protein editing technology into the surface modification of the encapsulin nanocarrier through molecular design; the prepared nanoparticle has a complementary split intein C fragment (split intein C, gp41-1-int C , abbreviated as int C , the nucleic acid sequence is shown in SEQ ID No.16)) Cargo molecules are mixed, intein is excised by itself, and the cargo molecules are covalently coupled to the surface of the nanoparticle.
  • the nanoparticle surface modification technology can effectively solve the problem.
  • the C-terminal metastability of encapsulin is limited by the specific and efficient delivery of different cargo molecules on the surface of encapsulin.
  • the technical solution provided by the present disclosure takes the GFP protein and the C-terminal amino acids 315-411 of the mouse cytomegalovirus (mouse cytomegalovirus, MCMV) M44 protein (referred to as the M44 protein, which is the marker of the MCMV replication center, or marker) as models, and uses the new
  • the developed nano-delivery technology delivers the aforementioned cargo proteins, and inoculates mice to induce high-titer specific antibodies. Then, using halo co-crosslinking technology and antigen-antibody high-affinity interaction, GFP and M44-related IgG antibodies were successfully purified, which can be used for western blotting detection and immunofluorescence (immunofluorescence, IF) analysis.
  • IF immunofluorescence
  • the nano-formulations of the present disclosure can efficiently display antigen proteins, immune enhancers, polypeptide epitopes and antibodies on the surface of nano particles, and are used in the field of enhanced preventive subunit vaccines and personalized polypeptide epitope tumor vaccines and antibody-conjugated drugs. .
  • the present disclosure solves the effect of the loaded protein on the stability of the protein self-assembled nanoparticle, and realizes that the nano preparation can efficiently deliver the antigen and the immune enhancer at the same time, and has good specificity.
  • Figure 1 is a schematic diagram of the modified encapsulin nanocarrier based on the method of intein-mediated protein editing and shearing;
  • Figure 2 is the molecular modification of encapsulin and the purification and analysis of recombinant nanoparticles
  • Figure 3 is based on the intein-mediated protein editing and shearing technology to efficiently load GFP protein on the surface of encapsulin.
  • Figure 4 is based on intein-mediated protein editing and shearing technology to efficiently load 2xstrep2 polypeptide on the surface of encapsulin.
  • Figure 5 is based on the intein-mediated protein editing and shearing technology to efficiently load the C-terminal amino acids 315-411 of MCMV on the surface of encapsulin.
  • Figure 6 is an enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) titration of cargo molecule-related IgG antibody titers.
  • Figure 7 is a schematic diagram of the principle of purifying cargo molecule-related antibodies based on halo covalent cross-linking technology and antigen-antibody interaction.
  • Figure 8 shows the preparation of halo TM -halo-GFP and the purification of GFP-related antibodies.
  • Figure 9 shows the preparation of halo TM -halo-M44 and the purification of M44 related antibodies.
  • Figure 10 is the analysis of the function of newly purified GFP and M44-related antibodies by western blotting (WB).
  • Fig. 11 is the function test of newly purified GFP antibody by immunofluorescence (IF) analysis.
  • Figure 12 is a functional test of the newly purified M44 antibody by IF analysis.
  • Figure 13 is a schematic diagram of the construction principle of the nanocarrier module.
  • Figure 14 is an analysis diagram showing that the introduction of the linker gbl-intein C does not affect the stability of the nanoparticles
  • Figure 14a is the electrophoresis of the three recombinant proteins
  • Figure 14b is the electrophoresis of gb1-intein C- HFT and gb1-intein C- PFT after precipitation with ammonium sulfate
  • Figure 14c is the electrophoresis of the purified gb1-intein C- HFT
  • Figure 14d is purified by gel filtration gb1-intein C -HFT UV gel
  • Figure 14e electron micrograph of purified gb1-intein C -HFT detecting a TEM
  • FIG. 14f is a TEM examination of purified gb1-intein C -PFT SEM picture.
  • Figure 15 is an analysis diagram of GFP protein and strep2 after being efficiently covalently coupled to HFT;
  • Figure 15a is the electrophoresis diagram of the GFP-HFT content of the target product detected by the Coomassie brilliant blue method (CB assay);
  • Figure 15b is the electrophoresis diagram of the coupling efficiency of GFP and HFT using Western-blotting detection (WB assay);
  • Figure 15c is the Coomassie brilliant blue The electrophoresis diagram of the content of strep2-HFT of the target product by the method;
  • Figure 15d is the electrophoresis diagram of the coupling efficiency of strep2 polypeptide and HFT using Western-blotting.
  • Figure 16 is an analysis diagram showing that loading GFP protein and strep2 polypeptide by means of self-shearing does not affect the stability of the nanoparticle structure
  • Figure 16a is a Coomassie brilliant blue electrophoresis analysis diagram of gel filtration purified GFP-HFT
  • Figure 16b is a UV curve diagram of gel filtration purified GFP-HFT
  • Figure 16c is an electron microscope image of purified GFP-HFT detected by TEM
  • 16d is an electrophoretic analysis chart of gel filtration purified strep2-HFT
  • FIG. 16e is a UV curve chart of gel filtration purification of strep2-HFT
  • FIG. 16f is an electron micrograph of TEM detection and purification of strep2-HFT.
  • FIG. 17 is a schematic diagram of preparing different nanodevices by splicing nanoparticles and exogenous proteins provided by the present disclosure through intein-mediated protein editing technology.
  • Figure 18 is an analysis diagram showing that nanoparticles can efficiently load polypeptide epitopes and flagellin;
  • Figure 18a is the gel filtration purification electrophoresis analysis diagram of the nano vaccine SP70-HFT;
  • Figure 18b is the UV curve diagram of the gel filtration purification nano vaccine SP70-HFT;
  • Figure 18c is the transmission electron microscope image of the nano vaccine SP70-HFT;
  • Figure 18d is SP70 and CBLB co-delivery nano-preparation SP70-CBLB-HFT gel filtration purification electrophoresis analysis diagram;
  • Figure 18e is the gel filtration purification co-delivery nano-preparation SP70-CBLB-HFT UV curve diagram;
  • Figure 18f is the co-delivery nano-preparation SP70 -CBLB-HFT transmission electron microscope image;
  • Figure 18g is the gel filtration purification electrophoresis analysis of the nano adjuvant CBLB-HFT;
  • Figure 18h is the UV curve of the gel filtration purification nano adjuvant CBLB-HFT;
  • Figure 18i is the nano adjuvant Transmission electron micrograph
  • Figure 19 is an analysis diagram of whether modular nanocarriers can efficiently load CpG
  • Figure 19a shows the electrophoresis analysis of rhizavidin-HFT gel filtration purification
  • Figure 19b shows the UV curve of rhizavidin-HFT gel filtration purification
  • Figure 19c shows the transmission electron microscope image of rhizavidin-HFT
  • Figure 19d shows the SP70-rhizavidin-HFT Gel filtration purification electrophoresis analysis chart
  • Figure 19e is the UV curve of SP70-rhizavidin-HFT gel filtration purification
  • Figure 19f is the transmission electron microscope image of SP70-rhizavidin-HFT
  • Figure 19g is the two-dimensional type of SP70-rhizavidin-HFT Average image
  • Figure 19h is the electrophoresis analysis image of target protein rhizavidin-HFT combined with biotinylated CpG.
  • Figure 20 is an analysis diagram of the influence of different vaccine formulations on the immune effect using the SP70 epitope as a model
  • Figures 20a-c are ELISA analysis of different delivery of CpG adjuvants and CBLB-induced IgG titers;
  • Figure 20d in vitro cell titration analysis of antibody neutralization titers induced by different vaccine formulations;
  • Figure 20e is the immune response induced by co-delivery of nano formulations.
  • Figure 21 is an analysis diagram of protein editing and cutting technology that can efficiently load HA antigen on HFT/PFT carriers;
  • Figure 21a is the electrophoresis diagram of recombinant gene HA-HFT expression analysis
  • Figure 21b is the electrophoretic analysis diagram of gel filtration purification of HA-HFT prepared based on protein editing shearing technology
  • Figure 21c is the UV curve of gel filtration purification of HA-HFT Figure
  • Figure 21d is a transmission electron microscope image of HA-HFT
  • Figure 21e is an electrophoresis analysis image of HA-PFT prepared based on protein editing and shearing technology purified by gel filtration
  • Figure 21f is a UV curve of HA-PFT purified by gel filtration Figure
  • Figure 21g is a transmission electron microscope image of HA-PFT.
  • Figure 22 is an analysis diagram of nano adjuvant CpG-HFT enhancing HA specific humoral immune response and regulating IgG type.
  • Figure 22a shows that ELISA analysis reveals that both nanocarriers and nanoadjuvants can enhance HA specific humoral immune response
  • Figure 22b-d shows that ELISA analysis reveals that vaccine-induced antibodies can specifically recognize H1N1(P), H3N2, and H7H9 strains, and nano
  • the antibody recognition ability induced by the adjuvant and nano vaccine mixed preparation is higher than that of the nano vaccine itself.
  • the present disclosure provides a nanocarrier mediated by intein.
  • the nanocarrier is constructed through the following steps:
  • the C-terminus of the encapsulin protein is introduced by gene fusion method into three tandem proteins int N , gb1 and halo to form a recombinant protein encapsulin-int N -gb1-halo, the nucleic acid sequence of which is shown in SEQ ID No. 8;
  • the intein intein is self-excised, forming by-products int N -gb1-halo and gb1-int C , and encapsulin is covalently coupled with cargo to produce the target product encapsulin-cargo.
  • the nucleic acid sequence of the encapsulin protein is shown in SEQ ID No. 1
  • the nucleic acid sequence of int N is shown in SEQ ID No. 2
  • the nucleic acid sequence of gb1 is shown in SEQ ID No. 2.
  • ID No. 3 the nucleic acid sequence of halo is shown in SEQ ID No. 4
  • the nucleic acid sequence of int C is shown in SEQ ID No. 16.
  • the cargo is GFP or M44 or strep2.
  • nucleic acid sequence of the GFP is shown in SEQ ID No. 9.
  • nucleic acid sequence of M44 is shown in SEQ ID No. 10.
  • nucleic acid sequence of strep2 is shown in SEQ ID No. 11.
  • the above-mentioned intein-mediated nanocarrier, step 3) is encapsulin-int N -gb1-halo and gb1-int C -GFP in a concentration ratio of 1:1-2 After the two were incubated for 2 hours at room temperature, the target product encapsulin-GFP was separated by superose 6B increase molecular exclusion method.
  • the above-mentioned intein-mediated nanocarrier, step 3) is encapsulin-int N -gb1-halo and gb1-int C -strep2 in a concentration ratio of 1:1-3 After the two were incubated for 2 hours at room temperature, the target product encapsulin-strep2 was separated by superose 6B increase molecular exclusion method.
  • the above-mentioned intein-mediated nanocarrier, step 2) is encapsulin-int N -gb1-halo and gb1-int C -M44 in a concentration ratio of 1:1-2 After the two were incubated for 2 hours at room temperature, the target product encapsulin-M44 was separated by superose 6B increase molecular exclusion method.
  • the present disclosure also provides the application of the nanocarrier as described herein as a delivery cargo protein, and inoculating mice to induce high-titer specific antibodies.
  • the antibody purification method is:
  • the mixture is mixed with Promega halo TM beads, and the target product halo-cargo is covalently bound to halo TM beads. After centrifugation to discard the supernatant, the immunized serum is added. Affinity interaction is combined with halo TM -halo-cargo, other antibodies and proteins are separated after centrifugation and washing, and finally the target antibody is eluted with 200mM glycine pH2.8.
  • the preparation and purification method of halo TM -halo-GFP is as follows: halo-int N -gb1 and gb1-int C -GFP are placed at room temperature at a concentration ratio of 1:1-2 After incubating for 4 hours under the conditions, mix 250 ⁇ L of the reaction solution with 50 ⁇ L of the balanced Promega halo TM room temperature shaker for half an hour, then centrifuge, wash the beads with 500 ⁇ L of PBS to remove non-specific binding proteins; the serum after the second immunization; the serum and The flow out of halo TM -halo-GFP after incubation; first wash halo TM -halo-GFP with 500 ⁇ L PBST; then eluate the target antibody with 90 ⁇ L of 200 mM glycine pH2.8.
  • the method for preparing halo TM -halo-M44 and purifying M44 related antibodies is: halo-int N -gb1 and gb1-int C -M44 are in a concentration ratio of 1:1 -2 After incubating for 2-4 hours at room temperature, 250 ⁇ L of the reaction solution was mixed with 50 ⁇ L of the balanced Promega halo TM room temperature shaker for half an hour, and then centrifuged, and the beads were washed with 500 ⁇ L of PBS to remove non-specific binding proteins.
  • the induced antibody is used for WB and/or IF analysis.
  • the present disclosure provides a nanoformulation capable of simultaneously delivering antigens and immune enhancers.
  • the nanoformulation is constructed by the following steps: 1) The N-terminal of ferritin and the C-terminal of the linker gbl-intein are fused to form a recombinant The protein gbl-intein c- ferrtin self-assembles into 24-mer twenty-four mers to form nanoparticles;
  • the C-terminus of the foreign protein is fused with the N-terminus of the intein to form a recombinant protein, and the N-terminus of the intein specifically recognizes and excises the ferritin exposed on the surface of the nanoparticle;
  • the foreign protein is an immune enhancer or an antigen or a combination of the two.
  • the nucleic acid sequence of the gbl is shown in SEQ ID No. 22, and the nucleic acid sequence of the intein c is shown in SEQ ID No. 19.
  • the immune enhancer is flagellin or rhizavidin, a monomer analog of avidin.
  • the antigen is a protein antigen of a single polypeptide epitope or a protein antigen of different polypeptide epitopes.
  • the molar ratio is 1 to 3:5.
  • the ferritin is human-derived heavy-chain ferritin or Pyrococcus furiosus or other species-derived ferritin.
  • the C-terminus of the encapsulin protein (nucleic acid sequence is shown in SEQ ID No. 1) is introduced into int N (gp41-1-intein N , abbreviated as int N ) by gene fusion; the nucleic acid sequence is shown in SEQ ID No. 2 ), gb1 (nucleic acid sequence shown in SEQ ID No. 3) and halo (nucleic acid sequence shown in SEQ ID No. 4), a total of 3 tandem proteins form a recombinant protein (AB);
  • encapsulin-int N nucleic acid sequence shown in SEQ ID No. 5
  • encapsulin -int N -gb1 nucleic acid sequence is shown in SEQ ID No. 6
  • halo nucleic acid sequence is shown in SEQ ID No. 4
  • encapsulin-int N -halo nucleic acid sequence is shown in SEQ ID No. 7
  • encapsulin-int N -gb1-halo nucleic acid sequence is shown in SEQ ID No.
  • the percentage analysis graph is drawn with Graph Padprism5. After three repetitions, the two-tailed t test analysis found that the ratio of 4 recombinant protein supernatant was much higher than that of 2 and 3. The combination of surface soluble label gb1 and halo had a significant synergistic effect.
  • d 12% Tricine-SDS electrophoresis detection uses superose 6B increase molecular exclusion method to separate and purify recombinant protein 4. The number represents the collection tube number of the collector, and the collection volume of each collection tube is 1 mL.
  • e UV absorption spectrum of recombinant protein 4 purified by molecular exclusion method.
  • the horizontal axis is the collection volume, and the vertical axis is the absorbance of ultraviolet light at A280nm.
  • the target product is indicated by an arrow.
  • f Observation of recombinant protein 4 by transmission electron microscope, it is found that the protein exists in the form of spherical nanoparticles. The white scale is 20nm.
  • the cargo protein is easy to be exposed to the surface of the nanoparticle due to its large molecular weight. 60 recombinant protein monomers self-assemble into nanoparticles.
  • the exposed int N -gb1-halo(B) acts as a universal "adapter" with the cargo recombinant protein gb1 -After int C -cargo (CD) is mixed, molecular recombination occurs with the help of DTT;
  • the intein intein is self-resected to form a by-product.
  • the intein intein is self-resected to form the by-products int N -gb1-halo and gb1-int C (A and C), and encapsulin is covalently coupled with cargo for the purpose of production
  • the cargo protein cargo is GFP (nucleic acid sequence shown in SEQ ID No. 9) or M44 (M44 315-411aa, abbreviated as M44 nucleic acid sequence shown in SEQ ID No. 10) or strep2 (nucleic acid sequence shown in SEQ ID No. 11).
  • the initial reaction substrate concentrations are: encapsulin-int N -gb1-halo (nucleic acid sequence is shown in SEQ ID No. 8) 10 ⁇ M, gb1-int C -GFP (nucleic acid sequence is shown in SEQ ID No. 12) 20 ⁇ M.
  • the reaction substrate encapsulin-int N -gb1-halo no longer decreased significantly as time passed, and the target product encapsulin-GFP no longer increase.
  • the reaction efficiency is measured by calculating the reduction of the reaction substrate encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8).
  • the abscissa is the reaction time, and the ordinate is the shear efficiency.
  • the initial reaction substrate encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) has a concentration of 10 ⁇ M, and the reaction substrate gb1-int C -GFP (nucleic acid sequence is shown in SEQ ID No. 12) concentration, when the ratio of the two is 1:1, the reaction efficiency is higher than 90%.
  • Increasing the concentration of gb1-int C- GFP no longer promotes the production of more target products.
  • the abscissa represents the change in gb1-int C -GFP concentration, and the ordinate represents the reaction efficiency.
  • the shear efficiency calculation formula is the same as b.
  • Cut 2mL reaction system encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) concentration is 10 ⁇ M, gb1-int C -GFP concentration is 15 ⁇ M), use superose 6B after 2h at room temperature Increase molecular exclusion method to separate the target product encapsulin-GFP.
  • encapsulin-int N -gb1-halo nucleic acid sequence shown in SEQ ID No. 8
  • concentration 10 ⁇ M
  • gb1-int C -GFP concentration is 15 ⁇ M
  • use superose 6B after 2h at room temperature Increase molecular exclusion method to separate the target product encapsulin-GFP.
  • the abscissa represents the elution volume, and the ordinate represents the change in absorbance of ultraviolet light at A280nm.
  • the target product is indicated by an arrow.
  • the initial reaction substrate concentrations are: encapsulin-int N -gb1-halo (nucleic acid sequence is shown in SEQ ID No. 8) 10 ⁇ M, gb1-int C -strep2 (nucleic acid sequence is shown in SEQ ID No. 13) 30 ⁇ M.
  • the reaction substrate encapsulin-int N -gb1-halo no longer decreased significantly, and the target product encapsulin-strep2 no longer increase.
  • the reaction efficiency is measured by calculating the reduction ratio of the reaction substrate encapsulin-intein N- gb1-halo (nucleic acid sequence shown in SEQ ID No. 8).
  • the abscissa is the reaction time, and the ordinate is the shear efficiency.
  • the concentration of the initial reaction substrate encapsulin-int N -gb1-halo is 10 ⁇ M, and gradually increase the reaction substrate gb1-int C -strep2 (nucleic acid sequence shown in SEQ ID No. 13 ) Concentration, when the ratio of the two is 1:1, the reaction efficiency is greater than 90%. Increasing the concentration of gb1-int C- strep2 (nucleic acid sequence shown in SEQ ID No. 13) no longer promotes the production of more target products.
  • the abscissa represents the concentration change of gb1-int C- strep2 (nucleic acid sequence is shown in SEQ ID No. 13), and the ordinate represents the reaction efficiency.
  • the shear efficiency calculation formula is the same as b.
  • f UV absorption spectrum of the target product separated and purified by molecular exclusion method.
  • the abscissa represents the elution volume, and the ordinate represents the change in absorbance of ultraviolet light at A280nm.
  • the target product is indicated by an arrow.
  • the initial reaction substrate concentrations are: encapsulin-int N -gb1-halo (nucleic acid sequence is shown in SEQ ID No. 8) 10 ⁇ M, gb1-int C -M44 (nucleic acid sequence is shown in SEQ ID No. 14) 20 ⁇ M.
  • the reaction substrate encapsulin-int N- gb1-halo no longer decreased significantly as time passed, and the target product encapsulin-M44 no longer increase.
  • b Use ImageJ to calculate the spliced production change over time (three repetitions, the representative value of each point includes the average and standard deviation).
  • the reaction efficiency is measured by calculating the reduction ratio of the reaction substrate encapsulin-intein N -gb1-halo.
  • the abscissa is the reaction time, and the ordinate is the shear efficiency.
  • the concentration of the initial reaction substrate encapsulin-int N -gb1-halo was 10 ⁇ M, and the reaction substrate gb1-int C -M44 (nucleic acid sequence shown in SEQ ID No. 14) concentration, when the ratio of the two is 1:1, the reaction efficiency is higher than 90%.
  • Increasing the concentration of gb1-int C- M44 no longer promotes the production of more target products.
  • the abscissa represents the concentration change of gb1-int C- M44 (nucleic acid sequence is shown in SEQ ID No. 14), and the ordinate represents the reaction efficiency.
  • the shear efficiency calculation formula is the same as b.
  • f UV absorption spectrum of the target product separated and purified by molecular exclusion method.
  • the abscissa represents the elution volume, and the ordinate represents the change in absorbance of ultraviolet light at A280nm.
  • the target product is indicated by an arrow.
  • the molecular exclusion method involved in the separation of nanoparticle products in this disclosure is all carried out on the Clear-First 3000 system of Shanghai Flash Spectrum Co., Ltd. using a superose 6B increase chromatography column.
  • Use 12% Tricine-SDS-PAGE electrophoresis to detect the molecular exclusion method to purify the nanoparticle samples take 30 ⁇ L of the sample in the collection tube numbered 7-18 (collection volume of each tube is 1mL), add 10 ⁇ L of 4xSDS loading buffer and then 100°C Cook the sample for 10 minutes. Load 2 ⁇ L before and after cutting, and load 6 ⁇ L for each sample in the collection tube.
  • the horizontal axis of the spectrum of UV absorbance change with elution volume represents the elution volume
  • the vertical axis represents the absorbance at A280 nm.
  • the present disclosure relates to the induced expression of proteins: the recombinant genes used in the present disclosure are inserted into the pET28a vector, and transformed into BL21(DE3)plysS to induce expression.
  • encapsulin-int N nucleic acid sequence shown in SEQ ID No. 14
  • encapsulin-int N -gb1 fusion gene with NcoI and XhoI restriction sites encapsulin-int N -halo
  • encapsulin-int N -gb1-halo The nucleic acid sequence is shown in SEQ ID No. 8
  • the single clone was sequenced correctly and transformed into BL21(DE3)plysS to induce expression and purification.
  • Cargo molecule-related fusion genes gb1-intein C -2xstrep2 (abbreviated as strep2), gb1-intein C -GFP and gb1-intein C -M44 were also amplified by overlap, and then used EcoRI and XhoI as restriction sites for linear insertion
  • the transformed pET28a vector was transformed into Top10 for amplification.
  • the restriction sites of the halo-intein N- gb1 recombinant gene are NcoI and XhoI, and the cloning construction method is the same as above. All recombinant genes were converted to BL21(DE3)plysS.
  • LB When LB was cultured to an OD of 0.6-0.8, 0.2mM IPTG was added, cultured on a shaker at 25°C for 5 hours, and then centrifuged at 4000rpm and 4°C for 30 minutes, and the bacterial pellets were collected. Resuspend the bacteria with 30mL of PBS, centrifuge under the same conditions and discard the supernatant. The bacteria can be used for the next step of purification or long-term freezing at -80°C.
  • Nanocarrier-related proteins Resuspend 500 mL of LB culture bacteria with 30 mL of 50 mM Tris, pH 7.5, 150 mM NaCl (NT buffer), and then ultrasonically disrupt. After the sonication is over, centrifuge at 12000 rpm, 4°C for 30 minutes, and transfer the supernatant to a 50 mL centrifuge tube. Add 0.15g/mL ammonium sulfate solid, mix well on a shaker at 4°C for 15 minutes, and centrifuge at 12000rpm, 4°C for 30 minutes.
  • encapsulin-int N- gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) measured by BCA was 2.75 mg/mL, which can be used for in vitro shear analysis or cryopreservation at -80°C.
  • proteins are purified by Ni 2+ affinity chromatography to purify soluble proteins in the supernatant of the bacterial lysate.
  • the bacteria were resuspended in 30mL NT buffer and then sonicated for lysis. After centrifugation, the supernatant was mixed with 1mL N 2+ resin on a shaker at 4°C for 30 minutes. The non-specific binding protein flows out of the liquid by gravity. Next, add 20 mL of NT buffer containing 10 mM, 20 mM imidazole to wash the resin to remove loosely bound proteins. Subsequently, 10 mL of NT buffer containing 500 mM imidazole was added for elution.
  • the protein concentrations measured by the BCA method were halo-intein N -gb1: 1.2 mg/ml; gb1-intein C -strep2: 1.8 mg/ml; gb1-intein C -GFP: 1.8 mg/ml, gb1-intein C -M44 : 1.92mg/ml.
  • the above-mentioned protein does not require dialysis and can be directly used for intein-mediated protein editing and shearing or long-term freezing at -80°C.
  • the present disclosure first explores the effect of different reaction times and substrate concentrations on reaction efficiency under a small volume condition (30 ⁇ L).
  • the initial reaction substrate encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) or halo-int N -gb1 nucleic acid sequence shown in SEQ ID No. 15) concentration is fixed at 10 ⁇ M, gb1-int
  • concentration range of C- cargo (cargo is GFP, strep2 or M44) is: 1-30 ⁇ M.
  • the initial reaction substrate concentration is fixed, with time as a variable, ranging from 1 minute to 16 hours, and it is found that all reactions are completed within 4 hours, and the efficiency is as high as 90%.
  • the efficiency of all reactions reaches the maximum, and the efficiency is as high as 90% or more. All reactions were performed at room temperature (around 25°C), and the reaction solution contained 2mM DTT (dithiothreitol). Take encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) or halo-int N -gb1 nucleic acid sequence shown in SEQ ID No. 15 as the target, and use ImageJ to calculate the reaction efficiency. The formula is : (1-Unreacted substrate/initial reaction substrate)*100.
  • Encapsulin-cargo enzyme-linked immunosorbent assay enzyme-linked immunosorbent assay, ELISA
  • titration of cargo molecule-related IgG antibody titer experiment see Figure 6.
  • Mouse immunization and enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) titration of cargo molecule-related IgG antibody titers: 5-8 weeks old female Balb/c mice were intraperitoneally inoculated with 10 ⁇ g of cargo protein (GFP or M44) ) Or the cargo protein delivered by the nanocarrier (encapsulin-GFP or encapsulin-M44), inoculated twice, two weeks apart. After two weeks of immunization, about 200 ⁇ L of blood was collected from the eye socket, placed at 37°C for 30 minutes, and then placed at 4°C overnight to fill the serum. Centrifuge at 5,000 rpm and 4°C for 30 minutes the next day, and collect serum.
  • ELISA enzyme-linked immunosorbent assay
  • mice were immunized intraperitoneally with 10 ⁇ g of protein antigen or antigen delivered by nanocarriers and were vaccinated twice, with an interval of two weeks each time. Two weeks after immunization, blood was collected from the orbit and the antibody titer was detected by ELISA.
  • a After one and two immunizations, encapsulin-GFP delivered by nanotechnology induces GFP-related antibody titer significantly higher than GFP protein (Mann Whitney test, nonparametric tests, p ⁇ 0.01), and the antibody titer is four times that of the latter .
  • the gb1-int C- GFP or gb1-intein C- M44 used can be edited and cut with the halo-int N- gb1 nucleic acid sequence as shown in SEQ ID No. 15) to produce recombinant protein halo-cargo (cargo is GFP or M44), gb1-int C and int N -gb1 exist as by-products.
  • the mixture is mixed with Promega halo TM beads, and the target product halo-cargo is covalently bound to halo TM beads. After centrifugation to discard the supernatant, the immunized serum was added.
  • Cargo molecule-related antibodies use the high-affinity interaction between antigen and antibody to bind to halo TM -halo-cargo, and other antibodies and proteins are separated after centrifugation and washing. Finally, the target antibody was eluted with 200mM glycine pH2.8.
  • the initial reaction substrate concentration is: halo-int N- gb1 nucleic acid sequence shown in SEQ ID No. 15) 10 ⁇ M, gb1-int C- GFP (nucleic acid sequence shown in SEQ ID No. 12) 20 ⁇ M.
  • the nucleic acid sequence of the reaction substrate halo-int N- gb1 no longer decreased significantly, and the target product halo-GFP no longer increased.
  • b Use ImageJ to calculate the spliced production change over time (three repetitions, the representative value of each point includes the average and standard deviation).
  • the reaction efficiency is measured by calculating the reduction ratio of the reaction substrate halo-int N- gb1 nucleic acid sequence (shown in SEQ ID No. 15). The abscissa is the reaction time, and the ordinate is the shear efficiency.
  • d Use ImageJ to calculate the change of the target product (spliced production) with the concentration of the reaction substrate (three repetitions, the representative value of each point includes the average and standard deviation).
  • the abscissa represents the change in gb1-int C -GFP concentration, and the ordinate represents the reaction efficiency.
  • the shear efficiency calculation formula is the same as b.
  • Tricine-SDS-PAGE detection uses newly prepared halo TM -halo-GFP to separate and purify GFP-related proteins from serum.
  • input serum after 2 immunizations; flow: flow out of serum after incubation with halo TM -halo-GFP; wash1-wash3: wash halo TM -halo-GFP with 500 ⁇ L PBST; elution: 90 ⁇ L 200mM glycine pH2.8 elution Antibody of interest (add 10 ⁇ L of 1M Tris, pH9.0 buffer in advance to EP tube to protect the antibody).
  • the initial reaction substrate concentrations are: halo-int N- gb1 nucleic acid sequence shown in SEQ ID No. 15) 10 ⁇ M, gb1-int C- M44 (nucleic acid sequence shown in SEQ ID No. 14) 20 ⁇ M. After the two were incubated at room temperature for 2 hours, the nucleic acid sequence of the reaction substrate halo-int N- gb1 (as shown in SEQ ID No. 15) no longer decreased significantly, and the target product halo-GFP no longer increased.
  • b Use ImageJ to calculate the spliced production over time (three repetitions, the representative value of each point includes the average and standard deviation).
  • the reaction efficiency is measured by calculating the reduction ratio of the reaction substrate halo-int N- gb1 nucleic acid sequence (shown in SEQ ID No. 15). The abscissa is the reaction time, and the ordinate is the shear efficiency.
  • d Use ImageJ to calculate the change of the target product (spliced production) with the concentration of the reaction substrate (three repetitions, the representative value of each point includes the average and standard deviation).
  • the abscissa represents the concentration change of gb1-int C- M44 (nucleic acid sequence is shown in SEQ ID No. 14), and the ordinate represents the reaction efficiency.
  • the shear efficiency calculation formula is the same as b.
  • Tricine-SDS-PAGE detection uses newly prepared halo TM -halo-M44 to separate and purify M44 related proteins from serum.
  • Input serum after 2 immunizations; flow: flow out of serum after incubation with halo TM -halo-M44; wash1-wash3: wash halo TM -halo-M44 with 500 ⁇ L PBST; elution: 90 ⁇ L of 200mM glycine pH2.8 elution Antibody of interest (add 10 ⁇ L of 1M Tris, pH9.0 buffer in advance to EP tube to protect the antibody).
  • the pLKO vector was used to overexpress GFP protein and M44 protein in 293T cells, and then specific GFP(a) and M44(b) bands were detected with newly purified GFP and M44 antibodies, indicating that the newly prepared antibody can be used for WB analysis.
  • the pLKO vector was used to overexpress 3xflag-GFP protein in 293T cells, and both the GFP antibody (mouse anti-GFP) and flag antibody (rabbit anti-flag) prepared by the present disclosure could specifically recognize the GFP protein.
  • the fluorescence of the corresponding secondary antibodies anti-mouse (labeled with 555nm fluorescein) and anti-rabbit (labeled with 647nm fluorescein) highly coincides with the fluorescence position of the GFP protein itself, and the intensity is similar.
  • Immunofluorescence (IF) analysis shows that the antibodies prepared in the present disclosure have high specificity and can be used for IF detection, see FIG. 11.
  • MCMV Sgfp with a fluorescent label
  • MOI multiplicity of infection
  • a 24-hour IF analysis revealed that the M44-related antibodies prepared in the present disclosure can recognize the replication center of the virus (M44 is MCMV replication center marker), refer to Figure 12.
  • the antibodies prepared in the present disclosure can be used for WB and IF analysis.
  • Step 1 The recombinant protein gbl-intein c- ferrtin is formed by fusing the N-terminus of human heavy chain of human ferrtin (HFT) with the C-terminus of the linker gbl-intein and then self-assembled into twenty-four
  • the aggregates constitute nanoparticles with a diameter of 18 nm.
  • Step 2 The C-terminus of SP70 is fused with the N-terminus of the intein to form a recombinant protein.
  • the N-terminus of the intein specifically recognizes and excises the ferritin exposed on the surface of the nanoparticle; 30 ⁇ M foreign protein and 30 ⁇ M ferritin are covalently Cross-link to generate nano-formulations.
  • Step 1 Fuse the N-terminus of the pyrococcus furiosus heavy chain of human ferrtin (PFT) and the C-terminus of the linker gbl-intein to form a recombinant protein gbl-intein c- ferrtin and then self-assemble into two Tetrapamers constitute nanoparticles with a diameter of 18 nm.
  • PFT pyrococcus furiosus heavy chain of human ferrtin
  • gbl-intein linker gbl-intein
  • Step 2 The C-terminus of flagellin is fused with the N-terminus of the intein to form a recombinant protein, and the N-terminus of the intein specifically recognizes and excises the ferritin exposed on the surface of the nanoparticle; 60 ⁇ M foreign protein and 30 ⁇ M ferritin Valence cross-linking to generate nano formulations.
  • Step 1 Fusion of the N-terminus of the human heavy chain ferritin and the C-terminus of the linker gbl-intein to form a recombinant protein gbl-intein c- ferrtin and then self-assemble into twenty-tetramers to form nanoparticles with a diameter of 18nm.
  • Step 2 The same amount of CpG and the C-terminus of the influenza virus hemagglutinin stem region are simultaneously fused to the N-terminus of the intein to form a recombinant protein, and the N-terminus of the intein is specifically recognized and excised and exposed to the nano Ferritin on the surface of the particles; 45 ⁇ M foreign protein is covalently cross-linked with 30 ⁇ M ferritin to generate a nanoformulation.
  • the experiments and examples of the present disclosure involve the gene synthesis of the following proteins and polypeptides in Shanghai Tolo Harbor Biotechnology Co., Ltd., and primer synthesis in Nanjing GenScript Biotechnology Co., Ltd.
  • GFP SEQ ID NO.17 strep2 SEQ ID NO.18 gp41-1-intein C (referred to as the intein C) SEQ ID NO.19 gp41-1-intein N , (abbreviated as intein N ) SEQ ID NO.20 SP70 SEQ ID NO.21 gb1 SEQ ID NO.22 Rhizavidin SEQ ID NO.23 CBLB SEQ ID NO.24 H1HA10 SEQ ID NO.25 HFT SEQ ID NO.26 PFT SEQ ID NO.27 GFP-intein N -gb1 SEQ ID NO.28 strep2-gp41-1-intein N -gb1 SEQ ID NO.29 SP70-gp41-1-intein N -gb1 SEQ ID NO.30 rhizavidin-gp41-1-intein N -gb1 SEQ ID NO.31 CBLB-gp41-1-intein N SEQ ID NO.32 SP70-CBLB-gp41-1
  • HFT is a human heavy chain of human ferrtin
  • PFT is a heavy chain ferritin of Pyrococcus furiosus (pyrococcus furiosus heavy chain of human ferrtin).
  • GFP-intein N -gb1, strep2-gp41-1-intein N -gb1, SP70-gp41-1-intein N -gb1, rhizavidin-gp41-1-int N -gb1, CBLB-gp41-1-intein N , SP70-CBLB-gp41-1-intein N , H1HA10-gp41-1-intein N- gb1 are intein N- related "cargo" fusion proteins.
  • the gene of the "cargo" fusion protein is amplified by the overlap PCR method, and the 5'and 3'primers used have NcoI and XhoI restriction sites respectively.
  • the fusion gene was digested and inserted into the linearized pET28a vector with the same digestion, and transformed into E. coli JM109 for recombinant cloning construction.
  • gb1-gp41-1-intein C- HFT and gb1-gp41-1-intein C- PFT are intein C- related "carrier" fusion proteins.
  • the gene of the "vector" fusion protein is also amplified by the overlap PCR method.
  • the 5'primer and 3'primer have EcoRI and XhoI restriction sites respectively. After the digested fragment was inserted into the pET28a linearization vector, it was transformed into E. coli JM109.
  • the plasmid was extracted and transformed into E. coli BL21(DE3) plysS for protein expression. Pick a single monoclonal strain, inoculate it into 20ml of kanachloramphenicol bi-anti-LB medium, and cultivate overnight at 37°C on a shaker. On the second day, 1:25 inoculated into 500 ml of LB medium with kanachloramphenicol bi-antibody, cultured at 37°C and 250 rpm to an OD of 0.6-0.8, then 0.2mM IPTG was added for induction.
  • the bacteria were harvested by centrifugation at 4000 rpm, 4°C, and 30 minutes. The supernatant is discarded and the bacteria can be used for the next purification or frozen at -80°C for later use.
  • the primers used in this disclosure are used to prepare nanodevices with different functions.
  • the antigen or adjuvant protein purification involved in the present disclosure all proteins are purified by Ni 2+ affinity chromatography. Different recombinant proteins have slightly different purification methods because of their different properties and solubility. GFP-intein N- gb1, strep2-intein N- gb1, SP70-intein N -gb1, H1HA10-inteinN-gb1 and rhizavidin-inteinN-gb1 are soluble proteins, the supernatant of bacterial lysate can be directly added to Ni 2+ resin Carry out affinity chromatography purification.
  • the bacterial supernatant was lysed and mixed with 1ml resin on a shaker at 4°C for 30 minutes. Then, the non-specific binding protein flows out with the liquid gravity. Next, add 20ml NT buffer containing 10mM, 20mM, 40mM imidazole to wash the resin to remove non-specific binding proteins. Then, 500 mM imidazole was added to the NT buffer for elution. Finally, the eluted product was dialyzed overnight at 4°C in 1L NT buffer. Most of the CBLB-intein N and SP70-CBLB-intein N formed a precipitate.
  • CBLB-intein N and SP70-CBLB-intein N form inclusion body precipitates, which can be resuspended in NT buffer containing 2M urea, and the supernatant after centrifugation of the resuspension is purified by soluble protein purification.
  • intein was introduced into the Streptococcus G protein B1 domain tag (gb1) to construct the linker gb1-inteinC (shown as A in Figure 13).
  • the N-terminus of heavy chain ferritin (ferrti, shown as C in Figure 13) is genetically fused with the linker gb1-intein C to form the recombinant protein gb1-intein C- ferrtin.
  • the recombinant proteins gb1-intein C- HFT marked as 2 in Figure 14a-14b
  • gb1-intein C- PFT marked as 3 in Figure 14a-14b
  • the electrophoresis band at 35kDa-40kDa is very obvious, and the expression of recombinant proteins gb1-intein C- HFT and gb1-intein C- PFT are both significantly increased, and the solubility is high. It can be seen from the results that the introduction of gb1 can greatly increase the expression of heavy chain ferritin in cells and improve its solubility, which is conducive to the formation of large-scale production. Therefore, the recombinant protein gb1-intein C- ferrtin nanoparticles can be used to prepare nanocarrier platforms. feasibility.
  • Figure 14b is the electrophoresis diagram of gb1-intein C -HFT and gb1-intein C -PFT after precipitation with ammonium sulfate.
  • Figure 14c shows the electrophoresis after purification of gb1-intein C- HFT, in which pellet: ammonium sulfate enriched precipitation resuspended product, Purification of gb1-intein C- HFT by gel filtration: gel filtration Gel filtration method to purify gb1-intein C- HFT, gel filtration of gb1-intein C- HFT: Gel filtration to purify gb1-intein C- HFT, 9-17: Separate collection of gb1-intein C- HFT samples Number; Figure 14d is the UV image of the gel filtration purified gb1-intein C- HFT gel, the detection wavelength is 280nm, and the arrow points to the distribution position of gb1-intein C- HFT.
  • Figs. 14e and 14f The purified recombinant proteins gb1-intein C- HFT and gb1-intein C- PFT were observed by transmission electron microscope (TEM), the magnification of the transmission electron microscope was 110k, and the length of the ruler was 20nm.
  • Figs. 14e and 14f The results are shown in Figs. 14e and 14f.
  • Fig. 14e is the electron micrograph of the purified gb1-intein C- HFT by TEM (negative staining preparation);
  • Fig. 14f is the electron micrograph of the purified gb1-intein C- PFT by TEM;
  • Fig. 14e and Fig. 14f show that both gb1-intein C- HFT and gb1-intein C- PFT exist in the form of nanoparticles.
  • the ruler used to observe negatively stained samples by transmission electron microscopy all represents 20nm, and the arrow indicates the nanoparticle.
  • Both recombinant proteins exist in the form of 10-20nm spherical nanoparticles. It can be seen that the recombinant protein gb1-intein C- ferrtin forms nanoparticles through molecular self-assembly. It is determined that the recombinant proteins gb1-intein C- HFT and gb1-intein C- PFT can self-assemble into nanoparticles with a diameter of 18nm with 24 monomers.
  • gb1-intein C- HFT nanoparticles constructed in Example 4 were respectively contained with the exogenous protein GFP protein and strep2 polypeptide tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) Peptide-mediated protein splicing modification was performed to observe the stability of the nanoparticle.
  • strep2 polypeptide tag Trp-Ser-His-Pro-Gln-Phe-Glu-Lys
  • the C ends of the GFP protein and strep2 polypeptide tags were introduced into the linker intein N- gb1 through gene fusion, respectively, to form GFP-intein N- gb1 and strep2-intein N- gb1.
  • the target products GFP-HFT ( Figure 15a) and strep2-HFT ( Figure 15c) will increase.
  • Fix the amount of gb1-intein C -HFT increase the reaction concentration of GFP-intein N -gb1 or strep2-intein N -gb1, the reaction substrate gb1-intein C -HFT and GFP-intein N -gb1 or strep2-intein N -gb1
  • the reaction is maximized when the molar ratio is close to 1:1, the self-cleavage efficiency of the intein is near the highest, and the uncut gb1-intein C- HFT has almost no residue.
  • the GFP-labeled antibody, strep2-labeled antibody, and HFT antibody are used to detect changes in the reaction substrate and target product in the reaction system.
  • Western-blotting detection data show that the trans-shearing efficiency is very high and the reaction is complete ( Figure 15b and 15d). It can be seen that both GFP and strep2 can be efficiently and specifically covalently cross-linked to the HFT protein.
  • Fig. 16a is the Coomassie brilliant blue electrophoresis analysis diagram of gel filtration purified GFP-HFT
  • Fig. 16b is the UV curve diagram of gel filtration purified GFP-HFT
  • Fig. 16a is the Coomassie brilliant blue electrophoresis analysis diagram of gel filtration purified GFP-HFT
  • Fig. 16b is the UV curve diagram of gel filtration purified GFP-HFT
  • Fig. 16a is the Coomassie brilliant blue electrophoresis analysis diagram of gel filtration purified GFP-HFT
  • Fig. 16b is the UV curve diagram of gel filtration purified GFP-HFT
  • Fig. 16a is the Coomassie brilliant blue electrophoresis analysis diagram of gel filtration purified GFP-HFT
  • Fig. 16b is the UV curve diagram of gel filtration purified GFP-HFT
  • Fig. 16a is the Coomassie brilliant blue electrophoresis analysis diagram of gel filtration purified GFP-H
  • 16c is the TEM detection and purification The electron microscope image of GFP-HFT (negative staining);
  • Figure 16d is the electrophoresis analysis image of gel filtration purified strep2-HFT (Coomassie brilliant blue staining);
  • Figure 16e is the UV curve of gel filtration purified strep2-HFT;
  • 16f is the electron microscope image of the purified strep2-HFT detected by TEM (negative staining preparation); before: before the intein self-cleavage, after: after the intein self-cleavage, Figure 16a 8-19 and 16d 9- 20: Separately collect the number of the target product sample purified by gel filtration.
  • the proteins GFP-HFT and GFP-intein N- gb1 with very similar molecular weights can be separated well, and the elution position of the protein GFP-HFT is earlier than GFP-intein N- gb1, the arrow in Figure 16b
  • the peak of GFP-HFT indicates that the protein GFP-HFT exists as a macromolecular polymer.
  • the proteins strep2-HFT and strep2-intein N- gb1 with very similar molecular weights can also be separated well, and the elution position of the protein strep2-HFT is earlier than strep2-intein N- gb1, the arrow in Figure 16e
  • the peak of GFP-HFT indicates that the protein strrep2-HFT exists in the form of a macromolecular polymer.
  • the separated and purified target products GFP-HFT and strep2-HFT were observed by transmission electron microscope, and both GFP-HFT and strep2-HFT existed in the form of nanoparticles ( Figure 16c, 16f).
  • proteins or peptides can be efficiently sheared into HFT nanoparticles without affecting the stability of the nanoparticles, and the nanocarrier module platform can be efficiently covalently coupled with exogenous proteins.
  • enterovirus 71 enterovirus 71
  • enterovirus 71 enterovirus 71
  • nanocarrier module technology to allow polymorphic modification of the characteristics of nanoparticles, nanoparticles with different functions were prepared, in order to study the influence of different vaccine formulations on antigen immunogenicity, the establishment of nano adjuvants and vaccine-adjuvant co-delivery nano formulations And nano vaccines, see Figure 17 for details.
  • CpG or flagellin were used as adjuvants to prepare corresponding preparations.
  • Flagellin specifically uses CBLB.
  • CBLB is a truncated form of flagellin and has its adjuvant function.
  • Figure 18 The analysis diagram of the nanoparticle can efficiently load polypeptide epitopes and flagellin is shown in Figure 18:
  • Figure 18a is the gel filtration purification electrophoresis analysis diagram of the nano vaccine SP70-HFT;
  • Figure 18b is the gel filtration purification nano vaccine SP70-HFT
  • Figure 18c is the transmission electron micrograph of the nano vaccine SP70-HFT;
  • Figure 18d is the gel filtration purification electrophoresis analysis of the SP70 and CBLB co-delivered nano preparation SP70-CBLB-HFT;
  • Figure 18e is the gel filtration purification The UV curve of the delivery nano-preparation SP70-CBLB-HFT;
  • Figure 18f is the transmission electron microscope image of the co-delivery nano-preparation SP70-CBLB-HFT (negative staining);
  • Figure 18g is the gel filtration purification of the nano adjuvant CBLB-HFT Electrophoresis analysis diagram;
  • Figure 18h is the UV curve of
  • Nano vaccine 1 Intein is introduced into Streptococcus G protein B1 domain tag (gb1) to construct the linker gb1-intein C (shown as A in Figure 13). The N-terminal of human heavy chain ferritin HFT is genetically fused with the linker gb1-intein C to form the recombinant protein gb1-intein C- HFT. Twenty-four recombinant protein gb1-intein C- HFT molecules self-assembled into twenty-four polymer nanoparticles. 2Intein N- gb1 is introduced into the C-terminal of SP70 to form the protein SP70-intein N- gb1. In the 2mM DTT solution, the molar ratio is 1:1 by adding nanoparticles and protein SP70-intein N- gb1, and SP70-HFT nanoparticles are obtained by protein splicing, which is the nano vaccine delivery system.
  • Intein N is introduced into the C-end of CBLB to form the protein CBLB-intein N.
  • CBLB-intein N is added at a molar ratio of HFT to CBLB 1:1, and CBLB-HFT is obtained by protein splicing and self-assembly Form CBLB-HFT nanoparticles.
  • CpG cannot be directly covalently linked to HFT, but biotinylated CpG can be loaded onto the surface of nanoparticles through the high affinity binding of avidin-biotin. Biotinylated CpG can be loaded onto the surface of nanoparticles through the high affinity binding of avidin-biotin.
  • Intein N- gb1 is introduced into the C-terminus of avidin rhizavidin to form the protein rhizavidin-intein N- gb1.
  • Nanoparticles and rhizavidin-intein N- gb1 are added to the 2mM DTT solution to obtain rhizavidin-HFT through protein splicing and self-assembly Form rhizavidin-HFT nanoparticles.
  • Figure 19 The analysis diagram of whether the modular nanocarrier can efficiently load CpG is shown in Figure 19;
  • Figure 19a is the electrophoretic analysis diagram of rhizavidin-HFT gel filtration purification;
  • Figure 19b is the UV curve diagram of rhizavidin-HFT gel filtration purification;
  • 19c is the transmission electron microscope image of rhizavidin-HFT (negative staining);
  • Figure 19d is the gel filtration purification electrophoresis analysis image of SP70-rhizavidin-HFT;
  • Figure 19e is the UV curve of SP70-rhizavidin-HFT gel filtration purification;
  • Figure 19f is the transmission electron microscope image of SP70-rhizavidin-HFT (negative staining);
  • Figure 19g is the two-dimensional class average image of SP70-rhizavidin-HFT;
  • Figure 19h is the electrophoresis analysis image of target protein rhizavidin-HFT combined with biotinylated Cp
  • rhizavidin can be efficiently coupled to HFT, and the target product can be purified by gel filtration.
  • the rhizavidin-HFT purified by gel filtration was observed by transmission electron microscope in the form of spherical nanoparticles ( Figure 19c).
  • rhizavidin and SP70 are coupled at a molar ratio of 1:5, and it does not affect the nanoparticle structure, see Figure 19d-f for details.
  • the white circle indicates that the target protein self-assembles into spherical nano-sized particles, and the discretely distributed white spots in the circle are rhizavidin protein (the electron density is greater than the peptide) , And the average number of bright spots is consistent with the concentration of rhizavidin-intein N -gb1 and SP70-intein N -gb1 in the initial reaction.
  • the target product rhizavidin-HFT was combined with sufficient biotinylated CpG, the protein band migrated, revealing that CpG was successfully loaded.
  • the biotinylated CpG can be covalently attached to the nanoparticle by the following method.
  • the intein intein
  • the Streptococcus G protein B1 domain tag gb1
  • the N-terminal of human heavy chain ferritin HFT is genetically fused with the linker gb1-intein C to form the recombinant protein gb1-intein C- HFT.
  • Twenty-four recombinant protein gb1-intein C- HFT molecules self-assembled into twenty-four polymer nanoparticles.
  • 2Intein N- gb1 is introduced into the C-terminal of rhizavidin to form the protein rhizavidin-intein N- gb1.
  • the molar ratio is 1:1:1 by adding nanoparticles, biotinylated CpG and protein rhizavidin-intein N- gb1, obtained by protein splicing and the high affinity interaction between antibiotic protein and biotinylated CpG CpG-HFT nanoparticles are nano adjuvant drug delivery systems.
  • Figure 18a-b shows the results of Tricine-SDS-PAGE and UV curve analysis gel filtration separation and purification of the target product SP70-HFT.
  • the results show that this method can purify a relatively pure target product; before and after are respectively denoted as: samples before and after shearing, and numbers 8-19 represent the sample number (1ml/tube) of the collected sample for gel filtration purification.
  • Figure 18c TEM analysis target product SP70-HFT exists in the form of nanoparticles, and the arrow indicates the target product.
  • Figure 18d-f and Figure 18g-i are the purification and quality detection of the target product SP70-CBLB-HFT and CBLB-HFT by gel filtration, respectively.
  • Figures 18a, 18d and 18g show that the unsheared gb1-intein C- HFT bands are almost invisible, indicating that the self-shearing efficiency of the nanoparticles is high.
  • Figures 18c, 18f and 18i show that the three target products all exist in the form of nanoparticles, indicating that the nanoparticles provided in the present disclosure can be covalently cross-linked with antigens and adjuvants through self-shearing, thereby imparting different functions to the nanoparticles.
  • intein was introduced into the Streptococcus G protein B1 domain tag (gb1) to construct the linker gb1-intein C (shown as A in Figure 13).
  • the N-terminal of human heavy chain ferritin HFT is genetically fused with the linker gb1-intein C to form the recombinant protein gb1-intein C- HFT.
  • Twenty-four recombinant protein gb1-intein C- HFT molecules self-assembled into twenty-four polymer nanoparticles.
  • 2Intein N- gb1 is introduced into the C-terminal of SP70 to form the protein SP70-intein N- gb1.
  • the molar ratio of protein rhizavidin-intein N- gb1 and protein SP70-intein N- gb1 is 1:5, and the molar ratio of nanoparticles to the mixture of protein rhizavidin-intein N- gb1 and protein SP70-intein N- gb1 is 6. :1:5, the molar ratio of biotinylated CpG and protein rhizavidin-intein N- gb1 is 1:1.
  • SP70-CpG-HFT group used 0.4 ⁇ g/bottle of biotinylated CpG and 10 ⁇ g/bottle SP70-rhizavidin-HFT at a molar ratio of 1:1 to mix for 30 minutes in an ice bath to immunize mice.
  • SP70-HFT+CpG-HFT group used nano vaccine SP70-HFT and nano adjuvant CpG-HFT to immunize mice together, using doses of 10 ⁇ g/mouse and 1.6 ⁇ g/mouse respectively.
  • Nano-vaccine SP70-HFT group SP70-HFT immunized mice with a dose of 10 ⁇ g/mouse.
  • SP70-HFT+CpG group SP70-HFT and 0.4 ⁇ g/mouse of biotinylated CpG co-immunized mice.
  • Five mice in each group were vaccinated 3 times with an interval of 2 weeks between each inoculation. Two weeks after the third immunization, blood was collected from the orbit for immune analysis.
  • SP70-HFT Nano vaccine
  • SP70-HFT+CpG Nano vaccine and free CpG mixture
  • SP70-CpG-HFT SP70 and CpG co-delivery nano formulation
  • SP70-HFT+CpG-HFT Nano vaccine and nano adjuvant Agent combination preparation.
  • mice After inoculating mice with different dosage forms, the mice were vaccinated 3 times with an interval of 2 weeks. Blood was taken from the orbit for ELISA analysis after 2 weeks of the third immunization.
  • ELISA analysis of different ways to deliver CpG adjuvant induces IgG titers.
  • the low-dose free 0.4 ⁇ g/CpG has no obvious adjuvant effect
  • the nano adjuvant CpG-HFT has obvious boosting effect
  • the adjuvant effect of SP70-CpG-HFT co-delivery nano formulation is the best. It can be seen that the low-dose 0.4 ⁇ g/free CpG adjuvant has no obvious effect.
  • nano-adjuvant CpG-HFT delivered with nanoparticles and the antigen-adjuvant co-delivered nano-particle SP70-CpG-HFT adjuvant effect is significant, and the co-delivery of nano-formulations induces the most efficient B cell immune response. This is because the co-delivery of nanoformulations ensures that the antigen and the adjuvant are localized to the same antigen presenting cell (APC) to the fullest extent possible to fully exert the boosting effect of the adjuvant.
  • ELISA was used to detect IgG antibody titers of different subtypes (1:1000).
  • the IgG subtype analysis results showed that the co-delivery of the nanoformulation SP70-CpG-HFT also significantly enhanced the IgG titers of Th1 and Th2 types, as shown in Table 1. Th2 type IgG1 accounted for the highest proportion.
  • CpG As an adjuvant, CpG has a better boosting effect on B cell immune response than flagellin; comparing co-delivered CpG and flagellin, it is found that CpG induces higher antibody titers; see Figure 20c for details.
  • mice in each test group Take the sera of mice in each test group from points 1 to 2 of this experimental example, serially dilute the sera (50 ⁇ l) in a 2-fold gradient, and place them in a CO2 incubator at 37°C with 100TCID50 EV71G082 (50 ⁇ l) 1h later, 15,000 human embryo rhabdomyosarcoma cells (RD cells) were added. CPE was observed 3 days after infection, and the neutralizing antibody titer was counted.
  • RD cells human embryo rhabdomyosarcoma cells
  • the antibody neutralization titer analysis showed that the antibody titer of SP70-CpG-HFT, SP70-HFT+CpG-HFT, SP70-CBLB-HFT and SP70-HFT+CBLB-HFT were significantly higher than SP70-HFT, among them, the antibody titer induced by SP70-CpG-HFT is the highest.
  • Figure 20a-c shows the ELISA analysis of different ways of delivering CpG adjuvant and CBLB to induce IgG titers
  • Figure 20a shows The effect of different ways of delivering CpG on SP70IgG antibody titer.
  • the nano-vaccine mixed with free CpG SP70-HFT+CpG
  • nano-vaccine and nano-adjuvant CpG-HFT mixed preparation and SP70 and CpG co-delivery nano-preparation SP70-CpG-HFT can significantly increase the specific antibody titer.
  • the effect of co-delivery of nano preparations is higher than the antibody titer induced by the mixed preparation of nano vaccine and nano adjuvant.
  • Figure 20b reveals that when CBLB is used as an adjuvant, the co-delivery of the nanoformulation induces a higher specific antibody titre than the nanovaccine SP70-HFT and nanoadjuvant CBLB-HFT mixed formulation.
  • Figure 20c shows that the adjuvant effect of CpG is higher than that of CBLB.
  • Figure 20d In vitro cell titration analysis of antibody neutralization titers induced by different vaccine preparations. The results of antibody neutralization titers showed that the co-delivery of nanoformulations induced the highest antibody neutralization ability, and the neutralization ability of the antibodies stimulated by the nanovaccine and nanoadjuvant mixed formulation was the second, which was significantly higher than the nanovaccine itself. And CpG adjuvant induced antibody neutralization titer is better than CBLB adjuvant.
  • Figure 20e In vivo lethal protection analysis reveals that co-delivery of nanoformulations can provide the most effective immune protection (75%), followed by nanovaccine and nanoadjuvant (50%), both of which are higher than nanovaccine (25 %). The results of immunological analysis showed that the immune response induced by co-delivery of nano-formulations was the most effective, and the mixture of nano-adjuvant and nano-vaccine also had significant adjuvant effects.
  • Influenza virus hemagglutinin stem region is an important target of universal influenza vaccine, in which antibodies induced by H1HA10 trimer expressed by E. coli can neutralize different subtypes of strains.
  • H1HA10 abbreviated as HA
  • ferrtin were fused using the existing gene fusion method to obtain the recombinant protein HA-HFT.
  • Figure 21 the protein editing and shearing technology can efficiently load HA antigen on HFT/PFT vector
  • Figure 21a is the electrophoresis diagram of recombinant gene HA-HFT expression analysis; in the figure, control: pET28a vector blank control bacteria lysis All: bacterial lysate; up: bacterial lysate supernatant
  • HA-HFT direct gene fusion of HA (H1HA10, abbreviated as HA) with HFT to form a recombinant protein.
  • Figure 21b is an electrophoretic analysis diagram of gel filtration purification of HA-HFT prepared based on protein editing and shearing technology.
  • Figure 21c shows the UV curve of HA-HFT purified by gel filtration.
  • Figure 21d is a transmission electron microscope image of HA-HFT (negative staining).
  • Figure 21e is an electrophoretic analysis diagram of gel filtration purification of HA-PFT prepared based on protein editing and shearing technology.
  • Figure 21f shows the UV curve of HA-PFT purified by gel filtration.
  • Figure 21g is a transmission electron microscope image of HA-PFT (negative staining).
  • the supernatant of the bacterial lysate does not contain the recombinant protein HA-HFT, and it can be seen that the recombinant protein forms an inclusion body precipitate.
  • HA-HFT prepared based on protein editing and shearing exists in the form of nanoparticles.
  • HA-PFT prepared based on protein editing and shearing also exists in the form of nanoparticles, which means that PFT can also carry HA .
  • mice in each group were immunized three times, with an interval of 2 weeks between each inoculation, and blood was collected from the orbit for immunoassay after 3 weeks of immunization.
  • the HA group used H1HA10-intein N to immunize mice with a dose of 10 ⁇ g/mouse.
  • the HA-HFT+CpG-HFT group used the nano-vaccine HA-HFT and the nano-adjuvant CpG-HFT to immunize mice together, and the dosages were 10 ⁇ g/mouse and 1.6 ⁇ g/mouse respectively.
  • Nano-vaccine HA-HFT group HA-HFT immunized mice with a dose of 10 ⁇ g/mouse.
  • Nano-vaccine HA-PFT group HA-PFT immunized mice with a dose of 10 ⁇ g/mouse.
  • mice were immunized with the nano-vaccine HA-PFT and the nano-adjuvant CpG-HFT at the doses of 10 ⁇ g/mouse and 1.6 ⁇ g/mouse respectively.
  • Fig. 22a is an ELISA analysis revealing that both nanocarrier and nano adjuvant can enhance HA specific humoral immune response.
  • nano-vaccine HA-HFT can increase HA-specific IgG antibody titer by about 10 times, and the mixed preparation of nano-adjuvant CpG-HFT and nano-vaccine can further increase specific antibody titer (about 10 times).
  • the effect of PFT as a carrier is higher than that of HFT, which may be caused by the low homology of PFT and mouse ferritin, which has a certain adjuvant effect.
  • Human HFT is highly homologous to mouse ferritin (99% identical in amino acid sequence), and HFT protein itself has no adjuvant effect.
  • Figure 22b-d ELISA analysis revealed that vaccine-induced antibodies can specifically recognize H1N1(P), H3N2, and H7H9 strains, and the antibody recognition ability induced by the nano-adjuvant and nano-vaccine mixture is higher than the nano-vaccine itself.
  • the primary antibody is the serum of mice vaccinated with 3 doses of vaccine, and the secondary antibody is a mouse secondary antibody labeled with horseradish peroxidase HRP).
  • Figure 22a shows that the delivery of HA by nanoparticles significantly improves the body's B cell immune response in response to HA, which is 10 times higher than HA-induced antibody titer, and when used in combination with the nano adjuvant CpG-HFT, the specific antibody gradient is increased again by 10 times, further increasing Response level.
  • the antibody titer induced by PFT carrier is higher than that of HFT carrier, suggesting that PFT has low similarity with mouse ferrtin, and PFT carrier may have certain adjuvant function.
  • Figure 22b-d shows that HA-specific related antibodies can recognize influenza strains of different subtypes, including: H1NI (abbreviated P), H3N2 and H7N9 (Anhui strain, abbreviated Ahpri).
  • HA H1HA10-intein N ; HA-HFT: HFT nanoparticles deliver H1HA10; HA-HFT+CpG-HFT: HA-HFT and CpG-HFT combined preparation; HA-PFT: PFT nanoparticles deliver H1HA10; HA-PFT +CpG-HFT: HA-PFT and CpG-HFT combined preparation (Note: Since gb1 specifically binds to all types of IgG, H1HA10-intein N protein is used when detecting HA-specific antibody titer. Use protein editing scissors When cutting, gb1-based can significantly improve the solubility of the protein, so use H1HA10-intein N -gb1).
  • the nanocarrier module platform of the present disclosure can efficiently display antigen proteins, immune enhancers, and polypeptide epitopes on the surface of nanoparticles, and is used for the development of enhanced preventive subunit vaccines and personalized polypeptide epitope tumor vaccines. field.
  • the problem of the influence of loaded protein on the stability of protein self-assembled nanoparticles and the difficulty of co-delivering adjuvant and antigen molecules with nanoparticles is solved.
  • the present disclosure provides an intein-mediated nanocarrier and its application, and aims to provide a comprehensive use of genetic engineering technology and intein-mediated protein editing and shearing technology to perfectly solve the C-terminal metastability of encapsulin
  • the problem is to provide a universal nanocarrier.
  • the technical solution provided by the present disclosure successfully introduces the intein-mediated protein editing technology into the surface modification of the encapsulin nanocarrier through molecular design; the prepared nanoparticle and the N-terminus have a complementary split intein C fragment (split intein C) Cargo molecules are mixed, intein is self-removed, and cargo molecules are covalently coupled to the surface of nanoparticles.
  • the nanoparticle surface modification technology effectively solves the limitation of C-terminal metastability of encapsulin, and can deliver different cargo molecules specifically and efficiently on the surface of encapsulin.
  • the technical solution provided by the present disclosure uses the GFP protein and the C-terminal amino acids 315-411 of the murine cytomegalovirus M44 protein as models, and uses the newly developed nano-delivery technology to deliver the aforementioned cargo protein, and inoculate mice to induce high-titer specific antibodies; Using halo co-cross-linking technology and antigen-antibody high-affinity interaction, GFP and M44-related IgG antibodies were successfully purified, which can be used for western blotting detection and immunofluorescence (IF) analysis.
  • IF immunofluorescence
  • the present disclosure also provides a nanoformulation capable of simultaneously delivering antigen and immune enhancer.
  • the nanoformulation can efficiently deliver antigen and immune enhancer at the same time, and has good specificity.
  • the nano-formulations of the present disclosure can efficiently display antigen proteins, immune enhancers, polypeptide epitopes and antibodies on the surface of nano particles, and are used in the field of enhanced preventive subunit vaccines and personalized polypeptide epitope tumor vaccines and antibody-conjugated drugs. .
  • the present disclosure solves the effect of the loaded protein on the stability of the protein self-assembled nanoparticle, and realizes that the nano preparation can efficiently deliver the antigen and the immune enhancer at the same time, and has good specificity.

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Abstract

Provided are an intein-mediated nano-carrier and an application thereof. The nano-carrier is constructed by the following steps: introducing three tandem proteins, i.e., intN, gb1 and halo, into a C-terminal of an encapsulin protein by using a gene fusion method so as to form a recombinant protein encapsulin-intN-gb1-halo; subjecting 60 recombinant protein monomers to self-assembly so as to obtain nano-particles, exposed intN-gb1-halo thereof being taken as a universal "adapter", and after the adapter is mixed with cargo recombinant proteins gb1-intC and cargo, performing molecular recombination under the assistance of DTT; and subjecting intein to self-excision to form by-products intN-gb1-halo and gb1-intC, and subjecting encapsulin and the cargo to covalent coupling so as to produce a target product encapsulin-cargo. Also provided is a nano preparation capable of simultaneously delivering an antigen and an immunopotentiator, which formed by fusing an N-terminal of ferritin with a C-terminal of a connector gbl-intein to form a recombinant protein gbl-inteinc-ferrtin, performing self-assembly to obtain 24 polymers to form nanoparticles, fusing a C-terminal of an exogenous protein with an N-terminal of the intein to form the recombinant protein, enabling the N-terminal of the intein to specifically recognize and excise the ferritin exposed on the surface of the nanoparticles, and performing covalent cross-linking of the exogenous protein and the ferritin, wherein the exogenous protein is an immunopotentiator, or an antigen, or a combination of the immunopotentiator and the antigen.

Description

一种基于内含肽介导纳米载体及其应用与一种可同时递送抗原和免疫增强剂的纳米制剂An intein-mediated nano carrier and its application and a nano preparation capable of simultaneously delivering antigen and immune enhancer
相关申请的交叉引用Cross references to related applications
本申请要求于2019年5月21日提交中国专利局的申请号为201910421450.0、名称为“一种基于内含肽介导纳米载体及其应用”的中国专利申请以及于2019年5月21日提交中国专利局的申请号为201910421369.2、名称为“一种可同时递送抗原和免疫增强剂的纳米制剂”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application requires a Chinese patent application filed on May 21, 2019 with the application number of 201910421450.0, titled "A kind of intein-mediated nanocarrier and its application" and filed on May 21, 2019 The application number of the Chinese Patent Office is 201910421369.2, the priority of the Chinese patent application titled "A nano preparation capable of delivering antigens and immune enhancers at the same time", the entire content of which is incorporated into this application by reference.
技术领域Technical field
本公开涉及纳米载体和纳米制剂领域，特别是涉及基于内含肽介导的纳米载体及其构建方法和应用以及可同时递送抗原和免疫增强剂的纳米制剂。The present disclosure relates to the field of nanocarriers and nanoformulations, in particular to intein-mediated nanocarriers, construction methods and applications thereof, and nanoformulations capable of simultaneously delivering antigens and immune enhancers.
背景技术Background technique
近年研究发现六十个拷贝的encapsulin蛋白可自装配成直径28nm的球形纳米颗粒，在抗原递送和肿瘤治疗等领域展示出较大的应用潜力。该蛋白的C端是货物分子基因插入的主要位点，用以在纳米载体表面负载货物蛋白。然而该蛋白的C端处于“亚稳定性状态”，其C端基因融合的较小蛋白分子通常因重组蛋白无法正确组装(misassembly)进而限制其应用前景。随着蛋白质组学的飞速发展，越来越多的未知蛋白被发现，迫切需要制备这些蛋白相关抗体以研究其功能。In recent years, studies have found that sixty copies of encapsulin protein can self-assemble into spherical nanoparticles with a diameter of 28nm, showing great application potential in the fields of antigen delivery and tumor therapy. The C-terminus of the protein is the main site for the insertion of the cargo molecule gene, which is used to load the cargo protein on the surface of the nanocarrier. However, the C-terminus of the protein is in a "meta-stable state", and the smaller protein molecules of the C-terminus gene fusion are usually misassembly of the recombinant protein, which limits its application prospects. With the rapid development of proteomics, more and more unknown proteins have been discovered, and there is an urgent need to prepare these protein-related antibodies to study their functions.
纳米递送技术在疫苗设计、药物靶向性运输、活体成像诊断等生物医疗领域的重要性与日俱增。目前，已有研究利用encapsulin蛋白可包裹C端带有信号肽蛋白的性质，将之开发为货物运输载体，然而该应用严重受制于纳米颗粒内部空间容量。Encapsulin纳米载体的另外一个负载技术是将其表面氨基酸定点突变为赖氨酸或半胱氨酸，用以共价偶联负载分子。但是，化学偶联的方法存在效率较低、特异性较差、副反应产物存在安全隐患等缺点，限制其应用的广度。也有研究表明encapsulin的C端基因融合负载货物分子可展示至纳米载体表面，但是亦有研究表明C端融合基因未能展示至纳米颗粒表面，其不稳定性极大地影响该方法的通用性。简而言之，利用encapsulin纳米载体递送货物备受已有递送技术的制约。Nano-delivery technology is becoming increasingly important in biomedical fields such as vaccine design, targeted drug delivery, and live imaging diagnosis. At present, there have been studies using the properties of encapsulin protein to encapsulate the C-terminal protein with signal peptide and developing it as a cargo transport carrier. However, this application is severely restricted by the internal space capacity of nanoparticles. Another loading technology of Encapsulin nanocarriers is the site-directed mutation of amino acids on its surface to lysine or cysteine to covalently couple the load molecule. However, chemical coupling methods have disadvantages such as low efficiency, poor specificity, and potential safety hazards in side reaction products, which limit the breadth of its application. Studies have also shown that the C-terminal gene fusion cargo molecule of encapsulin can be displayed on the surface of nanocarriers, but other studies have shown that the C-terminal fusion gene cannot be displayed on the surface of nanoparticles, and its instability greatly affects the versatility of the method. In short, the use of encapsulin nanocarriers to deliver goods is restricted by existing delivery technologies.
在人类后基因组时代，研究基因编码的蛋白质功能的重要性与日俱增。随着经典的遗传方法(yeast two-hybrid(Y2H)酵母双杂交)和生化相关方法(co-immunoprecipitation(Co-IP)共免疫沉淀，affinity purification(AP)亲和纯及tandem affinity purification-mass spectrometry(TAP-MS)串联亲和纯化-质谱分析)的应用，越来越多的未知蛋白被发现。实验室的研究迫切需求这些新发现蛋白的相关抗体，以探索其功能。In the post-genome era of humans, the importance of studying the functions of proteins encoded by genes is increasing day by day. With the classic genetic method (yeast two-hybrid (Y2H) yeast two-hybrid) and biochemical related methods (co-immunoprecipitation (Co-IP) co-immunoprecipitation, affinity purification (AP) affinity purification and tandem affinity purification-mass spectrometry (TAP-MS) Tandem Affinity Purification-Mass Spectrometry) application, more and more unknown proteins have been discovered. Laboratory research urgently needs antibodies related to these newly discovered proteins to explore their functions.
疫苗是预防和控制传染性疾病最有效、最经济的医疗干预手段。减毒或灭活病原作为传统的经典疫苗取得巨大成功，帮助人类消灭天花和小儿麻痹症。然而，传统的疫苗存在安全隐患且无法有效预防具有高频突变特性的病毒，如HIV、流感病毒等。Vaccines are the most effective and economical means of medical intervention to prevent and control infectious diseases. Attenuated or inactivated pathogens have achieved great success as a traditional classic vaccine, helping humans eliminate smallpox and polio. However, traditional vaccines have potential safety hazards and cannot effectively prevent viruses with high-frequency mutation characteristics, such as HIV and influenza viruses.
目前，亚单位疫苗备受疫苗学家关注，但亚单位疫苗免疫原性差，诱导免疫应答较弱且持续时间短，限制其临床上的应用。At present, subunit vaccines have attracted much attention from vaccinologists, but subunit vaccines have poor immunogenicity, weak inducing immune responses and short duration, which limits their clinical applications.
纳米颗粒可通过基因工程的手段，设计成多功能载体，同时递送抗原和免疫增强剂。然而负载的抗原可能严重影响纳米颗粒组装，导致重组蛋白形成沉淀，需要一系列的复性-再组装才可重新形成纳米颗粒。此外，基因融合的方法难以将抗原分子和免疫增强剂分子同时整合至同一个纳米颗粒上。Nanoparticles can be designed as a multifunctional carrier by means of genetic engineering to deliver antigens and immune enhancers at the same time. However, the loaded antigen may seriously affect the assembly of nanoparticles, leading to the formation of precipitates of recombinant proteins, and a series of renaturation-reassembly is required to re-form nanoparticles. In addition, the method of gene fusion is difficult to integrate antigen molecules and immune enhancer molecules into the same nanoparticle at the same time.
化学偶联可便捷、多态性地修饰纳米颗粒，使其多功能化，实现在纳米颗粒上共递送免疫增强剂与抗原。但是该方法效率较低、特异性差，副反应可能伴有副作用。这些缺点很大程度上限制了纳米制剂的应用。Chemical coupling can conveniently and polymorphically modify the nanoparticle to make it multifunctional, and realize the co-delivery of immune enhancer and antigen on the nanoparticle. However, this method has low efficiency and poor specificity, and side effects may be accompanied by side effects. These shortcomings largely limit the application of nano-formulations.
发明内容Summary of the invention
针对上述问题，本公开的目的包括综合使用基因工程技术和内含肽介导的蛋白编辑剪切技术，完美解决encapsulin的C端亚稳定性问题，提供一种通用的纳米载体，可在encapsulin的表面特异、高效递送不同的货物分子。In view of the above problems, the purpose of this disclosure includes the comprehensive use of genetic engineering technology and intein-mediated protein editing and shearing technology to perfectly solve the C-terminal metastability problem of encapsulin, and provide a universal nanocarrier that can be used in encapsulin Surface-specific and efficient delivery of different cargo molecules.
为此，本公开提供的技术方案包括：To this end, the technical solutions provided by the present disclosure include:
本公开提供了一种基于内含肽介导的纳米载体，所述的纳米载体是通过下述步骤构建的：The present disclosure provides a nanocarrier mediated by intein. The nanocarrier is constructed through the following steps:
1)encapsulin蛋白的C端通过基因融合的方法引入int N、gb1和halo共3个串联蛋白形成重组蛋白encapsulin-int N-gb1-halo，其核酸序列如SEQ ID No.8所示； 1) The C-terminus of the encapsulin protein is introduced by gene fusion method into three tandem proteins int N , gb1 and halo to form a recombinant protein encapsulin-int N -gb1-halo, the nucleic acid sequence of which is shown in SEQ ID No. 8;
2)60个重组蛋白单体自装配成纳米颗粒，其暴露的int N-gb1-halo作为通用“适配器”，与货物重组蛋白gb1-int C和cargo混合后，在DTT的帮助下发生分子重组； 2) 60 recombinant protein monomers self-assemble into nanoparticles. The exposed int N- gb1-halo serves as a universal "adapter". After mixing with the cargo recombinant protein gb1-int C and cargo, molecular recombination occurs with the help of DTT ；
3)内含肽intein自我切除，形成副产物int N-gb1-halo和gb1-int C，而encapsulin与cargo共价偶联，生产目的产物encapsulin-cargo。 3) The intein intein is self-excised, forming by-products int N -gb1-halo and gb1-int C , and encapsulin is covalently coupled with cargo to produce the target product encapsulin-cargo.
可选地，所述encapsulin蛋白的核酸序列如SEQ ID No.1所示，所述int N的核酸序列如SEQ ID No.2所示，所述gb1的核酸序列如SEQ ID No.3所示，所述halo的核酸序列如SEQ ID No.4所示，所述int C的核酸序列如SEQ ID No.16所示。 Optionally, the nucleic acid sequence of the encapsulin protein is shown in SEQ ID No. 1, the nucleic acid sequence of int N is shown in SEQ ID No. 2, and the nucleic acid sequence of gb1 is shown in SEQ ID No. 3. The nucleic acid sequence of the halo is shown in SEQ ID No. 4, and the nucleic acid sequence of the int C is shown in SEQ ID No. 16.
可选地，所述的cargo为GFP或M44或者strep2。Optionally, the cargo is GFP or M44 or strep2.
可选地，所述GFP的核酸序列如SEQ ID No.9所示。Optionally, the nucleic acid sequence of the GFP is shown in SEQ ID No. 9.
可选地，所述M44的核酸序列如SEQ ID No.10所示。Optionally, the nucleic acid sequence of M44 is shown in SEQ ID No. 10.
可选地，所述strep2的核酸序列如SEQ ID No.11所示。Optionally, the nucleic acid sequence of strep2 is shown in SEQ ID No. 11.
可选地，上述的基于内含肽介导的纳米载体，步骤3)为encapsulin-int N-gb1-halo和gb1-int C-GFP按照浓度比例为1:1-2，两者在室温条件下孵育2小时后，用superose 6B increase分子排阻法分离目的产物encapsulin-GFP。 Optionally, the above-mentioned intein-mediated nanocarrier, step 3) is encapsulin-int N -gb1-halo and gb1-int C- GFP according to the concentration ratio of 1:1-2, and both are at room temperature. After incubating for 2 hours, the target product encapsulin-GFP was separated by superose 6B increase molecular exclusion method.
可选地，上述的基于内含肽介导的纳米载体，步骤3)为encapsulin-int N-gb1-halo和gb1-int C-strep2按照浓度比例为1:1-3，两者在室温条件下孵育2小时后，用superose 6B increase分子排阻法分离目的产物encapsulin-strep2。 Optionally, the above-mentioned intein-mediated nanocarrier, step 3) is encapsulin-int N -gb1-halo and gb1-int C -strep2 in a concentration ratio of 1:1-3, and both are at room temperature. After incubating for 2 hours, the target product encapsulin-strep2 was separated by superose 6B increase molecular exclusion method.
可选地，上述的基于内含肽介导的纳米载体，步骤2)为encapsulin-int N-gb1-halo和gb1-int C-M44按照浓度比例为1:1-2，两者在室温条件下孵育2小时后，用superose 6B increase分子排阻法分离目的产物encapsulin-M44。 Optionally, the above-mentioned intein-mediated nanocarrier, step 2) is encapsulin-int N -gb1-halo and gb1-int C -M44 in a concentration ratio of 1:1-2, and both are at room temperature. After incubating for 2 hours, the target product encapsulin-M44 was separated by superose 6B increase molecular exclusion method.
本公开还提供了上述纳米载体作为递送货物蛋白，接种小鼠诱导高滴度特异性抗体的应用。The present disclosure also provides the application of the above-mentioned nanocarrier as the delivery cargo protein and inoculating mice to induce high-titer specific antibodies.
可选地，上述的纳米载体作为递送货物蛋白，接种小鼠诱导高滴度特异性抗体的应用，所述的抗体的纯化方法为：Optionally, the above-mentioned nanocarrier is used as a cargo protein to be used to inoculate mice to induce high-titer specific antibodies. The antibody purification method is:
1)使用gb1-int C-GFP或gb1-intein C-M44与halo-int N-gb1蛋白进行蛋白编辑剪切，生成重组蛋白halo-cargo，以及副产物gb1-int C和int N-gb1； 1) Use gb1-int C -GFP or gb1-intein C -M44 and halo-int N -gb1 protein for protein editing and cutting to generate recombinant protein halo-cargo, and by-products gb1-int C and int N -gb1;
2)反应结束后，混合物与Promega halo TM beads混合，目的产物halo-cargo共价结合至halo TM beads，离心弃上清后，加入免疫后的血清，货物分子相关抗体利用抗原-抗体之间高亲和相互作用与halo TM-halo-cargo结合，其它抗体和蛋白通过离心、洗涤后分离，最后用200mM glycine pH2.8洗脱目的抗体。 2) After the reaction, the mixture is mixed with Promega halo TM beads, and the target product halo-cargo is covalently bound to halo TM beads. After centrifugation to discard the supernatant, the immunized serum is added. Affinity interaction is combined with halo TM -halo-cargo, other antibodies and proteins are separated after centrifugation and washing, and finally the target antibody is eluted with 200mM glycine pH2.8.
可选地，上述纳米载体作为递送货物蛋白，接种小鼠诱导高滴度特异性抗体的应用，所述的halo TM-halo-GFP的制备与纯化方法为：将halo-int N-gb1和gb1-int C-GFP按照浓度比1：1-2在室温条件下孵育4小时后，将250μL反应液与50μL平衡好的Promega halo TM室温摇床混合半个小时后离心，用500μL PBS洗涤beads，除去非特异性结合蛋白；免疫2次后的血清；血清与halo TM-halo-GFP孵育后的流出；先用500μL PBST洗涤halo TM-halo-GFP；再90μL的200mM glycine pH2.8洗脱目的抗体。 Optionally, the above-mentioned nanocarrier is used to deliver cargo protein and inoculate mice to induce high-titer specific antibodies. The method for preparing and purifying halo TM -halo-GFP is: combining halo-int N -gb1 and gb1 -int C -GFP was incubated for 4 hours at room temperature according to a concentration ratio of 1:1-2, 250 μL of the reaction solution was mixed with 50 μL of the balanced Promega halo TM room temperature shaker for half an hour and centrifuged, and the beads were washed with 500 μL of PBS. Removal of non-specific binding proteins; serum after 2 immunizations; effluent after incubation of serum with halo TM -halo-GFP; first wash halo TM -halo-GFP with 500 μL PBST; then 90 μL of 200 mM glycine pH 2.8 to elute the target antibody .
可选地，上述纳米载体作为货物蛋白，接种小鼠诱导高滴度特异性抗体的应用，所述的halo TM-halo-M44的制备及M44相关抗体的纯化方法为：将halo-int N-gb1和gb1-int C-M44按照浓度比1：1-2在室温条件下孵育2-4小时后，250μL反应液与50μL平衡好的Promega halo TM室温摇床混合半个小时，而后离心，用500μL PBS洗涤beads，除去非特异性结合蛋白，反应液与Promega halo TM混合后离心上清；PBS洗涤液洗涤3次，使得目的蛋白halo-GFP特异性结合至Promega halo TM beads；免疫2次后的血清；血清与halo TM-halo-M44孵育后的流出；先用500μL PBST洗涤halo TM-halo-M44；再90μL的200mM glycine pH2.8洗脱目的抗体。 Optionally, the above-mentioned nanocarrier is used as a cargo protein to inoculate mice to induce high-titer specific antibodies. The preparation method of halo TM -halo-M44 and the purification method of M44 related antibodies are: halo-int N- After incubating gb1 and gb1-int C- M44 at room temperature for 2-4 hours at a concentration ratio of 1:1-2, 250 μL of the reaction solution was mixed with 50 μL of the balanced Promega halo TM room temperature shaker for half an hour, and then centrifuged and used Wash the beads with 500 μL PBS to remove non-specific binding proteins. Mix the reaction solution with Promega halo TM and centrifuge the supernatant; wash 3 times with PBS washing solution to make the target protein halo-GFP specifically bind to Promega halo TM beads; Serum; the flow of serum after incubation with halo TM -halo-M44; first wash halo TM -halo-M44 with 500 μL PBST; then 90 μL of 200 mM glycine pH 2.8 to elute the target antibody.
可选地，上述的纳米载体作为递送货物蛋白，接种小鼠诱导高滴度特异性抗体的应用，诱导后的抗 体做为WB和/或IF分析。Optionally, the above-mentioned nanocarrier is used to deliver cargo protein, inoculate mice to induce high-titer specific antibodies, and the induced antibodies are used for WB and/or IF analysis.
本公开还提供了一种可同时递送抗原和免疫增强剂的纳米制剂，所述的纳米制剂是通过下述步骤构建的：1)由铁蛋白的N端与接头gbl-intein的C端融合形成重组蛋白gbl-intein c-ferrtin后自装配成二十四聚体构成纳米颗粒； The present disclosure also provides a nano-formulation capable of simultaneously delivering antigen and immune enhancer. The nano-formulation is constructed by the following steps: 1) The N-terminal of ferritin is fused with the C-terminal of the linker gbl-intein. The recombinant protein gbl-intein c- ferrtin self-assembles into twenty-four polymers to form nanoparticles;
2)外源蛋白的C端融合内含肽的N端形成重组蛋白，内含肽的N端特异性识别并切除暴露在所述纳米颗粒表面的铁蛋白；2) The C-terminus of the foreign protein is fused with the N-terminus of the intein to form a recombinant protein, and the N-terminus of the intein specifically recognizes and excises the ferritin exposed on the surface of the nanoparticle;
3)外源蛋白与铁蛋白共价交联获得所述纳米制剂；3) Covalent cross-linking of exogenous protein and ferritin to obtain the nanoformulation;
其中，所述的外源蛋白为免疫增强剂或抗原或二者组合。Wherein, the foreign protein is an immune enhancer or an antigen or a combination of the two.
可选地，所述gbl的核酸序列如SEQ ID No.22所示，所述intein c的核酸序列如SEQ ID No.19所示。 Optionally, the nucleic acid sequence of the gbl is shown in SEQ ID No. 22, and the nucleic acid sequence of the intein c is shown in SEQ ID No. 19.
可选地，所述的免疫增强剂为鞭毛蛋白或抗生物素蛋白单体类似物rhizavidin。Optionally, the immune enhancer is flagellin or rhizavidin, a monomer analog of avidin.
可选地，所述的抗原为单一多肽表位的蛋白抗原或不同多肽表位的蛋白抗原。Optionally, the antigen is a protein antigen of a single polypeptide epitope or a protein antigen of different polypeptide epitopes.
可选地，所述的外源蛋白为免疫增强剂与抗原二者组合时，其摩尔比为1～3:5。Optionally, when the exogenous protein is a combination of an immune enhancer and an antigen, the molar ratio is 1 to 3:5.
可选地，所述的铁蛋白为人源的重链铁蛋白或强烈火球菌的重链铁蛋白或其他物种来源的铁蛋白。Optionally, the ferritin is human-derived heavy-chain ferritin or Pyrococcus furiosus or ferritin derived from other species.
与现有技术相比，本公开提供的技术方案具有如下技术优点：Compared with the prior art, the technical solution provided by the present disclosure has the following technical advantages:
本公开提供的技术方案通过分子设计，成功将内含肽介导蛋白编辑技术引入encapsulin纳米载体的表面修饰；制备的纳米颗粒与N端带有互补的断裂内含肽C片段(split intein C，gp41-1-int C，简称int C，核酸序列如SEQ ID No.16所示))货物分子混合，intein自我切除，货物分子共价偶联至纳米颗粒表面，改纳米颗粒表面修饰技术有效解决encapsulin的C端亚稳定性的限制，可在encapsulin的表面特异、高效递送不同的货物分子。本公开提供的技术方案以GFP蛋白和鼠巨细胞病毒(mouse cytomegalovirus，MCMV)M44蛋白的C端氨基酸315-411(简称M44蛋白，该蛋白是MCMV复制中心标志物即marker)为模型，利用新开发的纳米递送技术递送上述货物蛋白，接种小鼠诱导高滴度的特异性抗体。而后利用halo共交联技术和抗原-抗体高亲和相互作用，成功纯化出GFP和M44相关IgG抗体，可用于western blotting检测和免疫荧光(immunofluorescence，IF)分析。本公开的纳米制剂可在纳米颗粒表面高效展示抗原蛋白、免疫增强剂、多肽表位和抗体，用于增强型的预防性亚单位疫苗和个性化多肽表位肿瘤疫苗研发及抗体偶联药物领域。本公开解决了负载蛋白对蛋白自装配纳米颗粒稳定性影响，实现了纳米制剂高效地同时递送抗原和免疫增强剂，同时且具有良好特异性。 The technical solution provided by the present disclosure successfully introduces the intein-mediated protein editing technology into the surface modification of the encapsulin nanocarrier through molecular design; the prepared nanoparticle has a complementary split intein C fragment (split intein C, gp41-1-int C , abbreviated as int C , the nucleic acid sequence is shown in SEQ ID No.16)) Cargo molecules are mixed, intein is excised by itself, and the cargo molecules are covalently coupled to the surface of the nanoparticle. The nanoparticle surface modification technology can effectively solve the problem. The C-terminal metastability of encapsulin is limited by the specific and efficient delivery of different cargo molecules on the surface of encapsulin. The technical solution provided by the present disclosure takes the GFP protein and the C-terminal amino acids 315-411 of the mouse cytomegalovirus (mouse cytomegalovirus, MCMV) M44 protein (referred to as the M44 protein, which is the marker of the MCMV replication center, or marker) as models, and uses the new The developed nano-delivery technology delivers the aforementioned cargo proteins, and inoculates mice to induce high-titer specific antibodies. Then, using halo co-crosslinking technology and antigen-antibody high-affinity interaction, GFP and M44-related IgG antibodies were successfully purified, which can be used for western blotting detection and immunofluorescence (immunofluorescence, IF) analysis. The nano-formulations of the present disclosure can efficiently display antigen proteins, immune enhancers, polypeptide epitopes and antibodies on the surface of nano particles, and are used in the field of enhanced preventive subunit vaccines and personalized polypeptide epitope tumor vaccines and antibody-conjugated drugs. . The present disclosure solves the effect of the loaded protein on the stability of the protein self-assembled nanoparticle, and realizes that the nano preparation can efficiently deliver the antigen and the immune enhancer at the same time, and has good specificity.
附图说明Description of the drawings
为了更清楚地说明本公开实施方式的技术方案，下面将对实施方式中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施方式，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。In order to explain the technical solutions of the embodiments of the present disclosure more clearly, the following will briefly introduce the drawings that need to be used in the embodiments. It should be understood that the following drawings only show certain embodiments of the present disclosure, and therefore do not It should be regarded as a limitation of the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative work.
图1是基于内含肽intein介导蛋白编辑剪切的方法修饰encapsulin纳米载体原理示意图；Figure 1 is a schematic diagram of the modified encapsulin nanocarrier based on the method of intein-mediated protein editing and shearing;
图2是encapsulin的分子改造及重组纳米颗粒的纯化与分析图；Figure 2 is the molecular modification of encapsulin and the purification and analysis of recombinant nanoparticles;
图3是基于内含肽介导蛋白编辑剪切技术在encapsulin表面高效负载GFP蛋白。Figure 3 is based on the intein-mediated protein editing and shearing technology to efficiently load GFP protein on the surface of encapsulin.
图4是基于内含肽介导蛋白编辑剪切技术在encapsulin表面高效负载2xstrep2多肽。Figure 4 is based on intein-mediated protein editing and shearing technology to efficiently load 2xstrep2 polypeptide on the surface of encapsulin.
图5是基于内含肽介导蛋白编辑剪切技术在encapsulin表面高效负载MCMV的M44蛋白C端氨基酸315-411。Figure 5 is based on the intein-mediated protein editing and shearing technology to efficiently load the C-terminal amino acids 315-411 of MCMV on the surface of encapsulin.
图6是酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)滴定货物分子相关IgG抗体滴度。Figure 6 is an enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) titration of cargo molecule-related IgG antibody titers.
图7是基于halo共价交联技术和抗原-抗体相互作用纯化货物分子相关抗体原理示意图。Figure 7 is a schematic diagram of the principle of purifying cargo molecule-related antibodies based on halo covalent cross-linking technology and antigen-antibody interaction.
图8是halo TM-halo-GFP的制备及GFP相关抗体的纯化。 Figure 8 shows the preparation of halo TM -halo-GFP and the purification of GFP-related antibodies.
图9是halo TM-halo-M44的制备及M44相关抗体的纯化。 Figure 9 shows the preparation of halo TM -halo-M44 and the purification of M44 related antibodies.
图10是用western blotting(WB)分析新纯化的GFP和M44相关抗体的功能。Figure 10 is the analysis of the function of newly purified GFP and M44-related antibodies by western blotting (WB).
图11是用immunofluorescence(IF)分析新纯化的GFP抗体功能检测。Fig. 11 is the function test of newly purified GFP antibody by immunofluorescence (IF) analysis.
图12是用IF分析新纯化的M44抗体功能检测。Figure 12 is a functional test of the newly purified M44 antibody by IF analysis.
图13为纳米载体模块构建原理的示意图。Figure 13 is a schematic diagram of the construction principle of the nanocarrier module.
图14为引入接头gbl-intein C不影响纳米颗粒稳定性的分析图； Figure 14 is an analysis diagram showing that the introduction of the linker gbl-intein C does not affect the stability of the nanoparticles;
图14a为三种重组蛋白的电泳图；图14b为gb1-intein C-HFT和gb1-intein C-PFT经硫酸铵沉淀后的电泳图；图14c为gb1-intein C-HFT纯化后的电泳图；图14d为凝胶过滤纯化gb1-intein C-HFT凝胶的UV图；图14e为TEM检测纯化的gb1-intein C-HFT的电镜图；图14f为TEM检测纯化的gb1-intein C-PFT的电镜图。 Figure 14a is the electrophoresis of the three recombinant proteins; Figure 14b is the electrophoresis of gb1-intein C- HFT and gb1-intein C- PFT after precipitation with ammonium sulfate; Figure 14c is the electrophoresis of the purified gb1-intein C- HFT ; Figure 14d is purified by gel filtration gb1-intein C -HFT UV gel; Figure 14e electron micrograph of purified gb1-intein C -HFT detecting a TEM; FIG. 14f is a TEM examination of purified gb1-intein C -PFT SEM picture.
图15为GFP蛋白和strep2高效共价偶联至HFT后的分析图；Figure 15 is an analysis diagram of GFP protein and strep2 after being efficiently covalently coupled to HFT;
图15a为考马斯亮蓝法(CB assay)检测目的产物GFP-HFT含量的电泳图；图15b为使用Western-blotting检测(WB assay)GFP与HFT偶联效率的电泳图；图15c为考马斯亮兰法检测目的产物strep2-HFT含量的电泳图；图15d为使用Western-blotting分析strep2多肽与HFT偶联效率的电泳图。Figure 15a is the electrophoresis diagram of the GFP-HFT content of the target product detected by the Coomassie brilliant blue method (CB assay); Figure 15b is the electrophoresis diagram of the coupling efficiency of GFP and HFT using Western-blotting detection (WB assay); Figure 15c is the Coomassie brilliant blue The electrophoresis diagram of the content of strep2-HFT of the target product by the method; Figure 15d is the electrophoresis diagram of the coupling efficiency of strep2 polypeptide and HFT using Western-blotting.
图16为通过自剪切的方式负载GFP蛋白和strep2多肽不影响纳米颗粒结构稳定性的分析图；Figure 16 is an analysis diagram showing that loading GFP protein and strep2 polypeptide by means of self-shearing does not affect the stability of the nanoparticle structure;
图16a为凝胶过滤纯化GFP-HFT的考马斯亮蓝染色法电泳分析图；图16b为凝胶过滤纯化GFP-HFT的UV曲线图；图16c为TEM检测纯化的GFP-HFT的电镜图；图16d为凝胶过滤纯化strep2-HFT的电泳分析图；图16e为凝胶过滤纯化strep2-HFT的UV曲线图；图16f为TEM检测纯化的strep2-HFT的电镜图。Figure 16a is a Coomassie brilliant blue electrophoresis analysis diagram of gel filtration purified GFP-HFT; Figure 16b is a UV curve diagram of gel filtration purified GFP-HFT; Figure 16c is an electron microscope image of purified GFP-HFT detected by TEM; 16d is an electrophoretic analysis chart of gel filtration purified strep2-HFT; FIG. 16e is a UV curve chart of gel filtration purification of strep2-HFT; FIG. 16f is an electron micrograph of TEM detection and purification of strep2-HFT.
图17为本公开提供的纳米颗粒与外源蛋白经内含肽介导蛋白编辑技术剪接制备不同纳米器件示意图。FIG. 17 is a schematic diagram of preparing different nanodevices by splicing nanoparticles and exogenous proteins provided by the present disclosure through intein-mediated protein editing technology.
图18为纳米颗粒可高效负载多肽表位和鞭毛蛋白的分析图；Figure 18 is an analysis diagram showing that nanoparticles can efficiently load polypeptide epitopes and flagellin;
图18a为纳米疫苗SP70-HFT的凝胶过滤纯化电泳分析图；图18b为凝胶过滤纯化纳米疫苗SP70-HFT的UV曲线图；图18c为纳米疫苗SP70-HFT的透射电镜图；图18d为SP70与CBLB共递送纳米制剂SP70-CBLB-HFT的凝胶过滤纯化电泳分析图；图18e为凝胶过滤纯化共递送纳米制剂SP70-CBLB-HFT的UV曲线图；图18f为共递送纳米制剂SP70-CBLB-HFT的透射电镜图；图18g为纳米佐剂CBLB-HFT的凝胶过滤纯化电泳分析图；图18h为凝胶过滤纯化纳米佐剂CBLB-HFT的UV曲线图；图18i为纳米佐剂CBLB-HFT的透射电镜图。Figure 18a is the gel filtration purification electrophoresis analysis diagram of the nano vaccine SP70-HFT; Figure 18b is the UV curve diagram of the gel filtration purification nano vaccine SP70-HFT; Figure 18c is the transmission electron microscope image of the nano vaccine SP70-HFT; Figure 18d is SP70 and CBLB co-delivery nano-preparation SP70-CBLB-HFT gel filtration purification electrophoresis analysis diagram; Figure 18e is the gel filtration purification co-delivery nano-preparation SP70-CBLB-HFT UV curve diagram; Figure 18f is the co-delivery nano-preparation SP70 -CBLB-HFT transmission electron microscope image; Figure 18g is the gel filtration purification electrophoresis analysis of the nano adjuvant CBLB-HFT; Figure 18h is the UV curve of the gel filtration purification nano adjuvant CBLB-HFT; Figure 18i is the nano adjuvant Transmission electron micrograph of agent CBLB-HFT.
图19为模块化纳米载体是否可高效负载CpG的分析图；Figure 19 is an analysis diagram of whether modular nanocarriers can efficiently load CpG;
图19a为rhizavidin-HFT的凝胶过滤纯化电泳分析图；图19b为rhizavidin-HFT凝胶过滤纯化的UV曲线图；图19c为rhizavidin-HFT的透射电镜图；图19d为SP70-rhizavidin-HFT的凝胶过滤纯化电泳分析图；图19e为SP70-rhizavidin-HFT凝胶过滤纯化的UV曲线图；图19f为SP70-rhizavidin-HFT的透射电镜图；图19g为SP70-rhizavidin-HFT的二维类平均图；图19h为目的蛋白rhizavidin-HFT结合生物素化CpG电泳分析图。Figure 19a shows the electrophoresis analysis of rhizavidin-HFT gel filtration purification; Figure 19b shows the UV curve of rhizavidin-HFT gel filtration purification; Figure 19c shows the transmission electron microscope image of rhizavidin-HFT; Figure 19d shows the SP70-rhizavidin-HFT Gel filtration purification electrophoresis analysis chart; Figure 19e is the UV curve of SP70-rhizavidin-HFT gel filtration purification; Figure 19f is the transmission electron microscope image of SP70-rhizavidin-HFT; Figure 19g is the two-dimensional type of SP70-rhizavidin-HFT Average image; Figure 19h is the electrophoresis analysis image of target protein rhizavidin-HFT combined with biotinylated CpG.
图20为以SP70表位为模型分析不同疫苗剂型对免疫效果的影响分析图；Figure 20 is an analysis diagram of the influence of different vaccine formulations on the immune effect using the SP70 epitope as a model;
图20a-c为ELISA分析不同方式递送CpG佐剂以及CBLB诱导IgG滴度；图20d体外细胞滴定分析不同疫苗制剂诱导的抗体中和效价；图20e为共递送纳米制剂诱导的免疫应答。Figures 20a-c are ELISA analysis of different delivery of CpG adjuvants and CBLB-induced IgG titers; Figure 20d in vitro cell titration analysis of antibody neutralization titers induced by different vaccine formulations; Figure 20e is the immune response induced by co-delivery of nano formulations.
图21为蛋白编辑剪切技术可在HFT/PFT载体上高效负载HA抗原分析图；Figure 21 is an analysis diagram of protein editing and cutting technology that can efficiently load HA antigen on HFT/PFT carriers;
图21a为重组基因HA-HFT表达分析电泳图；图21b为凝胶过滤纯化基于蛋白编辑剪切技术制备的HA-HFT的电泳分析图；图21c为凝胶过滤纯化的HA-HFT的UV曲线图；图21d为HA-HFT的透射电镜图；图21e为凝胶过滤纯化基于蛋白编辑剪切技术制备的HA-PFT的电泳分析图；图21f为凝胶过滤纯化的HA-PFT的UV曲线图；图21g为HA-PFT的透射电镜图。Figure 21a is the electrophoresis diagram of recombinant gene HA-HFT expression analysis; Figure 21b is the electrophoretic analysis diagram of gel filtration purification of HA-HFT prepared based on protein editing shearing technology; Figure 21c is the UV curve of gel filtration purification of HA-HFT Figure; Figure 21d is a transmission electron microscope image of HA-HFT; Figure 21e is an electrophoresis analysis image of HA-PFT prepared based on protein editing and shearing technology purified by gel filtration; Figure 21f is a UV curve of HA-PFT purified by gel filtration Figure; Figure 21g is a transmission electron microscope image of HA-PFT.
图22为纳米佐剂CpG-HFT增强HA特异性体液免疫应答并调控IgG类型的分析图。Figure 22 is an analysis diagram of nano adjuvant CpG-HFT enhancing HA specific humoral immune response and regulating IgG type.
图22a为ELISA分析揭示纳米载体和纳米佐剂均可增强HA特异性体液免疫应答；图22b-d为ELISA分析揭示疫苗诱导抗体可特异性识别H1N1(P)、H3N2和H7H9毒株，且纳米佐剂与纳米疫苗混合制剂诱导的抗体识别能力高于纳米疫苗本身。Figure 22a shows that ELISA analysis reveals that both nanocarriers and nanoadjuvants can enhance HA specific humoral immune response; Figure 22b-d shows that ELISA analysis reveals that vaccine-induced antibodies can specifically recognize H1N1(P), H3N2, and H7H9 strains, and nano The antibody recognition ability induced by the adjuvant and nano vaccine mixed preparation is higher than that of the nano vaccine itself.
具体实施方式Detailed ways
下面结合实验及实施例，对本公开的权利要求做进一步的详细说明，但不构成对本公开的任何限制，任何在本公开权利要求保护范围内所做的有限次修改，仍在本公开的权利要求保护范围内。实施方式中 未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。The following combines experiments and examples to further describe the claims of the present disclosure in detail, but it does not constitute any limitation to the present disclosure. Any limited modification made within the protection scope of the claims of the present disclosure is still in the claims of the present disclosure. Within the scope of protection. If specific conditions are not specified in the implementation mode, proceed in accordance with conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.
除非本文另有定义，否则结合本公开使用的科学和技术术语应具有本领域普通技术人员通常理解的含义。以下描述示例性方法和材料，但是与本文描述的那些类似或等同的方法和材料也可以用于本公开的实践或测试中。Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure.
本公开提供了一种基于内含肽介导的纳米载体，所述的纳米载体是通过下述步骤构建的：The present disclosure provides a nanocarrier mediated by intein. The nanocarrier is constructed through the following steps:
1)encapsulin蛋白的C端通过基因融合的方法引入int N、gb1和halo共3个串联蛋白形成重组蛋白encapsulin-int N-gb1-halo，其核酸序列如SEQ ID No.8所示； 1) The C-terminus of the encapsulin protein is introduced by gene fusion method into three tandem proteins int N , gb1 and halo to form a recombinant protein encapsulin-int N -gb1-halo, the nucleic acid sequence of which is shown in SEQ ID No. 8;
2)60个重组蛋白单体自装配成纳米颗粒，其暴露的int N-gb1-halo作为通用“适配器”，与货物重组蛋白gb1-int C和cargo混合后，在DTT的帮助下发生分子重组； 2) 60 recombinant protein monomers self-assemble into nanoparticles. The exposed int N- gb1-halo serves as a universal "adapter". After mixing with the cargo recombinant protein gb1-int C and cargo, molecular recombination occurs with the help of DTT ；
3)内含肽intein自我切除，形成副产物int N-gb1-halo和gb1-int C，而encapsulin与cargo共价偶联，生产目的产物encapsulin-cargo。 3) The intein intein is self-excised, forming by-products int N -gb1-halo and gb1-int C , and encapsulin is covalently coupled with cargo to produce the target product encapsulin-cargo.
在一种或多种实施方式中，所述encapsulin蛋白的核酸序列如SEQ ID No.1所示，所述int N的核酸序列如SEQ ID No.2所示，所述gb1的核酸序列如SEQ ID No.3所示，所述halo的核酸序列如SEQ ID No.4所示，所述int C的核酸序列如SEQ ID No.16所示。 In one or more embodiments, the nucleic acid sequence of the encapsulin protein is shown in SEQ ID No. 1, the nucleic acid sequence of int N is shown in SEQ ID No. 2, and the nucleic acid sequence of gb1 is shown in SEQ ID No. 2. ID No. 3, the nucleic acid sequence of halo is shown in SEQ ID No. 4, and the nucleic acid sequence of int C is shown in SEQ ID No. 16.
在一种或多种实施方式中，所述的cargo为GFP或M44或者strep2。In one or more embodiments, the cargo is GFP or M44 or strep2.
在一种或多种实施方式中，所述GFP的核酸序列如SEQ ID No.9所示。In one or more embodiments, the nucleic acid sequence of the GFP is shown in SEQ ID No. 9.
在一种或多种实施方式中，所述M44的核酸序列如SEQ ID No.10所示。In one or more embodiments, the nucleic acid sequence of M44 is shown in SEQ ID No. 10.
在一种或多种实施方式中，所述strep2的核酸序列如SEQ ID No.11所示。In one or more embodiments, the nucleic acid sequence of strep2 is shown in SEQ ID No. 11.
在一种或多种实施方式中，上述的基于内含肽介导的纳米载体，步骤3)为encapsulin-int N-gb1-halo和gb1-int C-GFP按照浓度比例为1:1-2，两者在室温条件下孵育2小时后，用superose 6B increase分子排阻法分离目的产物encapsulin-GFP。 In one or more embodiments, the above-mentioned intein-mediated nanocarrier, step 3) is encapsulin-int N -gb1-halo and gb1-int C -GFP in a concentration ratio of 1:1-2 After the two were incubated for 2 hours at room temperature, the target product encapsulin-GFP was separated by superose 6B increase molecular exclusion method.
在一种或多种实施方式中，上述的基于内含肽介导的纳米载体，步骤3)为encapsulin-int N-gb1-halo和gb1-int C-strep2按照浓度比例为1:1-3，两者在室温条件下孵育2小时后，用superose 6B increase分子排阻法分离目的产物encapsulin-strep2。 In one or more embodiments, the above-mentioned intein-mediated nanocarrier, step 3) is encapsulin-int N -gb1-halo and gb1-int C -strep2 in a concentration ratio of 1:1-3 After the two were incubated for 2 hours at room temperature, the target product encapsulin-strep2 was separated by superose 6B increase molecular exclusion method.
在一种或多种实施方式中，上述的基于内含肽介导的纳米载体，步骤2)为encapsulin-int N-gb1-halo和gb1-int C-M44按照浓度比例为1:1-2，两者在室温条件下孵育2小时后，用superose 6B increase分子排阻法分离目的产物encapsulin-M44。 In one or more embodiments, the above-mentioned intein-mediated nanocarrier, step 2) is encapsulin-int N -gb1-halo and gb1-int C -M44 in a concentration ratio of 1:1-2 After the two were incubated for 2 hours at room temperature, the target product encapsulin-M44 was separated by superose 6B increase molecular exclusion method.
本公开还提供了如本文所述的纳米载体作为递送货物蛋白，接种小鼠诱导高滴度特异性抗体的应用。The present disclosure also provides the application of the nanocarrier as described herein as a delivery cargo protein, and inoculating mice to induce high-titer specific antibodies.
在一种或多种实施方式中，所述的抗体的纯化方法为：In one or more embodiments, the antibody purification method is:
1)使用gb1-int C-GFP或gb1-intein C-M44与halo-int N-gb1蛋白进行蛋白编辑剪切，生成重组蛋白halo-cargo，以及副产物gb1-int C和int N-gb1； 1) Use gb1-int C -GFP or gb1-intein C -M44 and halo-int N -gb1 protein for protein editing and cutting to generate recombinant protein halo-cargo, and by-products gb1-int C and int N -gb1;
2)反应结束后，混合物与Promega halo TM beads混合，目的产物halo-cargo共价结合至halo TM beads，离心弃上清后，加入免疫后的血清，货物分子相关抗体利用抗原-抗体之间高亲和相互作用与halo TM-halo-cargo结合，其它抗体和蛋白通过离心、洗涤后分离，最后用200mM glycine pH2.8洗脱目的抗体。 2) After the reaction, the mixture is mixed with Promega halo TM beads, and the target product halo-cargo is covalently bound to halo TM beads. After centrifugation to discard the supernatant, the immunized serum is added. Affinity interaction is combined with halo TM -halo-cargo, other antibodies and proteins are separated after centrifugation and washing, and finally the target antibody is eluted with 200mM glycine pH2.8.
在一种或多种实施方式中，所述的halo TM-halo-GFP的制备与纯化方法为：将halo-int N-gb1和gb1-int C-GFP按照浓度比1：1-2在室温条件下孵育4小时后，将250μL反应液与50μL平衡好的Promega halo TM室温摇床混合半个小时后离心，用500μL PBS洗涤beads，除去非特异性结合蛋白；免疫2次后的血清；血清与halo TM-halo-GFP孵育后的流出；先用500μL PBST洗涤halo TM-halo-GFP；再90μL的200mM glycine pH2.8洗脱目的抗体。 In one or more embodiments, the preparation and purification method of halo TM -halo-GFP is as follows: halo-int N -gb1 and gb1-int C -GFP are placed at room temperature at a concentration ratio of 1:1-2 After incubating for 4 hours under the conditions, mix 250 μL of the reaction solution with 50 μL of the balanced Promega halo TM room temperature shaker for half an hour, then centrifuge, wash the beads with 500 μL of PBS to remove non-specific binding proteins; the serum after the second immunization; the serum and The flow out of halo TM -halo-GFP after incubation; first wash halo TM -halo-GFP with 500 μL PBST; then eluate the target antibody with 90 μL of 200 mM glycine pH2.8.
在一种或多种实施方式中，所述的halo TM-halo-M44的制备及M44相关抗体的纯化方法为：将halo-int N-gb1和gb1-int C-M44按照浓度比1：1-2在室温条件下孵育2-4小时后，250μL反应液与50μL平衡好的Promega halo TM室温摇床混合半个小时，而后离心，用500μL PBS洗涤beads，除去非特异性结合蛋白，反应液与Promega halo TM混合后离心上清；PBS洗涤液洗涤3次，使得目的蛋白halo-GFP特异性结合至Promega halo TM beads；免疫2次后的血清；血清与halo TM-halo-M44孵育后的流出；先用 500μL PBST洗涤halo TM-halo-M44；再90μL的200mM glycine pH2.8洗脱目的抗体。 In one or more embodiments, the method for preparing halo TM -halo-M44 and purifying M44 related antibodies is: halo-int N -gb1 and gb1-int C -M44 are in a concentration ratio of 1:1 -2 After incubating for 2-4 hours at room temperature, 250 μL of the reaction solution was mixed with 50 μL of the balanced Promega halo TM room temperature shaker for half an hour, and then centrifuged, and the beads were washed with 500 μL of PBS to remove non-specific binding proteins. Centrifuge the supernatant after mixing Promega halo TM ; wash 3 times with PBS washing solution to make the target protein halo-GFP specifically bind to Promega halo TM beads; serum after 2 immunizations; flow out after the serum is incubated with halo TM -halo-M44 ; First wash halo TM -halo-M44 with 500 μL PBST; then eluate the target antibody with 90 μL 200mM glycine pH2.8.
在一种或多种实施方式中，诱导后的抗体做为WB和/或IF分析。In one or more embodiments, the induced antibody is used for WB and/or IF analysis.
本公开提供了一种可同时递送抗原和免疫增强剂的纳米制剂，所述的纳米制剂是通过下述步骤构建的：1)由铁蛋白的N端与接头gbl-intein的C端融合形成重组蛋白gbl-intein c-ferrtin后自装配成24聚体二十四聚体构成纳米颗粒； The present disclosure provides a nanoformulation capable of simultaneously delivering antigens and immune enhancers. The nanoformulation is constructed by the following steps: 1) The N-terminal of ferritin and the C-terminal of the linker gbl-intein are fused to form a recombinant The protein gbl-intein c- ferrtin self-assembles into 24-mer twenty-four mers to form nanoparticles;
2)外源蛋白的C端融合内含肽的N端形成重组蛋白，内含肽的N端特异性识别并切除暴露在所述纳米颗粒表面的铁蛋白；2) The C-terminus of the foreign protein is fused with the N-terminus of the intein to form a recombinant protein, and the N-terminus of the intein specifically recognizes and excises the ferritin exposed on the surface of the nanoparticle;
3)外源蛋白与铁蛋白共价交联获得所述纳米制剂而得；3) Covalently cross-linked foreign protein and ferritin to obtain the nano-formulation;
其中，所述的外源蛋白为免疫增强剂或抗原或二者组合。在一种或多种实施方式中，所述gbl的核酸序列如SEQ ID No.22所示，所述intein c的核酸序列如SEQ ID No.19所示。 Wherein, the foreign protein is an immune enhancer or an antigen or a combination of the two. In one or more embodiments, the nucleic acid sequence of the gbl is shown in SEQ ID No. 22, and the nucleic acid sequence of the intein c is shown in SEQ ID No. 19.
在一种或多种实施方式中，所述的免疫增强剂为鞭毛蛋白或抗生物素蛋白单体类似物rhizavidin。In one or more embodiments, the immune enhancer is flagellin or rhizavidin, a monomer analog of avidin.
在一种或多种实施方式中，所述的抗原为单一多肽表位的蛋白抗原或不同多肽表位的蛋白抗原。In one or more embodiments, the antigen is a protein antigen of a single polypeptide epitope or a protein antigen of different polypeptide epitopes.
在一种或多种实施方式中，所述的外源蛋白为免疫增强剂与抗原二者组合时，其摩尔比为1～3:5。In one or more embodiments, when the exogenous protein is a combination of an immune enhancer and an antigen, the molar ratio is 1 to 3:5.
在一种或多种实施方式中，所述的铁蛋白为人源的重链铁蛋白或强烈火球菌的重链铁蛋白或其他物种来源的铁蛋白。In one or more embodiments, the ferritin is human-derived heavy-chain ferritin or Pyrococcus furiosus or other species-derived ferritin.
实施例1Example 1
基于内含肽intein介导蛋白编辑剪切的方法修饰encapsulin纳米载体的构建方法，其原理示意图参阅图1：Based on the method of intein-mediated protein editing and shearing, the construction method of modified encapsulin nanocarrier is modified. The schematic diagram of the construction method is shown in Figure 1:
1)encapsulin蛋白(核酸序列如SEQ ID No.1所示)的C端通过基因融合的方法引入int N(gp41-1-intein N，简写为int N；核酸序列如SEQ ID No.2所示)、gb1(核酸序列如SEQ ID No.3所示)和halo(核酸序列如SEQ ID No.4所示)共3个串联蛋白形成重组蛋白(A-B)； 1) The C-terminus of the encapsulin protein (nucleic acid sequence is shown in SEQ ID No. 1) is introduced into int N (gp41-1-intein N , abbreviated as int N ) by gene fusion; the nucleic acid sequence is shown in SEQ ID No. 2 ), gb1 (nucleic acid sequence shown in SEQ ID No. 3) and halo (nucleic acid sequence shown in SEQ ID No. 4), a total of 3 tandem proteins form a recombinant protein (AB);
在本申请中的研究中，设计了4个不同的encapsulin重组蛋白，其编号为1、2、3和4，分别对应：encapsulin-int N(核酸序列如SEQ ID No.5所示)、encapsulin-int N-gb1(核酸序列如SEQ ID No.6所示)、halo(核酸序列如SEQ ID No.4所示)、encapsulin-int N-halo(核酸序列如SEQ ID No.7所示)和encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)；参阅图2，encapsulin(核酸序列如SEQ ID No.1所示)与int N(核酸序列如SEQ ID No.2所示)融合表达生成包涵体沉淀(图2a)，引入辅助溶解蛋白gb1(核酸序列如SEQ ID No.3所示)或halo(核酸序列如SEQ ID No.4所示)可使部分重组蛋白可溶。两者联合使用则显著改善重组蛋白可溶性(图2a-c)。重组蛋白4的的上清加入0.15g/mL的硫酸铵沉淀用含10％甘油的PBS重悬溶解后，可用分子排阻法进一步分离纯化(图2d-e)。用透射电镜观察目的样品，我们发现重组蛋白4自装配为球形纳米颗粒(图2e)。a：本公开设计的4个不同的encapsulin(核酸序列如SEQ ID No.1所示)重组蛋白。b：小量诱导(1.5mL)表达上述4个重组蛋白，离心弃上清后用500μL PBS重悬，超声破碎后检测重组蛋白的可溶性。all：细菌裂解液；up：细菌裂解液离心后的上清。仅有4号重组蛋白可溶性高。c：构建4个重组蛋白可溶性分析。用ImageJ软件统计all和up的蛋白条带强度，而后计算上清中蛋白的百分比。计算公式：up百分比＝(up/all)*10。百分比分析图用Graph Padprism5绘画而成。三次重复，经two-tailed t test分析发现4重组蛋白上清比例远高于2和3，表面可溶标签gb1和halo联合使用具有显著协同作用。d：12％Tricine-SDS电泳检测使用superose 6B increase分子排阻法分离纯化重组蛋白4。数字编号代表收集器的收集管编号，每个收集管收集体积为1mL。e：分子排阻法纯化重组蛋白4的紫外吸光图谱。横轴为收集体积，纵轴为紫外光在A280nm的吸光度。目的产物用箭头标示。f：透射电镜观察重组蛋白4，发现该蛋白以球形纳米颗粒形式存在。白色标尺为20nm。 In the research in this application, 4 different encapsulin recombinant proteins were designed, numbered 1, 2, 3, and 4, corresponding to: encapsulin-int N (nucleic acid sequence shown in SEQ ID No. 5), encapsulin -int N -gb1 (nucleic acid sequence is shown in SEQ ID No. 6), halo (nucleic acid sequence is shown in SEQ ID No. 4), encapsulin-int N -halo (nucleic acid sequence is shown in SEQ ID No. 7) And encapsulin-int N -gb1-halo (nucleic acid sequence is shown in SEQ ID No. 8); see Figure 2, encapsulin (nucleic acid sequence is shown in SEQ ID No. 1) and int N (nucleic acid sequence is shown in SEQ ID No. 2) Fusion expression generates inclusion body precipitation (Figure 2a), and the introduction of auxiliary solubilization protein gb1 (nucleic acid sequence shown in SEQ ID No. 3) or halo (nucleic acid sequence shown in SEQ ID No. 4) can partially recombine The protein is soluble. The combined use of the two significantly improved the solubility of recombinant proteins (Figure 2a-c). After the supernatant of recombinant protein 4 was added 0.15g/mL ammonium sulfate, the precipitate was resuspended and dissolved in PBS containing 10% glycerol, and then further separated and purified by molecular exclusion method (Figure 2d-e). Observing the target sample with transmission electron microscope, we found that recombinant protein 4 self-assembled into spherical nanoparticles (Figure 2e). a: 4 different encapsulin (nucleic acid sequence shown in SEQ ID No. 1) recombinant proteins designed by the present disclosure. b: A small amount of induction (1.5 mL) of the above four recombinant proteins was induced, the supernatant was discarded by centrifugation, and the supernatant was resuspended in 500 μL PBS. The solubility of the recombinant protein was tested after ultrasonic disruption. all: bacterial lysate; up: supernatant after centrifugation of bacterial lysate. Only No. 4 recombinant protein is highly soluble. c: Construction of 4 recombinant protein solubility analysis. Use ImageJ software to count the protein band intensity of all and up, and then calculate the percentage of protein in the supernatant. Calculation formula: up percentage=(up/all)*10. The percentage analysis graph is drawn with Graph Padprism5. After three repetitions, the two-tailed t test analysis found that the ratio of 4 recombinant protein supernatant was much higher than that of 2 and 3. The combination of surface soluble label gb1 and halo had a significant synergistic effect. d: 12% Tricine-SDS electrophoresis detection uses superose 6B increase molecular exclusion method to separate and purify recombinant protein 4. The number represents the collection tube number of the collector, and the collection volume of each collection tube is 1 mL. e: UV absorption spectrum of recombinant protein 4 purified by molecular exclusion method. The horizontal axis is the collection volume, and the vertical axis is the absorbance of ultraviolet light at A280nm. The target product is indicated by an arrow. f: Observation of recombinant protein 4 by transmission electron microscope, it is found that the protein exists in the form of spherical nanoparticles. The white scale is 20nm.
2)货物蛋白由于分子量大，利于暴露至纳米颗粒表面，60个重组蛋白单体自装配成纳米颗粒，其暴露的int N-gb1-halo(B)作为通用“适配器”，与货物重组蛋白gb1-int C-cargo(C-D)混合后，在DTT的帮助下发生分子重组； 2) The cargo protein is easy to be exposed to the surface of the nanoparticle due to its large molecular weight. 60 recombinant protein monomers self-assemble into nanoparticles. The exposed int N -gb1-halo(B) acts as a universal "adapter" with the cargo recombinant protein gb1 -After int C -cargo (CD) is mixed, molecular recombination occurs with the help of DTT;
3)内含肽intein自我切除，形成副产物内含肽intein自我切除，形成副产物int N-gb1-halo和gb1-int C(A和C)，而encapsulin与cargo共价偶联，生产目的产物encapsulin-cargo(A-D)。 3) The intein intein is self-resected to form a by-product. The intein intein is self-resected to form the by-products int N -gb1-halo and gb1-int C (A and C), and encapsulin is covalently coupled with cargo for the purpose of production The product encapsulin-cargo (AD).
所述的货物蛋白cargo为GFP(核酸序列如SEQ ID No.9所示)或M44(M44 315-411aa，简写为M44核酸序列如SEQ ID No.10所示)或者strep2(核酸序列如SEQ ID No.11所示)。The cargo protein cargo is GFP (nucleic acid sequence shown in SEQ ID No. 9) or M44 (M44 315-411aa, abbreviated as M44 nucleic acid sequence shown in SEQ ID No. 10) or strep2 (nucleic acid sequence shown in SEQ ID No. 11).
具体地说：Specifically:
A)基于内含肽介导蛋白编辑剪切技术在encapsulin表面高效负载GFP蛋白的方法如下，参阅图3。A) The method for efficiently loading GFP protein on the surface of encapsulin based on intein-mediated protein editing and shearing technology is as follows, see Figure 3.
首先开展体外小量剪切(总体积30μL)分析实验，探索合理的剪切条件。First, carry out a small amount of shear in vitro (total volume 30μL) analysis experiment to explore reasonable shear conditions.
a：初始反应底物浓度分别为：encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)10μM，gb1-int C-GFP(核酸序列如SEQ ID No.12所示)20μM。两者在室温条件下孵育2小时后，随着时间延长反应底物encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)不再明显减少，目的产物encapsulin-GFP不再增加。 a: The initial reaction substrate concentrations are: encapsulin-int N -gb1-halo (nucleic acid sequence is shown in SEQ ID No. 8) 10 μM, gb1-int C -GFP (nucleic acid sequence is shown in SEQ ID No. 12) 20μM. After the two were incubated for 2 hours at room temperature, the reaction substrate encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) no longer decreased significantly as time passed, and the target product encapsulin-GFP no longer increase.
b：用ImageJ计算剪切产物(spliced production)随时间变化(三次重复，各点代表值包含平均值和标准方差)。b: Use ImageJ to calculate the spliced production over time (three repetitions, the representative value of each point includes the average and standard deviation).
通过计算反应底物encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)的减少量来衡量反应效率。横坐标为反应时间，纵坐标为剪切效率。计算公式为：spliced production＝(1-未反应底物/初始反应底物)*100。经半定量计算，2小时后反应效果高达90％以上。 The reaction efficiency is measured by calculating the reduction of the reaction substrate encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8). The abscissa is the reaction time, and the ordinate is the shear efficiency. The calculation formula is: spliced production=(1-unreacted substrate/initial reaction substrate)*100. After semi-quantitative calculation, the reaction effect reached more than 90% after 2 hours.
c：探索不同gb1-int C-GFP(核酸序列如SEQ ID No.12所示)浓度对反应效率的影响。 c: Explore the effect of different gb1-int C- GFP (nucleic acid sequence as shown in SEQ ID No. 12) concentration on the reaction efficiency.
初始反应底物encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)浓度为10μM，以两倍梯度增加反应底物gb1-int C-GFP(核酸序列如SEQ ID No.12所示)浓度，当两者比例为1:1时反应效率高于90％。再增加gb1-int C-GFP(核酸序列如SEQ ID No.12所示)浓度不再促进更多目的产物生成。 The initial reaction substrate encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) has a concentration of 10 μM, and the reaction substrate gb1-int C -GFP (nucleic acid sequence is shown in SEQ ID No. 12) concentration, when the ratio of the two is 1:1, the reaction efficiency is higher than 90%. Increasing the concentration of gb1-int C- GFP (nucleic acid sequence shown in SEQ ID No. 12) no longer promotes the production of more target products.
d：用ImageJ计算剪切产物(spliced production)随反应底物浓度变化(三次重复，各点代表值包含平均值和标准方差)。d: Use ImageJ to calculate the spliced production changes with the concentration of the reaction substrate (three repetitions, the representative value of each point includes the average and standard deviation).
横坐标代表gb1-int C-GFP浓度变化，纵坐标代表反应效率。剪切效率计算公式与b相同。 The abscissa represents the change in gb1-int C -GFP concentration, and the ordinate represents the reaction efficiency. The shear efficiency calculation formula is the same as b.
e：剪切2mL的反应体系(encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)浓度为10μM，gb1-int C-GFP浓度为15μM)，室温2h后用superose 6B increase分子排阻法分离目的产物encapsulin-GFP。before：反应前，after：剪切后；不同数字代表收集管编号，每管收集1mL。 e: Cut 2mL reaction system (encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) concentration is 10μM, gb1-int C -GFP concentration is 15μM), use superose 6B after 2h at room temperature Increase molecular exclusion method to separate the target product encapsulin-GFP. Before: before reaction, after: after cutting; different numbers represent the collection tube number, each tube collects 1mL.
f：分子排阻法分离纯化目的产物紫外吸光图谱。f: UV absorption spectrum of the target product separated and purified by molecular exclusion method.
横坐标代表洗脱体积，纵坐标代表紫外光在A280nm处的吸光度变化。目的产物用箭头标示。The abscissa represents the elution volume, and the ordinate represents the change in absorbance of ultraviolet light at A280nm. The target product is indicated by an arrow.
g：用透射电镜观察发现目的产物encapsulin-GFP维持球形纳米颗粒形状。白色标尺代表20nm。g: Observed by transmission electron microscope, it is found that the target product encapsulin-GFP maintains the shape of spherical nanoparticles. The white scale represents 20nm.
B)基于内含肽介导蛋白编辑剪切技术在encapsulin表面高效负载2xstrep2多肽(简写为strep2)的具体方法如下，参阅图4：B) The specific method for efficiently loading 2xstrep2 polypeptide (abbreviated as strep2) on the surface of encapsulin based on the intein-mediated protein editing and shearing technology is as follows, see Figure 4:
先开展体外小量剪切(总体积30μL)分析实验，探索合理的剪切条件。First carry out in vitro small shear (total volume 30μL) analysis experiment to explore reasonable shear conditions.
a：初始反应底物浓度分别为：encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)10μM，gb1-int C-strep2(核酸序列如SEQ ID No.13所示)30μM。两者在室温条件下孵育2小时后，随着时间延长反应底物encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)不再明显减少，目的产物encapsulin-strep2不再增加。 a: The initial reaction substrate concentrations are: encapsulin-int N -gb1-halo (nucleic acid sequence is shown in SEQ ID No. 8) 10μM, gb1-int C -strep2 (nucleic acid sequence is shown in SEQ ID No. 13) 30μM. After the two were incubated at room temperature for 2 hours, the reaction substrate encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) no longer decreased significantly, and the target product encapsulin-strep2 no longer increase.
b：用ImageJ计算目的产物(spliced production)随时间变化(三次重复，各点代表值包含平均值和标准方差)。b: Use ImageJ to calculate the spliced production over time (three repetitions, the representative value of each point includes the average and standard deviation).
通过计算反应底物encapsulin-intein N-gb1-halo(核酸序列如SEQ ID No.8所示)减少比例来衡量反应效率。横坐标为反应时间，纵坐标为剪切效率。计算公式为：spliced production＝(1-未反应底物/初始反应底物)*100。经半定量计算，2小时后反应效果高达90％以上。 The reaction efficiency is measured by calculating the reduction ratio of the reaction substrate encapsulin-intein N- gb1-halo (nucleic acid sequence shown in SEQ ID No. 8). The abscissa is the reaction time, and the ordinate is the shear efficiency. The calculation formula is: spliced production=(1-unreacted substrate/initial reaction substrate)*100. After semi-quantitative calculation, the reaction effect reached more than 90% after 2 hours.
c：探索不同gb1-int C-strep2(核酸序列如SEQ ID No.13所示)浓度对反应效率的影响。 c: Explore the effect of different gb1-int C- strep2 (nucleic acid sequence shown in SEQ ID No. 13) concentration on reaction efficiency.
初始反应底物encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)浓度为10μM，逐步增加反应底物gb1-int C-strep2(核酸序列如SEQ ID No.13所示)浓度，当两者比例为1:1时反应效率大于90％。再增加gb1-int C-strep2(核酸序列如SEQ ID No.13所示)浓度不再促进更多目的产物生成。 The concentration of the initial reaction substrate encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) is 10 μM, and gradually increase the reaction substrate gb1-int C -strep2 (nucleic acid sequence shown in SEQ ID No. 13 ) Concentration, when the ratio of the two is 1:1, the reaction efficiency is greater than 90%. Increasing the concentration of gb1-int C- strep2 (nucleic acid sequence shown in SEQ ID No. 13) no longer promotes the production of more target products.
d：用ImageJ计算剪切产物(spliced production)随反应底物浓度变化(三次重复，各点代表值包含 平均值和标准方差)。d: Use ImageJ to calculate the spliced production change with the concentration of the reaction substrate (three repetitions, the representative value of each point includes the average and standard deviation).
横坐标代表gb1-int C-strep2(核酸序列如SEQ ID No.13所示)浓度变化，纵坐标代表反应效率。剪切效率计算公式与b相同。 The abscissa represents the concentration change of gb1-int C- strep2 (nucleic acid sequence is shown in SEQ ID No. 13), and the ordinate represents the reaction efficiency. The shear efficiency calculation formula is the same as b.
e：剪切2mL的反应体系encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)浓度为10μM，gb1-int C-strep2(核酸序列如SEQ ID No.13所示)浓度为30μM)，室温2h后用superose 6B increase分子排阻法分离目的产物encapsulin-strep2。before：反应前，after：剪切后；不同数字代表收集管编号，每管收集1mL。 e: Cut 2mL of the reaction system encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) at a concentration of 10 μM, gb1-int C -strep2 (nucleic acid sequence shown in SEQ ID No. 13) The concentration is 30μM). After 2h at room temperature, the target product encapsulin-strep2 is separated by superose 6B increase molecular exclusion method. Before: before reaction, after: after cutting; different numbers represent the collection tube number, each tube collects 1mL.
f：分子排阻法分离纯化目的产物紫外吸光图谱。横坐标代表洗脱体积，纵坐标代表紫外光在A280nm处的吸光度变化。目的产物用箭头标示。f: UV absorption spectrum of the target product separated and purified by molecular exclusion method. The abscissa represents the elution volume, and the ordinate represents the change in absorbance of ultraviolet light at A280nm. The target product is indicated by an arrow.
g：用透射电镜观察发现目的产物encapsulin-strep2保持球形纳米颗粒形状。白色标尺代表20nm。g: Observed by transmission electron microscope, it is found that the target product encapsulin-strep2 maintains the shape of spherical nanoparticles. The white scale represents 20nm.
C)基于内含肽介导蛋白编辑剪切技术在encapsulin表面高效负载MCMV的M44蛋白C端氨基酸315-411(简称M44蛋白)的具体方法如下，参阅图5：C) The specific method for efficiently loading the C-terminal amino acids 315-411 (M44 protein for short) of MCMV M44 protein on the surface of encapsulin based on intein-mediated protein editing and shearing technology is as follows, refer to Figure 5:
我们先开展体外小量剪切(总体积30μL)分析实验，探索合理的剪切条件。We first carry out in vitro small shear (total volume 30μL) analysis experiment to explore reasonable shear conditions.
a：初始反应底物浓度分别为：encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)10μM，gb1-int C-M44(核酸序列如SEQ ID No.14所示)20μM。两者在室温条件下孵育2小时后，随着时间延长反应底物encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)不再明显减少，目的产物encapsulin-M44不再增加。 a: The initial reaction substrate concentrations are: encapsulin-int N -gb1-halo (nucleic acid sequence is shown in SEQ ID No. 8) 10μM, gb1-int C -M44 (nucleic acid sequence is shown in SEQ ID No. 14) 20μM. After the two were incubated for 2 hours at room temperature, the reaction substrate encapsulin-int N- gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) no longer decreased significantly as time passed, and the target product encapsulin-M44 no longer increase.
b：用ImageJ计算剪切产物(spliced production)随时间变化(三次重复，各点代表值包含平均值和标准方差)。通过计算反应底物encapsulin-intein N-gb1-halo减少比例来衡量反应效率。横坐标为反应时间，纵坐标为剪切效率。计算公式为：spliced production＝(1-未反应底物/初始反应底物)*100。经半定量计算，2小时后反应效果高达90％以上。 b: Use ImageJ to calculate the spliced production change over time (three repetitions, the representative value of each point includes the average and standard deviation). The reaction efficiency is measured by calculating the reduction ratio of the reaction substrate encapsulin-intein N -gb1-halo. The abscissa is the reaction time, and the ordinate is the shear efficiency. The calculation formula is: spliced production=(1-unreacted substrate/initial reaction substrate)*100. After semi-quantitative calculation, the reaction effect reached more than 90% after 2 hours.
c：探索不同gb1-int C-M44(核酸序列如SEQ ID No.14所示)浓度对反应效率的影响。 c: Explore the effect of different gb1-int C- M44 (nucleic acid sequence as shown in SEQ ID No. 14) concentration on reaction efficiency.
初始反应底物encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)浓度为10μM，两倍梯度逐步增加反应底物gb1-int C-M44(核酸序列如SEQ ID No.14所示)浓度，当两者比例为1:1时反应效率高于90％。再增加gb1-int C-M44(核酸序列如SEQ ID No.14所示)浓度不再促进更多目的产物生成。 The concentration of the initial reaction substrate encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) was 10 μM, and the reaction substrate gb1-int C -M44 (nucleic acid sequence shown in SEQ ID No. 14) concentration, when the ratio of the two is 1:1, the reaction efficiency is higher than 90%. Increasing the concentration of gb1-int C- M44 (nucleic acid sequence shown in SEQ ID No. 14) no longer promotes the production of more target products.
d：用ImageJ计算剪切产物(spliced production)随反应底物浓度变化(三次重复，各点代表值包含平均值和标准方差)。d: Use ImageJ to calculate the spliced production changes with the concentration of the reaction substrate (three repetitions, the representative value of each point includes the average and standard deviation).
横坐标代表gb1-int C-M44(核酸序列如SEQ ID No.14所示)浓度变化，纵坐标代表反应效率。剪切效率计算公式与b相同。 The abscissa represents the concentration change of gb1-int C- M44 (nucleic acid sequence is shown in SEQ ID No. 14), and the ordinate represents the reaction efficiency. The shear efficiency calculation formula is the same as b.
e：剪切2mL的反应体系(encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)浓度为10μM，gb1-int C-M44(核酸序列如SEQ ID No.14所示)浓度为15μM)，室温2h后用superose 6B increase分子排阻法分离目的产物encapsulin-M44。before：反应前，after：剪切后；不同数字代表收集管编号，每管收集1mL。 e: Cut 2mL reaction system (encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) at a concentration of 10 μM, gb1-int C -M44 (nucleic acid sequence shown in SEQ ID No. 14 ) The concentration is 15 μM), and the target product encapsulin-M44 is separated by superose 6B increase molecular exclusion method after 2 hours at room temperature. Before: before reaction, after: after cutting; different numbers represent the collection tube number, each tube collects 1mL.
f：分子排阻法分离纯化目的产物紫外吸光图谱。横坐标代表洗脱体积，纵坐标代表紫外光在A280nm处的吸光度变化。目的产物用箭头标示。f: UV absorption spectrum of the target product separated and purified by molecular exclusion method. The abscissa represents the elution volume, and the ordinate represents the change in absorbance of ultraviolet light at A280nm. The target product is indicated by an arrow.
g：用透射电镜观察发现目的产物encapsulin-M44以纳米颗粒形式存在。白色标尺代表20nm。g: Observed by transmission electron microscope, the target product encapsulin-M44 is found in the form of nanoparticles. The white scale represents 20nm.
本公开中涉及的分子排阻法分离纳米颗粒产物，均使用superose 6B increase层析柱在上海闪谱有限公司Clear-First 3000系统上开展。用12％的Tricine-SDS-PAGE电泳检测分子排阻法纯化纳米颗粒样品，取编号为7-18的收集管(每管收集体积1mL)中的样品30μL，加入10μL的4xSDS loading buffer后100℃煮样10分钟。剪切前、后上样2μL，收集管每个样品上样6μL。紫外吸光度随洗脱体积变化的图谱的横轴代表洗脱体积，纵轴代表A280nm吸光度。The molecular exclusion method involved in the separation of nanoparticle products in this disclosure is all carried out on the Clear-First 3000 system of Shanghai Flash Spectrum Co., Ltd. using a superose 6B increase chromatography column. Use 12% Tricine-SDS-PAGE electrophoresis to detect the molecular exclusion method to purify the nanoparticle samples, take 30μL of the sample in the collection tube numbered 7-18 (collection volume of each tube is 1mL), add 10μL of 4xSDS loading buffer and then 100℃ Cook the sample for 10 minutes. Load 2μL before and after cutting, and load 6μL for each sample in the collection tube. The horizontal axis of the spectrum of UV absorbance change with elution volume represents the elution volume, and the vertical axis represents the absorbance at A280 nm.
本公开中涉及蛋白的诱导表达：本公开中使用的重组基因均插入pET28a载体中，转化至BL21(DE3)plysS中诱导表达。The present disclosure relates to the induced expression of proteins: the recombinant genes used in the present disclosure are inserted into the pET28a vector, and transformed into BL21(DE3)plysS to induce expression.
纳米载体相关的重组蛋白涉及的基因在金斯瑞生物技术公司(南京)合成，而后用overlap的方法进行扩增。encapsulin-int N(核酸序列如SEQ ID No.14所示)、encapsulin-int N-gb1融合基因用NcoI和XhoI酶切位点，encapsulin-int N-halo、encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)使用NcoI和 Hind III酶切后，与相关限制性内切酶线性化的pET28a载体连接后转化至Top10感受态进行克隆扩增。单克隆经测序正确后转化至BL21(DE3)plysS诱导表达纯化。 The genes involved in the nanocarrier-related recombinant proteins were synthesized at GenScript Biotechnology Company (Nanjing), and then amplified by the overlap method. encapsulin-int N (nucleic acid sequence shown in SEQ ID No. 14), encapsulin-int N -gb1 fusion gene with NcoI and XhoI restriction sites, encapsulin-int N -halo, encapsulin-int N -gb1-halo ( The nucleic acid sequence is shown in SEQ ID No. 8) After digestion with NcoI and Hind III, it is ligated with the pET28a vector linearized by related restriction enzymes and transformed into Top10 competent for cloning amplification. The single clone was sequenced correctly and transformed into BL21(DE3)plysS to induce expression and purification.
货物分子相关融合基因gb1-intein C-2xstrep2(简写为strep2)、gb1-intein C-GFP和gb1-intein C-M44亦通过overlap扩增目的基因，而后用EcoRI和XhoI作为酶切位点插入线性化的pET28a载体中，再转化至Top10扩增。halo-intein N-gb1重组基因所用酶切位点为NcoI和XhoI，克隆构建方法同上。所有重组基因转换至BL21(DE3)plysS，在LB培养至OD为0.6-0.8时，加入0.2mM IPTG，25℃摇床培养5小时，而后4000rpm、4℃离心30分钟后，收集细菌沉淀。用30mL的PBS重悬细菌，相同条件离心后弃上清，细菌可用于下一步纯化或-80℃长期冷冻。 Cargo molecule-related fusion genes gb1-intein C -2xstrep2 (abbreviated as strep2), gb1-intein C -GFP and gb1-intein C -M44 were also amplified by overlap, and then used EcoRI and XhoI as restriction sites for linear insertion The transformed pET28a vector was transformed into Top10 for amplification. The restriction sites of the halo-intein N- gb1 recombinant gene are NcoI and XhoI, and the cloning construction method is the same as above. All recombinant genes were converted to BL21(DE3)plysS. When LB was cultured to an OD of 0.6-0.8, 0.2mM IPTG was added, cultured on a shaker at 25°C for 5 hours, and then centrifuged at 4000rpm and 4°C for 30 minutes, and the bacterial pellets were collected. Resuspend the bacteria with 30mL of PBS, centrifuge under the same conditions and discard the supernatant. The bacteria can be used for the next step of purification or long-term freezing at -80°C.
本公开中纳米载体相关蛋白纯化：用30mL的50mM Tris，pH 7.5，150mM NaCl(NT buffer)重悬500mL的LB培养细菌，而后超声破碎。超声结束后，12000rpm、4℃、30分钟离心，上清转移至50mL的离心管中。加入0.15g/mL的硫酸铵固体，在4℃摇床混匀15分钟后，12000rpm、4℃、30分钟离心。弃上清，往离心管中加入10ml的含10％甘油的PBS，在4℃冷库摇床缓慢溶解沉淀产物。BCA测得encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)浓度为2.75mg/mL，可用来开展体外剪切分析或-80℃冻存。 Purification of nanocarrier-related proteins in the present disclosure: Resuspend 500 mL of LB culture bacteria with 30 mL of 50 mM Tris, pH 7.5, 150 mM NaCl (NT buffer), and then ultrasonically disrupt. After the sonication is over, centrifuge at 12000 rpm, 4°C for 30 minutes, and transfer the supernatant to a 50 mL centrifuge tube. Add 0.15g/mL ammonium sulfate solid, mix well on a shaker at 4°C for 15 minutes, and centrifuge at 12000rpm, 4°C for 30 minutes. Discard the supernatant, add 10 ml of PBS containing 10% glycerol to the centrifuge tube, and slowly dissolve the precipitated product on a shaker in a cold storage at 4°C. The concentration of encapsulin-int N- gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) measured by BCA was 2.75 mg/mL, which can be used for in vitro shear analysis or cryopreservation at -80°C.
本公开中其它蛋白纯化：所有的蛋白均采用Ni 2+亲和层析的方法纯化细菌裂解液上清中可溶蛋白。30mL NT buffer重悬细菌后超声破碎裂解，离心后上清与1mL的N 2+树脂在4℃摇床上混匀30分钟。非特异性结合蛋白随液体以重力方式流出。接着，加入20mL含10mM、20mM咪唑的NT buffer洗涤树脂，去掉结合不紧密蛋白。随后，加入10mL含500mM咪唑的NT buffer洗脱。BCA法测得蛋白浓度分别为halo-intein N-gb1:1.2mg/ml；gb1-intein C-strep2：1.8mg/ml；gb1-intein C-GFP：1.8mg/ml，gb1-intein C-M44:1.92mg/ml。上述蛋白不需要透析，可直接用于内含肽介导的蛋白编辑剪切或-80℃长期冻存。 Purification of other proteins in the present disclosure: All proteins are purified by Ni 2+ affinity chromatography to purify soluble proteins in the supernatant of the bacterial lysate. The bacteria were resuspended in 30mL NT buffer and then sonicated for lysis. After centrifugation, the supernatant was mixed with 1mL N 2+ resin on a shaker at 4°C for 30 minutes. The non-specific binding protein flows out of the liquid by gravity. Next, add 20 mL of NT buffer containing 10 mM, 20 mM imidazole to wash the resin to remove loosely bound proteins. Subsequently, 10 mL of NT buffer containing 500 mM imidazole was added for elution. The protein concentrations measured by the BCA method were halo-intein N -gb1: 1.2 mg/ml; gb1-intein C -strep2: 1.8 mg/ml; gb1-intein C -GFP: 1.8 mg/ml, gb1-intein C -M44 : 1.92mg/ml. The above-mentioned protein does not require dialysis and can be directly used for intein-mediated protein editing and shearing or long-term freezing at -80°C.
本公开中体外分析内含肽介导蛋白编辑剪切效率：本公开先在小量体积条件(30μL)下探索不同反应时间与底物浓度对反应效率的影响。初始反应底物encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)或halo-int N-gb1核酸序列如SEQ ID No.15所示)浓度固定为10μM，gb1-int C-cargo(cargo为GFP、strep2或M44)浓度范围为：1～30μM。探索反应时间对反应效率影响，初始反应底物浓度固定，以时间为变量，范围为1分钟-16小时，发现所有反应在4小时内完成，效率高达90％以上。探索底物浓度对反应效率的影响，固定encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)或halo-int N-gb1核酸序列如SEQ ID No.15所示)浓度固定为10μM，逐步增加gb1-int C-cargo浓度。当底物gb1-int C-cargo超过10μM时，所有反应效率达最大，效率高达90％以上。所有反应均在室温(25℃左右)进行，反应溶液含2mM DTT(二硫苏糖醇)。以encapsulin-int N-gb1-halo(核酸序列如SEQ ID No.8所示)或halo-int N-gb1核酸序列如SEQ ID No.15所示)为目标，用ImageJ计算反应效率，公式为：(1-未反应底物/初始反应底物)*100。 In the present disclosure, in vitro analysis of intein-mediated protein editing and shearing efficiency: The present disclosure first explores the effect of different reaction times and substrate concentrations on reaction efficiency under a small volume condition (30 μL). The initial reaction substrate encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) or halo-int N -gb1 nucleic acid sequence shown in SEQ ID No. 15) concentration is fixed at 10 μM, gb1-int The concentration range of C- cargo (cargo is GFP, strep2 or M44) is: 1-30μM. Exploring the effect of reaction time on reaction efficiency, the initial reaction substrate concentration is fixed, with time as a variable, ranging from 1 minute to 16 hours, and it is found that all reactions are completed within 4 hours, and the efficiency is as high as 90%. To explore the influence of substrate concentration on reaction efficiency, fix the concentration of encapsulin-int N- gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) or halo-int N- gb1 nucleic acid sequence shown in SEQ ID No. 15) Fix it to 10μM and gradually increase the concentration of gb1-int C- cargo. When the substrate gb1-int C- cargo exceeds 10 μM, the efficiency of all reactions reaches the maximum, and the efficiency is as high as 90% or more. All reactions were performed at room temperature (around 25°C), and the reaction solution contained 2mM DTT (dithiothreitol). Take encapsulin-int N -gb1-halo (nucleic acid sequence shown in SEQ ID No. 8) or halo-int N -gb1 nucleic acid sequence shown in SEQ ID No. 15 as the target, and use ImageJ to calculate the reaction efficiency. The formula is : (1-Unreacted substrate/initial reaction substrate)*100.
实施例2Example 2
encapsulin-cargo酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)滴定货物分子相关IgG抗体滴度实验，参阅图6。Encapsulin-cargo enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) titration of cargo molecule-related IgG antibody titer experiment, see Figure 6.
小鼠免疫及酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)滴定货物分子相关IgG抗体滴度：对5-8周龄的雌性Balb/c小鼠腹腔接种10μg的货物蛋白(GFP或M44)或纳米载体递送的货物蛋白(encapsulin-GFP或encapsulin-M44)，共接种两次，间隔两周。免疫两周后眼眶采取200μL左右血液，37℃放置30分钟后4℃放置过夜以充分析出血清。第二天5,000rpm、4℃离心30分钟，取血清。1:1000稀释血清，而后以10倍梯度稀释血清来滴定货物分子相关抗体滴度。往高吸附96孔板内加入PBS稀释的抗原，GFP蛋白每孔25ng，M44蛋白每孔50ng，4℃过夜吸附。第二天用PBST洗涤三次后加入含5％脱脂牛奶的PBS在37℃培养箱中封闭45分钟。PBST洗涤三次后加入50μL、10倍梯度稀释的抗体，37℃培养箱中孵育1小时。PBST洗涤三次后加入1:5000的HRP修饰的anti-mouse，37℃培养箱中孵育1小时。PBST洗涤三次后加入TMB显色2-5分钟，再用1M硫酸终止反应。最后在酶标仪中检测450nm处吸光值，本公开以吸光值>0.2认定为阳性。特异性抗体滴度用Graph Padprism5分组(grouped)绘画，蛋白与纳米载体递送蛋白诱导的抗体滴度相关性用Mann Whitney test，nonparametric  tests进行显著性分析。Mouse immunization and enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) titration of cargo molecule-related IgG antibody titers: 5-8 weeks old female Balb/c mice were intraperitoneally inoculated with 10 μg of cargo protein (GFP or M44) ) Or the cargo protein delivered by the nanocarrier (encapsulin-GFP or encapsulin-M44), inoculated twice, two weeks apart. After two weeks of immunization, about 200 μL of blood was collected from the eye socket, placed at 37°C for 30 minutes, and then placed at 4°C overnight to fill the serum. Centrifuge at 5,000 rpm and 4°C for 30 minutes the next day, and collect serum. Dilute the serum at 1:1000, and then dilute the serum with a 10-fold gradient to titrate the antibody titers related to the cargo molecule. Add PBS diluted antigen to the high adsorption 96-well plate, 25ng GFP protein per well, 50ng M44 protein per well, overnight at 4°C. After washing with PBST three times on the next day, PBS containing 5% skim milk was added, and the cells were blocked in a 37°C incubator for 45 minutes. After washing three times with PBST, add 50μL, 10-fold dilution of the antibody, and incubate in a 37°C incubator for 1 hour. After washing with PBST three times, adding 1:5000 HRP-modified anti-mouse, and incubating in a 37°C incubator for 1 hour. After washing three times with PBST, TMB was added for color development for 2-5 minutes, and then the reaction was terminated with 1M sulfuric acid. Finally, the absorbance value at 450nm is detected in the microplate reader, and the present disclosure regards the absorbance value>0.2 as positive. Specific antibody titers are drawn by Graph Padprism5 grouped, and the correlation between protein and antibody titers induced by nanocarrier-delivered protein is analyzed by Mann Whitney test and nonparametric tests.
结果：腹腔免疫小鼠10μg的蛋白抗原或纳米载体递送的抗原，接种2次，每次间隔两周。免疫两周后眼眶采血，ELISA检测抗体滴度。a：一次和两次免疫后，用纳米技术递送的encapsulin-GFP诱导GFP相关抗体滴度显著高于GFP蛋白(Mann Whitney test，nonparametric tests,p<0.01)，抗体滴度是后者的四倍。b：一次和两次免疫后，encapsulin-M44诱导M44相关抗体滴度是M44蛋白的5倍，引起免疫应答水平显著高于后者(Mann Whitney test，nonparametric tests,p<0.01)。显著性统计用Graph Padprism5进行分析。Results: The mice were immunized intraperitoneally with 10 μg of protein antigen or antigen delivered by nanocarriers and were vaccinated twice, with an interval of two weeks each time. Two weeks after immunization, blood was collected from the orbit and the antibody titer was detected by ELISA. a: After one and two immunizations, encapsulin-GFP delivered by nanotechnology induces GFP-related antibody titer significantly higher than GFP protein (Mann Whitney test, nonparametric tests, p<0.01), and the antibody titer is four times that of the latter . b: After one and two immunizations, encapsulin-M44 induced M44-related antibody titer 5 times that of M44 protein, which caused an immune response level significantly higher than the latter (Mann Whitney test, nonparametric tests, p<0.01). Significance statistics are analyzed with Graph Padprism5.
实施例3Example 3
基于halo共价交联技术和抗原-抗体相互作用纯化货物分子相关抗体原理示意图，参阅图7。Based on halo covalent cross-linking technology and antigen-antibody interaction to purify cargo molecule-related antibodies, refer to Figure 7.
使用的gb1-int C-GFP或gb1-intein C-M44可与halo-int N-gb1核酸序列如SEQ ID No.15所示)蛋白进行蛋白编辑剪切，生成重组蛋白halo-cargo(cargo为GFP或M44)，gb1-int C和int N-gb1以副产物的形式存在。反应结束后，混合物与Promega halo TM beads混合，目的产物halo-cargo共价结合至halo TM beads。离心弃上清后，加入免疫后的血清。货物分子相关抗体利用抗原-抗体之间高亲和相互作用与halo TM-halo-cargo结合，其它抗体和蛋白通过离心、洗涤后分离。最后用200mM glycine pH2.8洗脱目的抗体。 The gb1-int C- GFP or gb1-intein C- M44 used can be edited and cut with the halo-int N- gb1 nucleic acid sequence as shown in SEQ ID No. 15) to produce recombinant protein halo-cargo (cargo is GFP or M44), gb1-int C and int N -gb1 exist as by-products. After the reaction, the mixture is mixed with Promega halo TM beads, and the target product halo-cargo is covalently bound to halo TM beads. After centrifugation to discard the supernatant, the immunized serum was added. Cargo molecule-related antibodies use the high-affinity interaction between antigen and antibody to bind to halo TM -halo-cargo, and other antibodies and proteins are separated after centrifugation and washing. Finally, the target antibody was eluted with 200mM glycine pH2.8.
上述的halo TM-halo-GFP的制备及GFP相关抗体的纯化方法如下，参阅图8： The preparation of halo TM -halo-GFP and the purification method of GFP-related antibodies are as follows, see Figure 8:
首先，通过体外剪切分析实验，探索合适的反应时间和底物浓度。First, through in vitro shear analysis experiments, explore the appropriate reaction time and substrate concentration.
a：初始反应底物浓度分别为：halo-int N-gb1核酸序列如SEQ ID No.15所示)10μM，gb1-int C-GFP(核酸序列如SEQ ID No.12所示)20μM。两者在室温条件下孵育4小时后，随着时间延长反应底物halo-int N-gb1核酸序列如SEQ ID No.15所示)不再明显减少，目的产物halo-GFP不再增加。 a: The initial reaction substrate concentration is: halo-int N- gb1 nucleic acid sequence shown in SEQ ID No. 15) 10 μM, gb1-int C- GFP (nucleic acid sequence shown in SEQ ID No. 12) 20 μM. After the two were incubated at room temperature for 4 hours, the nucleic acid sequence of the reaction substrate halo-int N- gb1 (as shown in SEQ ID No. 15) no longer decreased significantly, and the target product halo-GFP no longer increased.
b：用ImageJ计算剪切产物(spliced production)随时间变化(三次重复，各点代表值包含平均值和标准方差)。通过计算反应底物halo-int N-gb1核酸序列如SEQ ID No.15所示)减少比例来衡量反应效率。横坐标为反应时间，纵坐标为剪切效率。计算公式为：spliced production＝(1-未反应底物/初始反应底物)*100。经半定量计算，4小时后反应效果高达90％以上。 b: Use ImageJ to calculate the spliced production change over time (three repetitions, the representative value of each point includes the average and standard deviation). The reaction efficiency is measured by calculating the reduction ratio of the reaction substrate halo-int N- gb1 nucleic acid sequence (shown in SEQ ID No. 15). The abscissa is the reaction time, and the ordinate is the shear efficiency. The calculation formula is: spliced production=(1-unreacted substrate/initial reaction substrate)*100. After semi-quantitative calculation, the reaction effect reached more than 90% after 4 hours.
c：探索不同gb1-int C-GFP(核酸序列如SEQ ID No.12所示)浓度对反应效率的影响。初始反应底物halo-intein N-gb1浓度为10μM，两倍梯度逐步增加反应底物gb1-int C-GFP浓度，当两者比例为1:1时反应效率达高于90％。再增加gb1-int C-GFP浓度不再促进更多目的产物生成。 c: Explore the effect of different gb1-int C- GFP (nucleic acid sequence as shown in SEQ ID No. 12) concentration on the reaction efficiency. The initial reaction substrate halo-intein N- gb1 concentration is 10μM, and the two-fold gradient gradually increases the reaction substrate gb1-int C- GFP concentration. When the ratio of the two is 1:1, the reaction efficiency is higher than 90%. Increasing the concentration of gb1-int C -GFP no longer promotes the production of more target products.
d：用ImageJ计算目的产物(spliced production)随反应底物浓度变化(三次重复，各点代表值包含平均值和标准方差)。横坐标代表gb1-int C-GFP浓度变化，纵坐标代表反应效率。剪切效率计算公式与b相同。 d: Use ImageJ to calculate the change of the target product (spliced production) with the concentration of the reaction substrate (three repetitions, the representative value of each point includes the average and standard deviation). The abscissa represents the change in gb1-int C -GFP concentration, and the ordinate represents the reaction efficiency. The shear efficiency calculation formula is the same as b.
e：12％Tricine-SDS-PAGE电泳分析halo-GFP与Promega halo TM结合。室温孵育(halo-int N-gb1核酸序列如SEQ ID No.15所示)浓度为10μM，gb1-int C-GFP(核酸序列如SEQ ID No.12所示)浓度为20μM)4小时后，250μL反应液与50μL平衡好的Promega halo TM室温摇床混合半个小时。而后离心，用500μL PBS洗涤beads，除去非特异性结合蛋白。before：剪切前；after：剪切后；flow：反应液与Promega halo TM混合后离心上清；wash1-wash3：3次PBS洗涤液。结果表明目的蛋白halo-GFP特异性结合至Promega halo TM beads。 e: 12% Tricine-SDS-PAGE electrophoresis analysis of halo-GFP binding to Promega halo TM . After incubating at room temperature (halo-int N -gb1 nucleic acid sequence shown in SEQ ID No. 15) at a concentration of 10 μM, gb1-int C -GFP (nucleic acid sequence shown in SEQ ID No. 12) at a concentration of 20 μM) for 4 hours, 250μL of reaction solution was mixed with 50μL of balanced Promega halo TM room temperature shaker for half an hour. After centrifugation, the beads were washed with 500 μL PBS to remove non-specific binding proteins. Before: before shearing; after: after shearing; flow: centrifuge the supernatant after mixing the reaction solution with Promega halo TM ; wash1-wash3: 3 times PBS washing solution. The results show that the target protein halo-GFP specifically binds to Promega halo TM beads.
f：12％Tricine-SDS-PAGE检测用新制备的halo TM-halo-GFP从血清中分离纯化GFP相关蛋白。input：免疫2次后的血清；flow：血清与halo TM-halo-GFP孵育后的流出；wash1-wash3：用500μL PBST洗涤halo TM-halo-GFP；elution：90μL的200mM glycine pH2.8洗脱目的抗体(EP管提前添加10μL 1M Tris，pH9.0缓冲液以保护抗体)。 f: 12% Tricine-SDS-PAGE detection uses newly prepared halo TM -halo-GFP to separate and purify GFP-related proteins from serum. input: serum after 2 immunizations; flow: flow out of serum after incubation with halo TM -halo-GFP; wash1-wash3: wash halo TM -halo-GFP with 500 μL PBST; elution: 90 μL 200mM glycine pH2.8 elution Antibody of interest (add 10μL of 1M Tris, pH9.0 buffer in advance to EP tube to protect the antibody).
上述的halo TM-halo-M44的制备及M44相关抗体的纯化方法为，参阅图9： The preparation method of halo TM -halo-M44 and the purification method of M44-related antibodies are as shown in Figure 9:
首先，体外剪切分析实验，探索合适的反应时间和底物浓度。First, in vitro shear analysis experiments to explore the appropriate reaction time and substrate concentration.
a：初始反应底物浓度分别为：halo-int N-gb1核酸序列如SEQ ID No.15所示)10μM，gb1-int C-M44(核酸序列如SEQ ID No.14所示)20μM。两者在室温条件下孵育2小时后，随着时间延长反应底物halo-int N-gb1核酸序列如SEQ ID No.15所示)不再明显减少，目的产物halo-GFP不再增加。 a: The initial reaction substrate concentrations are: halo-int N- gb1 nucleic acid sequence shown in SEQ ID No. 15) 10 μM, gb1-int C- M44 (nucleic acid sequence shown in SEQ ID No. 14) 20 μM. After the two were incubated at room temperature for 2 hours, the nucleic acid sequence of the reaction substrate halo-int N- gb1 (as shown in SEQ ID No. 15) no longer decreased significantly, and the target product halo-GFP no longer increased.
b：用ImageJ计算目的产物(spliced production)随时间变化(三次重复，各点代表值包含平均值和 标准方差)。通过计算反应底物halo-int N-gb1核酸序列如SEQ ID No.15所示)减少比例来衡量反应效率。横坐标为反应时间，纵坐标为剪切效率。计算公式为：spliced production＝(1-未反应底物/初始反应底物)*100。经半定量计算，2小时后反应效果高达90％以上。 b: Use ImageJ to calculate the spliced production over time (three repetitions, the representative value of each point includes the average and standard deviation). The reaction efficiency is measured by calculating the reduction ratio of the reaction substrate halo-int N- gb1 nucleic acid sequence (shown in SEQ ID No. 15). The abscissa is the reaction time, and the ordinate is the shear efficiency. The calculation formula is: spliced production=(1-unreacted substrate/initial reaction substrate)*100. After semi-quantitative calculation, the reaction effect reached more than 90% after 2 hours.
c：探索不同gb1-int C-M44(核酸序列如SEQ ID No.14所示)浓度对反应效率的影响。初始反应底物halo-intein N-gb1浓度为10μM，两倍梯度逐步增加反应底物gb1-int C-M44(核酸序列如SEQ ID No.14所示)浓度，当两者比例为1:1时反应效率达最大。再增加gb1-int C-M44(核酸序列如SEQ ID No.14所示)浓度不再促进更多目的产物生成。 c: Explore the effect of different gb1-int C- M44 (nucleic acid sequence as shown in SEQ ID No. 14) concentration on reaction efficiency. The initial reaction substrate halo-intein N -gb1 concentration is 10μM, and the two-fold gradient gradually increases the reaction substrate gb1-int C -M44 (nucleic acid sequence shown in SEQ ID No.14) concentration, when the ratio of the two is 1:1 When the reaction efficiency is maximized. Increasing the concentration of gb1-int C- M44 (nucleic acid sequence shown in SEQ ID No. 14) no longer promotes the production of more target products.
d：用ImageJ计算目的产物(spliced production)随反应底物浓度变化(三次重复，各点代表值包含平均值和标准方差)。横坐标代表gb1-int C-M44(核酸序列如SEQ ID No.14所示)浓度变化，纵坐标代表反应效率。剪切效率计算公式与b相同。 d: Use ImageJ to calculate the change of the target product (spliced production) with the concentration of the reaction substrate (three repetitions, the representative value of each point includes the average and standard deviation). The abscissa represents the concentration change of gb1-int C- M44 (nucleic acid sequence is shown in SEQ ID No. 14), and the ordinate represents the reaction efficiency. The shear efficiency calculation formula is the same as b.
e：12％Tricine-SDS-PAGE电泳分析halo-M44与Promega halo TM结合。室温孵育(halo-int N-gb1核酸序列如SEQ ID No.15所示)浓度为10μM，gb1-int C-M44(核酸序列如SEQ ID No.14所示)浓度为20μM)4小时后，250μL反应液与50μL平衡好的Promega halo TM室温摇床混合半个小时。而后离心，用500μL PBS洗涤beads，除去非特异性结合蛋白。before：剪切前；after：剪切后；flow：反应液与Promega halo TM混合后离心上清；wash1-wash3：3次PBS洗涤液。结果显示目的蛋白halo-GFP特异性结合至Promega halo TM beads。 e: 12% Tricine-SDS-PAGE electrophoresis analysis of the binding of halo-M44 and Promega halo TM . After incubating at room temperature (halo-int N- gb1 nucleic acid sequence shown in SEQ ID No. 15) at a concentration of 10 μM, gb1-int C -M44 (nucleic acid sequence shown in SEQ ID No. 14) at a concentration of 20 μM) for 4 hours, 250μL of reaction solution was mixed with 50μL of balanced Promega halo TM room temperature shaker for half an hour. After centrifugation, the beads were washed with 500 μL PBS to remove non-specific binding proteins. Before: before shearing; after: after shearing; flow: centrifuge the supernatant after mixing the reaction solution with Promega halo TM ; wash1-wash3: 3 times PBS washing solution. The results show that the target protein halo-GFP specifically binds to Promega halo TM beads.
f：12％Tricine-SDS-PAGE检测用新制备的halo TM-halo-M44从血清中分离纯化M44相关蛋白。input：免疫2次后的血清；flow：血清与halo TM-halo-M44孵育后的流出；wash1-wash3：用500μL PBST洗涤halo TM-halo-M44；elution：90μL的200mM glycine pH2.8洗脱目的抗体(EP管提前添加10μL 1M Tris，pH9.0缓冲液以保护抗体)。 f: 12% Tricine-SDS-PAGE detection uses newly prepared halo TM -halo-M44 to separate and purify M44 related proteins from serum. Input: serum after 2 immunizations; flow: flow out of serum after incubation with halo TM -halo-M44; wash1-wash3: wash halo TM -halo-M44 with 500 μL PBST; elution: 90 μL of 200mM glycine pH2.8 elution Antibody of interest (add 10μL of 1M Tris, pH9.0 buffer in advance to EP tube to protect the antibody).
其中：halo-GFP或halo-M44与Promega halo TM的共价交联：250μL的halo-int N-gb1核酸序列如SEQ ID No.15所示)(10μM)与gb1-int C-GFP/M44(20μM)室温孵育4小时后，加入用PBS平衡好的50μL Promega halo TMbeads室温摇床混合半个小时。而后离心弃上清，再用500μL PBS洗涤三次即可完成halo TM-halo-GFP/M44的制备。最后用500μL PBS保存halo TM-halo-GFP/M44，4℃放置。 Among them: covalent cross-linking of halo-GFP or halo-M44 and Promega halo TM : 250 μL of halo-int N -gb1 nucleic acid sequence shown in SEQ ID No. 15 (10 μM) and gb1-int C -GFP/M44 (20μM) After incubating for 4 hours at room temperature, add 50μL Promega halo TM beads equilibrated with PBS on a shaker at room temperature for half an hour. Then the supernatant was discarded by centrifugation, and then washed three times with 500 μL PBS to complete the preparation of halo TM -halo-GFP/M44. Finally, store halo TM -halo-GFP/M44 with 500 μL PBS, and place at 4°C.
7.基于抗原-抗体相互作用纯化目的抗体：弃halo TM-halo-GFP/M44的PBS保存液，加入12,000rpm、4℃离心10分钟的血清，4℃摇床混匀1小时后离心弃上清。加入1ml的PBST洗涤3次除去非特定结合蛋白或抗体，最后加入90μL的200mM glycine pH2.8洗脱目的抗体。在新的EP管中加入10μL 1M Tris，pH 9.0，而加入抗体洗脱液。halo TM-halo-GFP/M44可反复使用，4℃放置2个月仍稳定。 7. Purification of the target antibody based on antigen-antibody interaction: discard the halo TM -halo-GFP/M44 PBS storage solution, add 12,000 rpm, 4 ℃ centrifugation 10 minutes of serum, 4 ℃ shaker and mix for 1 hour, then centrifuge and discard. clear. Add 1ml of PBST to wash 3 times to remove non-specific binding protein or antibody, and finally add 90μL of 200mM glycine pH2.8 to elute the target antibody. Add 10 μL of 1M Tris, pH 9.0 to a new EP tube, and add the antibody eluate. Halo TM -halo-GFP/M44 can be used repeatedly, and it is stable even after 2 months at 4℃.
用western blotting(WB)分析新纯化的GFP和M44相关抗体的功能。用pLKO载体在293T细胞中过量表达GFP蛋白和M44蛋白，而后用新纯化的GFP和M44抗体检测到特异的GFP(a)和M44(b)条带，表明新制备的抗体可用于WB分析，参阅图10。在293T细胞中用pLKO载体过表达3xflag-GFP蛋白，用本公开制备的GFP抗体(鼠源anti-GFP)和flag抗体(兔源anti-flag)均能特异识别GFP蛋白。相应的二抗anti-mouse(带555nm荧光素标记)和anti-rabbit(带647nm荧光素标记)所发荧光与GFP蛋白本身的荧光位置高度重合，且强度相若。免疫荧光(immunofluorescence，IF)分析表明本公开制备的抗体特异性高，可用于IF检测，参阅图11。用MCMV(带有荧光标记的SMgfp)以病毒感染复数(multiplicity of infection，MOI)为1感染MEF10.1细胞，24小时IF分析发现本公开制备的M44相关抗体可识别病毒的复制中心(M44为MCMV复制中心marker)，参阅图12。综合而言，本公开制备的抗体可用于WB和IF分析。Western blotting (WB) was used to analyze the functions of the newly purified GFP and M44-related antibodies. The pLKO vector was used to overexpress GFP protein and M44 protein in 293T cells, and then specific GFP(a) and M44(b) bands were detected with newly purified GFP and M44 antibodies, indicating that the newly prepared antibody can be used for WB analysis. Refer to Figure 10. The pLKO vector was used to overexpress 3xflag-GFP protein in 293T cells, and both the GFP antibody (mouse anti-GFP) and flag antibody (rabbit anti-flag) prepared by the present disclosure could specifically recognize the GFP protein. The fluorescence of the corresponding secondary antibodies anti-mouse (labeled with 555nm fluorescein) and anti-rabbit (labeled with 647nm fluorescein) highly coincides with the fluorescence position of the GFP protein itself, and the intensity is similar. Immunofluorescence (IF) analysis shows that the antibodies prepared in the present disclosure have high specificity and can be used for IF detection, see FIG. 11. MCMV (SMgfp with a fluorescent label) was used to infect MEF10.1 cells with a multiplicity of infection (MOI) of 1. A 24-hour IF analysis revealed that the M44-related antibodies prepared in the present disclosure can recognize the replication center of the virus (M44 is MCMV replication center marker), refer to Figure 12. In summary, the antibodies prepared in the present disclosure can be used for WB and IF analysis.
尽管已经示出和描述了本公开的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本公开的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本公开的范围由所附权利要求及其等同物限定。Although the embodiments of the present disclosure have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, and substitutions can be made to these embodiments without departing from the principle and spirit of the present disclosure. And variations, the scope of the present disclosure is defined by the appended claims and their equivalents.
实施例4Example 4
步骤1：由人源的重链铁蛋白(human heavy chain of human ferrtin，简写HFT)的N端与接头gbl-intein的C端融合形成重组蛋白gbl-intein c-ferrtin后自装配成二十四聚体构成直径为18nm的纳米颗粒。 Step 1: The recombinant protein gbl-intein c- ferrtin is formed by fusing the N-terminus of human heavy chain of human ferrtin (HFT) with the C-terminus of the linker gbl-intein and then self-assembled into twenty-four The aggregates constitute nanoparticles with a diameter of 18 nm.
步骤2：SP70的C端融合内含肽的N端形成重组蛋白，内含肽的N端特异性识别并切除暴露在所述纳米颗粒表面的铁蛋白；30μM外源蛋白与30μM铁蛋白共价交联，生成纳米制剂。Step 2: The C-terminus of SP70 is fused with the N-terminus of the intein to form a recombinant protein. The N-terminus of the intein specifically recognizes and excises the ferritin exposed on the surface of the nanoparticle; 30μM foreign protein and 30μM ferritin are covalently Cross-link to generate nano-formulations.
实施例5Example 5
步骤1：由强烈火球菌的重链铁蛋白(pyrococcus furiosus heavy chain of human ferrtin，简写PFT)的N端与接头gbl-intein的C端融合形成重组蛋白gbl-intein c-ferrtin后自装配成二十四聚体构成直径为18nm的纳米颗粒。 Step 1: Fuse the N-terminus of the pyrococcus furiosus heavy chain of human ferrtin (PFT) and the C-terminus of the linker gbl-intein to form a recombinant protein gbl-intein c- ferrtin and then self-assemble into two Tetrapamers constitute nanoparticles with a diameter of 18 nm.
步骤2：鞭毛蛋白的C端融合内含肽的N端形成重组蛋白，内含肽的N端特异性识别并切除暴露在所述纳米颗粒表面的铁蛋白；60μM外源蛋白与30μM铁蛋白共价交联，生成纳米制剂。Step 2: The C-terminus of flagellin is fused with the N-terminus of the intein to form a recombinant protein, and the N-terminus of the intein specifically recognizes and excises the ferritin exposed on the surface of the nanoparticle; 60μM foreign protein and 30μM ferritin Valence cross-linking to generate nano formulations.
实施例6Example 6
步骤1：由人源的重链铁蛋白的N端与接头gbl-intein的C端融合形成重组蛋白gbl-intein c-ferrtin后自装配成二十四聚体构成直径为18nm的纳米颗粒。 Step 1: Fusion of the N-terminus of the human heavy chain ferritin and the C-terminus of the linker gbl-intein to form a recombinant protein gbl-intein c- ferrtin and then self-assemble into twenty-tetramers to form nanoparticles with a diameter of 18nm.
步骤2：将同物质的量的CpG和流感病毒血凝素茎部区的C端同时融合内含肽的N端形成重组蛋白，内含肽的N端特异性识别并切除暴露在所述纳米颗粒表面的铁蛋白；45μM外源蛋白与30μM铁蛋白共价交联，生成纳米制剂。Step 2: The same amount of CpG and the C-terminus of the influenza virus hemagglutinin stem region are simultaneously fused to the N-terminus of the intein to form a recombinant protein, and the N-terminus of the intein is specifically recognized and excised and exposed to the nano Ferritin on the surface of the particles; 45μM foreign protein is covalently cross-linked with 30μM ferritin to generate a nanoformulation.
本公开的实验及实施例中涉及以下蛋白质、多肽的基因合成在上海吐露港生物科技有限公司，引物合成在南京金斯瑞生物科技有限公司。The experiments and examples of the present disclosure involve the gene synthesis of the following proteins and polypeptides in Shanghai Tolo Harbor Biotechnology Co., Ltd., and primer synthesis in Nanjing GenScript Biotechnology Co., Ltd.
GFPGFP SEQ ID NO.17SEQ ID NO.17 strep2strep2 SEQ ID NO.18SEQ ID NO.18
gp41-1-intein C(简称intein C) gp41-1-intein C (referred to as the intein C) SEQ ID NO.19SEQ ID NO.19 gp41-1-intein N，(简称intein N) gp41-1-intein N , (abbreviated as intein N ) SEQ ID NO.20SEQ ID NO.20
SP70SP70 SEQ ID NO.21SEQ ID NO.21 gb1gb1 SEQ ID NO.22SEQ ID NO.22
RhizavidinRhizavidin SEQ ID NO.23SEQ ID NO.23 CBLBCBLB SEQ ID NO.24SEQ ID NO.24
H1HA10H1HA10 SEQ ID NO.25SEQ ID NO.25 HFTHFT SEQ ID NO.26SEQ ID NO.26
PFTPFT SEQ ID NO.27SEQ ID NO.27 GFP-intein N-gb1 GFP-intein N -gb1 SEQ ID NO.28SEQ ID NO.28
strep2-gp41-1-intein N-gb1 strep2-gp41-1-intein N -gb1 SEQ ID NO.29SEQ ID NO.29 SP70-gp41-1-intein N-gb1 SP70-gp41-1-intein N -gb1 SEQ ID NO.30SEQ ID NO.30
rhizavidin-gp41-1-intein N-gb1 rhizavidin-gp41-1-intein N -gb1 SEQ ID NO.31SEQ ID NO.31 CBLB-gp41-1-intein N CBLB-gp41-1-intein N SEQ ID NO.32SEQ ID NO.32
SP70-CBLB-gp41-1-intein N SP70-CBLB-gp41-1-intein N SEQ ID NO.33SEQ ID NO.33 H1HA10-gp41-1-intein N-gb1 H1HA10-gp41-1-intein N -gb1 SEQ ID NO.34SEQ ID NO.34
gb1-gp41-1-intein C-HFT gb1-gp41-1-intein C -HFT SEQ ID NO.35SEQ ID NO.35 gb1-gp41-1-intein C-PFT gb1-gp41-1-intein C -PFT SEQ ID NO.36SEQ ID NO.36
其中，HFT为人源的重链铁蛋白(human heavy chain of human ferrtin)，PFT为强烈火球菌的重链铁蛋白(pyrococcus furiosus heavy chain of human ferrtin)。Among them, HFT is a human heavy chain of human ferrtin, and PFT is a heavy chain ferritin of Pyrococcus furiosus (pyrococcus furiosus heavy chain of human ferrtin).
本公开中GFP-intein N-gb1、strep2-gp41-1-intein N-gb1、SP70-gp41-1-intein N-gb1、rhizavidin-gp41-1-int N-gb1、CBLB-gp41-1-intein N、SP70-CBLB-gp41-1-intein N、H1HA10-gp41-1-intein N-gb1为intein N相关的“货物”融合蛋白。“货物”融合蛋白的基因通过overlap PCR方法扩增目的基因，使用的5’引物和3’引物分别带有NcoI和XhoI酶切位点。融合基因经酶切后插入相同酶切的线性化pET28a载体，转化至大肠杆菌JM109进行重组克隆构建。gb1-gp41-1-intein C-HFT、gb1-gp41-1-intein C-PFT为intein C相关的“载体”融合蛋白。“载体”融合蛋白的基因亦通过overlap PCR方法扩增目的基因，5’引物和3’引物分别带有EcoRI和XhoI酶切位点。酶切后的片段插入pET28a线性化载体后，转化大肠杆菌JM109。上述重组基因经测序正确后，提取质粒转化至大肠杆菌BL21(DE3)plysS进行蛋白表达。挑取单克隆菌株，接种至20ml的卡纳氯霉素双抗LB培养基中，37℃摇床过夜培养。第二天1:25接种至500ml的卡纳氯霉素双抗的LB培养基，37℃、250rpm培养至OD为0.6-0.8时，加入0.2mM IPTG进行诱导。25℃、250rpm诱导5h后，4000rpm、4℃、30分钟离心收菌。弃上清，菌可用于下一步纯化或-80℃冻存备用。 In this disclosure, GFP-intein N -gb1, strep2-gp41-1-intein N -gb1, SP70-gp41-1-intein N -gb1, rhizavidin-gp41-1-int N -gb1, CBLB-gp41-1-intein N , SP70-CBLB-gp41-1-intein N , H1HA10-gp41-1-intein N- gb1 are intein N- related "cargo" fusion proteins. The gene of the "cargo" fusion protein is amplified by the overlap PCR method, and the 5'and 3'primers used have NcoI and XhoI restriction sites respectively. The fusion gene was digested and inserted into the linearized pET28a vector with the same digestion, and transformed into E. coli JM109 for recombinant cloning construction. gb1-gp41-1-intein C- HFT and gb1-gp41-1-intein C- PFT are intein C- related "carrier" fusion proteins. The gene of the "vector" fusion protein is also amplified by the overlap PCR method. The 5'primer and 3'primer have EcoRI and XhoI restriction sites respectively. After the digested fragment was inserted into the pET28a linearization vector, it was transformed into E. coli JM109. After the above recombinant gene was sequenced correctly, the plasmid was extracted and transformed into E. coli BL21(DE3) plysS for protein expression. Pick a single monoclonal strain, inoculate it into 20ml of kanachloramphenicol bi-anti-LB medium, and cultivate overnight at 37°C on a shaker. On the second day, 1:25 inoculated into 500 ml of LB medium with kanachloramphenicol bi-antibody, cultured at 37°C and 250 rpm to an OD of 0.6-0.8, then 0.2mM IPTG was added for induction. After induction at 25°C and 250 rpm for 5 hours, the bacteria were harvested by centrifugation at 4000 rpm, 4°C, and 30 minutes. The supernatant is discarded and the bacteria can be used for the next purification or frozen at -80°C for later use.
本公开重组“载体”融合蛋白基因使用的引物及“货物”融合蛋白的基因使用的引物详见表1：The primers used in the recombinant "vector" fusion protein gene of the present disclosure and the primers used in the "cargo" fusion protein gene are shown in Table 1:
表1：Table 1:
Figure PCTCN2020091636-appb-000001
Figure PCTCN2020091636-appb-000001
Figure PCTCN2020091636-appb-000002
Figure PCTCN2020091636-appb-000002
注：本公开中使用的引物，用于制备不同功能的纳米器件。Note: The primers used in this disclosure are used to prepare nanodevices with different functions.
本公开涉及的抗原或佐剂蛋白纯化：所有的蛋白均采用Ni 2+亲和层析的方法进行纯化。不同的重组蛋白，因其性质不同，溶解度不同，采用的纯化方法略有差异。GFP-intein N-gb1、strep2-intein N-gb1、SP70-intein N-gb1、H1HA10-inteinN-gb1和rhizavidin-inteinN-gb1为可溶性蛋白，细菌裂解液上清液可直接加入Ni 2+树脂中开展亲和层析纯化。30ml NT buffer重悬后裂解细菌的上清，与1ml的树脂在4℃摇床上混匀30分钟。而后，非特异性结合蛋白随液体以重力方式流出。接着，加入含10mM、20mM、40mM咪唑的20ml NT buffer洗涤树脂，去掉非特异性结合蛋白。而后，加入500mM咪唑的NT buffer洗脱。最后，洗脱产物在1L的NT buffer中4℃透析过夜。CBLB-intein N和SP70-CBLB-intein N大部分形成沉淀，沉淀用含2M尿素的NT buffer重悬后，细菌裂解液上清液用可溶蛋白纯化的方法纯化。CBLB-intein N和SP70-CBLB-intein N大部分形成包涵体沉淀，可用含2M尿素的NT buffer重悬后，重悬液离心后上清用可溶蛋白纯化的方法纯化。 The antigen or adjuvant protein purification involved in the present disclosure: all proteins are purified by Ni 2+ affinity chromatography. Different recombinant proteins have slightly different purification methods because of their different properties and solubility. GFP-intein N- gb1, strep2-intein N- gb1, SP70-intein N -gb1, H1HA10-inteinN-gb1 and rhizavidin-inteinN-gb1 are soluble proteins, the supernatant of bacterial lysate can be directly added to Ni 2+ resin Carry out affinity chromatography purification. After resuspending in 30ml NT buffer, the bacterial supernatant was lysed and mixed with 1ml resin on a shaker at 4°C for 30 minutes. Then, the non-specific binding protein flows out with the liquid gravity. Next, add 20ml NT buffer containing 10mM, 20mM, 40mM imidazole to wash the resin to remove non-specific binding proteins. Then, 500 mM imidazole was added to the NT buffer for elution. Finally, the eluted product was dialyzed overnight at 4°C in 1L NT buffer. Most of the CBLB-intein N and SP70-CBLB-intein N formed a precipitate. After the precipitate was resuspended in NT buffer containing 2M urea, the supernatant of the bacterial lysate was purified by soluble protein purification. Most of CBLB-intein N and SP70-CBLB-intein N form inclusion body precipitates, which can be resuspended in NT buffer containing 2M urea, and the supernatant after centrifugation of the resuspension is purified by soluble protein purification.
实验例1纳米载体模块平台构建Experimental example 1 Nano-carrier module platform construction
①内含肽(inteinC)与HFT进行基因融合，获得重组蛋白inteinC-ferrtin(图14a中标记为1)。① Gene fusion of intein (inteinC) and HFT to obtain recombinant protein inteinC-ferrtin (marked as 1 in Figure 14a).
②内含肽(intein)引入链球菌G蛋白B1结构域标签(gb1)构建接头gb1-inteinC(图13中示为A)。重链铁蛋白(ferrti，图13中示为C)的N端与接头gb1-intein C进行基因融合构成重组蛋白gb1-intein C-ferrtin。分别以HFT和PFT构建重组蛋白gb1-intein C-HFT(图14a-14b中标记为2)和gb1-intein C-PFT(图14a-14b中标记为3)。在图14等本申请的各图中，本公开中所有UV profile of gel filtration的横坐标代表洗脱体积，纵坐标代表紫外光A280nm处的吸光度变化。 ②The intein (intein) was introduced into the Streptococcus G protein B1 domain tag (gb1) to construct the linker gb1-inteinC (shown as A in Figure 13). The N-terminus of heavy chain ferritin (ferrti, shown as C in Figure 13) is genetically fused with the linker gb1-intein C to form the recombinant protein gb1-intein C- ferrtin. The recombinant proteins gb1-intein C- HFT (marked as 2 in Figure 14a-14b) and gb1-intein C- PFT (marked as 3 in Figure 14a-14b) were constructed with HFT and PFT, respectively. In Figure 14 and other figures of this application, the abscissa of all UV profile of gel filtration in this disclosure represents the elution volume, and the ordinate represents the change in absorbance at A280 nm of ultraviolet light.
取上述三种重组蛋白细菌裂解进行Tricine-SDS-PAGE电泳，结果如图14a所示，其中，control：pET28a载体空白对照菌裂解液，1：intein C-ferrtin，2：gb1-intein C-HFT，3：gb1-intein C-PFT，all：细菌裂解液，up：上清液。从图14a可见，35kDa处电泳细菌裂解液有明显条带，但相应位置上清中几乎不可见蛋白条带，表明重组蛋白intein C-ferrtin形成包涵体。35kDa-40kDa处电泳带非常明显，重组蛋白 gb1-intein C-HFT和gb1-intein C-PFT表达量均显著增高，且可溶性高。由结果可见，引入gb1能够极大地提高重链铁蛋白在细胞内的表达并改善其可溶性，有利于形成规模性制备，故重组蛋白gb1-intein C-ferrtin纳米颗粒具有用于制备纳米载体平台的可行性。 The above three recombinant proteins were lysed by bacteria for Tricine-SDS-PAGE electrophoresis. The results are shown in Figure 14a, where control: pET28a vector blank control bacterial lysate, 1: intein C -ferrtin, 2: gb1-intein C -HFT , 3: gb1-intein C -PFT, all: bacterial lysate, up: supernatant. It can be seen from Figure 14a that there are obvious bands in the electrophoresis bacterial lysate at 35kDa, but there are almost no protein bands in the supernatant at the corresponding position, indicating that the recombinant protein intein C- ferrtin forms inclusion bodies. The electrophoresis band at 35kDa-40kDa is very obvious, and the expression of recombinant proteins gb1-intein C- HFT and gb1-intein C- PFT are both significantly increased, and the solubility is high. It can be seen from the results that the introduction of gb1 can greatly increase the expression of heavy chain ferritin in cells and improve its solubility, which is conducive to the formation of large-scale production. Therefore, the recombinant protein gb1-intein C- ferrtin nanoparticles can be used to prepare nanocarrier platforms. feasibility.
用30ml的50mM Tris，pH 7.5，150mM NaCl(NT buffer)重悬500ml的LB培养细菌，而后超声破碎。超声结束后，12000rpm、4℃、30分钟离心，上清转移至50ml的离心管中。加入0.15g/ml的硫酸铵固体，在4℃摇床混匀15分钟后，12000rpm、4℃、30分钟离心。弃上清，得硫酸铵沉淀产物(图14b-14c中标记为pellet)。用10ml的NT buffer重悬硫酸铵沉淀产物，在1L的NT buff中4℃透析过夜。BCA测得gb1-intein C-HFT和gb1-intein C-PFT浓度分别为10mg/ml和5mg/ml。硫酸铵沉淀产物用50mM pH8.0 Tris及150mM Nacl(NT buffer)重悬后，在上海闪谱生物科技有限公司ClearFirst-3000型蛋白纯化系统上利用superpose 6 Increase凝胶过滤分离纯化，并进行Tricine-SDS-PAGE进行电泳观察纯度，纯化结果见图14b-14d，其中，图14b为gb1-intein C-HFT和gb1-intein C-PFT经硫酸铵沉淀后的电泳图，其中，up：上清液，pellet：硫酸铵沉淀产物；图14c为gb1-intein C-HFT纯化后的电泳图，其中，pellet：硫酸铵富集沉淀重悬产物，Purification of gb1-intein C-HFT by gel filtration：凝胶过滤方法纯化gb1-intein C-HFT，gel filtration of gb1-intein C-HFT：凝胶过滤纯化gb1-intein C-HFT，9-17：分管收集凝胶过滤纯化gb1-intein C-HFT样品的编号；图14d为凝胶过滤纯化gb1-intein C-HFT凝胶的UV图，检测波长为280nm，箭头所指的是gb1-intein C-HFT的分布位置。 Resuspend 500 ml of LB culture bacteria with 30 ml of 50 mM Tris, pH 7.5, 150 mM NaCl (NT buffer), and then ultrasonically disrupt. After sonication, centrifuge at 12000 rpm, 4°C, 30 minutes, and transfer the supernatant to a 50 ml centrifuge tube. Add 0.15g/ml ammonium sulfate solid, mix well on a shaker at 4°C for 15 minutes, and centrifuge at 12000rpm, 4°C for 30 minutes. The supernatant was discarded to obtain the ammonium sulfate precipitation product (labeled pellet in Figure 14b-14c). Resuspend the ammonium sulfate precipitation product in 10ml NT buffer and dialyze it overnight at 4°C in 1L NT buff. The concentrations of gb1-intein C- HFT and gb1-intein C- PFT measured by BCA were 10mg/ml and 5mg/ml, respectively. After the ammonium sulfate precipitated product was resuspended in 50mM pH8.0 Tris and 150mM Nacl (NT buffer), it was separated and purified by superpose 6 Increase gel filtration on the ClearFirst-3000 protein purification system of Shanghai Shanpu Biotechnology Co., Ltd., and then Tricine -SDS-PAGE was performed to observe the purity by electrophoresis. The purification results are shown in Figures 14b-14d. Figure 14b is the electrophoresis diagram of gb1-intein C -HFT and gb1-intein C -PFT after precipitation with ammonium sulfate. Up: supernatant Solution, pellet: ammonium sulfate precipitation product; Figure 14c shows the electrophoresis after purification of gb1-intein C- HFT, in which pellet: ammonium sulfate enriched precipitation resuspended product, Purification of gb1-intein C- HFT by gel filtration: gel filtration Gel filtration method to purify gb1-intein C- HFT, gel filtration of gb1-intein C- HFT: Gel filtration to purify gb1-intein C- HFT, 9-17: Separate collection of gb1-intein C- HFT samples Number; Figure 14d is the UV image of the gel filtration purified gb1-intein C- HFT gel, the detection wavelength is 280nm, and the arrow points to the distribution position of gb1-intein C- HFT.
通过透射电镜(transmission electron microscope，TEM)观察纯化后的重组蛋白gb1-intein C-HFT和gb1-intein C-PFT，透射电镜放大倍数为110k，标尺长度为20nm。结果详见图14e及14f，其中，图14e为TEM检测纯化的gb1-intein C-HFT的电镜图(负染制样)；图14f为TEM检测纯化的gb1-intein C-PFT的电镜图；图14e和图14f显示gb1-intein C-HFT和gb1-intein C-PFT均以纳米颗粒形式存在，本公开中透射电镜观察负染样品所用标尺均代表20nm，箭头标示的为纳米颗粒。 The purified recombinant proteins gb1-intein C- HFT and gb1-intein C- PFT were observed by transmission electron microscope (TEM), the magnification of the transmission electron microscope was 110k, and the length of the ruler was 20nm. The results are shown in Figs. 14e and 14f. Fig. 14e is the electron micrograph of the purified gb1-intein C- HFT by TEM (negative staining preparation); Fig. 14f is the electron micrograph of the purified gb1-intein C- PFT by TEM; Fig. 14e and Fig. 14f show that both gb1-intein C- HFT and gb1-intein C- PFT exist in the form of nanoparticles. In the present disclosure, the ruler used to observe negatively stained samples by transmission electron microscopy all represents 20nm, and the arrow indicates the nanoparticle.
两种重组蛋白均以10-20nm的球形纳米颗粒形式存在，可见重组蛋白gb1-intein C-ferrtin通过分子自组装形成纳米颗粒。经测定，重组蛋白gb1-intein C-HFT和gb1-intein C-PFT均能以24单体自组装成直径为18nm纳米颗粒。 Both recombinant proteins exist in the form of 10-20nm spherical nanoparticles. It can be seen that the recombinant protein gb1-intein C- ferrtin forms nanoparticles through molecular self-assembly. It is determined that the recombinant proteins gb1-intein C- HFT and gb1-intein C- PFT can self-assemble into nanoparticles with a diameter of 18nm with 24 monomers.
实验例2纳米载体模块平台可与外源蛋白高效共价偶联Experimental Example 2 The nanocarrier module platform can be efficiently covalently coupled with foreign proteins
以实施例4构建的重组蛋白gb1-intein C-HFT纳米颗粒为例，分别与外源蛋白GFP蛋白和strep2多肽标签(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys)进行内含肽介导蛋白剪接修饰，观察该纳米颗粒的稳定性。 Taking the recombinant protein gb1-intein C- HFT nanoparticles constructed in Example 4 as an example, they were respectively contained with the exogenous protein GFP protein and strep2 polypeptide tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) Peptide-mediated protein splicing modification was performed to observe the stability of the nanoparticle.
1.GFP蛋白和strep2多肽标签的C端分别通过基因融合引入接头intein N-gb1，分别构成GFP-intein N-gb1和strep2-intein N-gb1。 1. The C ends of the GFP protein and strep2 polypeptide tags were introduced into the linker intein N- gb1 through gene fusion, respectively, to form GFP-intein N- gb1 and strep2-intein N- gb1.
2.取2μl浓度为10mg/ml的重组蛋白gb1-intein C-HFT母液加入DTT中，在室温下1小时内，在DTT中逐渐增加浓度为3mg/ml的GFP-intein N-gb1蛋白母液或5mg/ml的strep2-intein N-gb1蛋白母液，以3μl、6μl、9μl、12μl、15μl、18μl、21μl、24μl递增添加，并观察目的产物GFP-HFT或strep2-HFT的量。反应条件：反应体系总体积为30μl，2mM DTT，室温1小时。然后分别进行考马斯亮蓝法检测(CB assay)以及western blotting检测(WB assay) 2. Take 2μl of recombinant protein gb1-intein C- HFT mother solution with a concentration of 10mg/ml and add it to DTT, and gradually increase the concentration of 3mg/ml GFP-intein N- gb1 protein mother solution in DTT within 1 hour at room temperature. The 5mg/ml strep2-intein N- gb1 protein mother solution was added in increments of 3μl, 6μl, 9μl, 12μl, 15μl, 18μl, 21μl, 24μl, and the amount of the target product GFP-HFT or strep2-HFT was observed. Reaction conditions: The total volume of the reaction system is 30μl, 2mM DTT, room temperature for 1 hour. Then perform Coomassie brilliant blue detection (CB assay) and western blotting detection (WB assay)
3.检测结果3. Test results
如图15a及15c所示，随着GFP蛋白和strep2标签的量的逐渐增加，会促进目的产物GFP-HFT(图15a)和strep2-HFT(图15c)的量增加。固定gb1-intein C-HFT的量，增加GFP-intein N-gb1或strep2-intein N-gb1反应浓度，反应底物gb1-intein C-HFT与GFP-intein N-gb1或strep2-intein N-gb1的摩尔比例接近1:1时反应达最大程度，内含肽自剪切效率接近最高，未被剪切gb1-intein C-HFT几乎无残留。反应完成后，用GFP标签抗体、strep2标签抗体和HFT抗体分别检测反应体系中各反应底物和目的产物的变化。Western-blotting检测数据表明反式剪切效率非常高，反应彻底(图15b和15d)。可见，GFP及strep2均能被高效、特异地共价交联至HFT蛋白上。 As shown in Figures 15a and 15c, with the gradual increase in the amount of GFP protein and strep2 tag, the target products GFP-HFT (Figure 15a) and strep2-HFT (Figure 15c) will increase. Fix the amount of gb1-intein C -HFT, increase the reaction concentration of GFP-intein N -gb1 or strep2-intein N -gb1, the reaction substrate gb1-intein C -HFT and GFP-intein N -gb1 or strep2-intein N -gb1 The reaction is maximized when the molar ratio is close to 1:1, the self-cleavage efficiency of the intein is near the highest, and the uncut gb1-intein C- HFT has almost no residue. After the reaction is completed, the GFP-labeled antibody, strep2-labeled antibody, and HFT antibody are used to detect changes in the reaction substrate and target product in the reaction system. Western-blotting detection data show that the trans-shearing efficiency is very high and the reaction is complete (Figure 15b and 15d). It can be seen that both GFP and strep2 can be efficiently and specifically covalently cross-linked to the HFT protein.
反应结束后，所得目的产物在上海闪谱生物科技有限公司ClearFirst-3000型蛋白纯化系统上利用superpose 6 increase开展凝胶过滤加以纯化。结果详见图16，其中，图16a为凝胶过滤纯化GFP-HFT的考马斯亮蓝染色法电泳分析图；图16b为凝胶过滤纯化GFP-HFT的UV曲线图；图16c为TEM检测纯化的GFP-HFT的电镜图(负染制样)；图16d为凝胶过滤纯化strep2-HFT的电泳分析图(考马斯亮蓝 染色)；图16e为凝胶过滤纯化strep2-HFT的UV曲线图；图16f为TEM检测纯化的strep2-HFT的电镜图(负染制样)；before：内含肽自剪切前，after：内含肽自剪切后，图16a 8-19及16d中的9-20：分管收集凝胶过滤纯化的目的产物样品的编号。After the reaction, the obtained target product is purified by superpose 6 increase on the ClearFirst-3000 protein purification system of Shanghai Shanpu Biotechnology Co., Ltd. to carry out gel filtration. The results are shown in Fig. 16. Fig. 16a is the Coomassie brilliant blue electrophoresis analysis diagram of gel filtration purified GFP-HFT; Fig. 16b is the UV curve diagram of gel filtration purified GFP-HFT; Fig. 16c is the TEM detection and purification The electron microscope image of GFP-HFT (negative staining); Figure 16d is the electrophoresis analysis image of gel filtration purified strep2-HFT (Coomassie brilliant blue staining); Figure 16e is the UV curve of gel filtration purified strep2-HFT; 16f is the electron microscope image of the purified strep2-HFT detected by TEM (negative staining preparation); before: before the intein self-cleavage, after: after the intein self-cleavage, Figure 16a 8-19 and 16d 9- 20: Separately collect the number of the target product sample purified by gel filtration.
如图16a，16b所示，分子量极为接近的蛋白GFP-HFT和GFP-intein N-gb1可以很好地分开，且蛋白GFP-HFT洗脱位置比GFP-intein N-gb1早，图16b中箭头所指为GFP-HFT的峰，显示该蛋白GFP-HFT以大分子聚合物的形式存在。图16d，16e所示，分子量极为接近的蛋白strep2-HFT和strep2-intein N-gb1也可以很好地分开，且蛋白strep2-HFT洗脱位置比strep2-intein N-gb1早，图16e中箭头所指为GFP-HFT的峰，显示该蛋白strep2-HFT以大分子聚合物的形式存在。将分离纯化的目的产物GFP-HFT和strep2-HFT通过透射电镜观察，GFP-HFT和strep2-HFT均以纳米颗粒的形式存在(图16c，16f)。综上，蛋白或多肽可高效剪切至HFT纳米颗粒，且不影响纳米颗粒稳定性，纳米载体模块平台可与外源蛋白高效共价偶联。 As shown in Figures 16a and 16b, the proteins GFP-HFT and GFP-intein N- gb1 with very similar molecular weights can be separated well, and the elution position of the protein GFP-HFT is earlier than GFP-intein N- gb1, the arrow in Figure 16b The peak of GFP-HFT indicates that the protein GFP-HFT exists as a macromolecular polymer. As shown in Figures 16d and 16e, the proteins strep2-HFT and strep2-intein N- gb1 with very similar molecular weights can also be separated well, and the elution position of the protein strep2-HFT is earlier than strep2-intein N- gb1, the arrow in Figure 16e The peak of GFP-HFT indicates that the protein strrep2-HFT exists in the form of a macromolecular polymer. The separated and purified target products GFP-HFT and strep2-HFT were observed by transmission electron microscope, and both GFP-HFT and strep2-HFT existed in the form of nanoparticles (Figure 16c, 16f). In summary, proteins or peptides can be efficiently sheared into HFT nanoparticles without affecting the stability of the nanoparticles, and the nanocarrier module platform can be efficiently covalently coupled with exogenous proteins.
实验例3纳米载体模块平台的多态性Experimental example 3 Polymorphism of nanocarrier module platform
以人肠道病毒71型(enterovirus 71)的SP70中和性表位为抗原模型，探讨不同的给药系统与佐剂对抗原免疫应答的影响。利用纳米载体模块技术允许多态性地修饰纳米颗粒的特点，制备了不同功能的纳米颗粒，为研究不同疫苗剂型对抗原免疫原性的影响，建立纳米佐剂、疫苗-佐剂共递送纳米制剂和纳米疫苗，详见图17。Taking human enterovirus 71 (enterovirus 71) SP70 neutralizing epitope as an antigen model, the effects of different drug delivery systems and adjuvants on antigen immune responses were discussed. Using nanocarrier module technology to allow polymorphic modification of the characteristics of nanoparticles, nanoparticles with different functions were prepared, in order to study the influence of different vaccine formulations on antigen immunogenicity, the establishment of nano adjuvants and vaccine-adjuvant co-delivery nano formulations And nano vaccines, see Figure 17 for details.
以CpG或鞭毛蛋白(flagellin)为佐剂分别制备相应制剂。鞭毛蛋白具体采用CBLB。CBLB是鞭毛蛋白的一个截短体，具备其佐剂功能。CpG or flagellin (flagellin) were used as adjuvants to prepare corresponding preparations. Flagellin specifically uses CBLB. CBLB is a truncated form of flagellin and has its adjuvant function.
纳米颗粒可高效负载多肽表位和鞭毛蛋白的分析图详见图18：其中，图18a为纳米疫苗SP70-HFT的凝胶过滤纯化电泳分析图；图18b为凝胶过滤纯化纳米疫苗SP70-HFT的UV曲线图；图18c为纳米疫苗SP70-HFT的透射电镜图；图18d为SP70与CBLB共递送纳米制剂SP70-CBLB-HFT的凝胶过滤纯化电泳分析图；图18e为凝胶过滤纯化共递送纳米制剂SP70-CBLB-HFT的UV曲线图；图18f为共递送纳米制剂SP70-CBLB-HFT的透射电镜图(负染制样)；图18g为纳米佐剂CBLB-HFT的凝胶过滤纯化电泳分析图；图18h为凝胶过滤纯化纳米佐剂CBLB-HFT的UV曲线图；图18i为纳米佐剂CBLB-HFT的透射电镜图(负染制样)；before：内含肽自剪切前，after：内含肽剪切后，图18a、18d及18g中的8-19：分管收集凝胶过滤纯化的目的产物样品的编号。The analysis diagram of the nanoparticle can efficiently load polypeptide epitopes and flagellin is shown in Figure 18: Figure 18a is the gel filtration purification electrophoresis analysis diagram of the nano vaccine SP70-HFT; Figure 18b is the gel filtration purification nano vaccine SP70-HFT Figure 18c is the transmission electron micrograph of the nano vaccine SP70-HFT; Figure 18d is the gel filtration purification electrophoresis analysis of the SP70 and CBLB co-delivered nano preparation SP70-CBLB-HFT; Figure 18e is the gel filtration purification The UV curve of the delivery nano-preparation SP70-CBLB-HFT; Figure 18f is the transmission electron microscope image of the co-delivery nano-preparation SP70-CBLB-HFT (negative staining); Figure 18g is the gel filtration purification of the nano adjuvant CBLB-HFT Electrophoresis analysis diagram; Figure 18h is the UV curve of the purified nano-adjuvant CBLB-HFT by gel filtration; Figure 18i is the transmission electron microscope image of the nano-adjuvant CBLB-HFT (prepared by negative staining); before: self-cleavage of intein Before, after: After the intein is cut, 8-19 in Figures 18a, 18d and 18g: collect the number of the target product sample purified by gel filtration.
1.纳米疫苗：①内含肽(intein)引入链球菌G蛋白B1结构域标签(gb1)构建接头gb1-intein C(图13中示为A)。人源重链铁蛋白HFT的N端与接头gb1-intein C进行基因融合构成重组蛋白gb1-intein C-HFT。24个重组蛋白gb1-intein C-HFT分子自组装成二十四聚体纳米颗粒。②SP70的C端引入intein N-gb1构成蛋白SP70-intein N-gb1。在2mM DTT溶液中，按摩尔比为1:1添加纳米颗粒和蛋白SP70-intein N-gb1，经蛋白剪接得到SP70-HFT纳米颗粒，即为纳米疫苗给药系统。 1. Nano vaccine: ① Intein is introduced into Streptococcus G protein B1 domain tag (gb1) to construct the linker gb1-intein C (shown as A in Figure 13). The N-terminal of human heavy chain ferritin HFT is genetically fused with the linker gb1-intein C to form the recombinant protein gb1-intein C- HFT. Twenty-four recombinant protein gb1-intein C- HFT molecules self-assembled into twenty-four polymer nanoparticles. ②Intein N- gb1 is introduced into the C-terminal of SP70 to form the protein SP70-intein N- gb1. In the 2mM DTT solution, the molar ratio is 1:1 by adding nanoparticles and protein SP70-intein N- gb1, and SP70-HFT nanoparticles are obtained by protein splicing, which is the nano vaccine delivery system.
2.纳米佐剂：2. Nano adjuvant:
2.1 CBLB的C端引入intein N构成蛋白CBLB-intein N，在2mM DTT溶液中，按HFT与CBLB摩尔比1:1添加纳米颗粒和CBLB-intein N，经蛋白质剪接获得CBLB-HFT，并自组装形成CBLB-HFT纳米颗粒。 2.1 Intein N is introduced into the C-end of CBLB to form the protein CBLB-intein N. In a 2mM DTT solution, nanoparticles and CBLB-intein N are added at a molar ratio of HFT to CBLB 1:1, and CBLB-HFT is obtained by protein splicing and self-assembly Form CBLB-HFT nanoparticles.
2.2 CpG不能直接与HFT共价连接，但生物素化的CpG可通过抗生物素-生物素的高亲结合作用，负载至纳米颗粒表面。生物素化的CpG可通过抗生物素-生物素的高亲结合作用，负载至纳米颗粒表面。抗生物素蛋白rhizavidin的C端引入intein N-gb1构成蛋白rhizavidin-intein N-gb1，在2mM DTT溶液中，添加纳米颗粒和rhizavidin-intein N-gb1，经蛋白质剪接获得rhizavidin-HFT，并自组装形成rhizavidin-HFT纳米颗粒。 2.2 CpG cannot be directly covalently linked to HFT, but biotinylated CpG can be loaded onto the surface of nanoparticles through the high affinity binding of avidin-biotin. Biotinylated CpG can be loaded onto the surface of nanoparticles through the high affinity binding of avidin-biotin. Intein N- gb1 is introduced into the C-terminus of avidin rhizavidin to form the protein rhizavidin-intein N- gb1. Nanoparticles and rhizavidin-intein N- gb1 are added to the 2mM DTT solution to obtain rhizavidin-HFT through protein splicing and self-assembly Form rhizavidin-HFT nanoparticles.
模块化纳米载体是否可高效负载CpG的分析图详见图19；其中，图19a为rhizavidin-HFT的凝胶过滤纯化电泳分析图；图19b为rhizavidin-HFT凝胶过滤纯化的UV曲线图；图19c为rhizavidin-HFT的透射电镜图(负染制样)；图19d为SP70-rhizavidin-HFT的凝胶过滤纯化电泳分析图；图19e为SP70-rhizavidin-HFT凝胶过滤纯化的UV曲线图；图19f为SP70-rhizavidin-HFT的透射电镜图(负染制样)；图19g为SP70-rhizavidin-HFT的二维类平均图；图19h为目的蛋白rhizavidin-HFT结合生物素化CpG电泳分析图；before：内含肽自剪切前，after：内含肽剪切后，图19a、19d及19g中的8-19编号： 分管收集凝胶过滤纯化的目的产物样品的编号。The analysis diagram of whether the modular nanocarrier can efficiently load CpG is shown in Figure 19; Figure 19a is the electrophoretic analysis diagram of rhizavidin-HFT gel filtration purification; Figure 19b is the UV curve diagram of rhizavidin-HFT gel filtration purification; 19c is the transmission electron microscope image of rhizavidin-HFT (negative staining); Figure 19d is the gel filtration purification electrophoresis analysis image of SP70-rhizavidin-HFT; Figure 19e is the UV curve of SP70-rhizavidin-HFT gel filtration purification; Figure 19f is the transmission electron microscope image of SP70-rhizavidin-HFT (negative staining); Figure 19g is the two-dimensional class average image of SP70-rhizavidin-HFT; Figure 19h is the electrophoresis analysis image of target protein rhizavidin-HFT combined with biotinylated CpG ; Before: before the intein self-cleavage, after: after the intein is cut, the numbers 8-19 in Figure 19a, 19d and 19g: the numbers of the target product samples collected by the gel filtration purification in separate tubes.
如图19a-b所示：rhizavidin可高效偶联至HFT，目的产物可用凝胶过滤加以纯化。凝胶过滤纯化的rhizavidin-HFT经透射电镜观察以球形纳米颗粒的形式存在(图19c)。且rhizavidin与SP70以1:5的摩尔比例偶联，亦不影响纳米颗粒结构，详见图19d-f。如图19g所示，二维类平均(2D-class average)分析，白色圆圈表示目的蛋白自装配成球形纳米尺寸的颗粒，圆圈中离散分布的白色亮斑为rhizavidin蛋白(电子密度比多肽大)，且亮斑平均数目与初始反应的rhizavidin-intein N-gb1和SP70-intein N-gb1的浓度一致。如图19h所示，目的产物rhizavidin-HFT与足量的生物素化的CpG结合后，蛋白条带迁移，揭示CpG被成功负载。 As shown in Figure 19a-b: rhizavidin can be efficiently coupled to HFT, and the target product can be purified by gel filtration. The rhizavidin-HFT purified by gel filtration was observed by transmission electron microscope in the form of spherical nanoparticles (Figure 19c). And rhizavidin and SP70 are coupled at a molar ratio of 1:5, and it does not affect the nanoparticle structure, see Figure 19d-f for details. As shown in Figure 19g, the 2D-class average analysis, the white circle indicates that the target protein self-assembles into spherical nano-sized particles, and the discretely distributed white spots in the circle are rhizavidin protein (the electron density is greater than the peptide) , And the average number of bright spots is consistent with the concentration of rhizavidin-intein N -gb1 and SP70-intein N -gb1 in the initial reaction. As shown in Figure 19h, after the target product rhizavidin-HFT was combined with sufficient biotinylated CpG, the protein band migrated, revealing that CpG was successfully loaded.
因此，生物素化的CpG可通过以下方法与纳米颗粒共价连接。如图18所示，①内含肽(intein)引入链球菌G蛋白B1结构域标签(gb1)构建接头gb1-intein C(图13中示为A)。人源重链铁蛋白HFT的N端与接头gb1-intein C进行基因融合构成重组蛋白gb1-intein C-HFT。24个重组蛋白gb1-intein C-HFT分子自组装成二十四聚体纳米颗粒。②rhizavidin的C端引入intein N-gb1构成蛋白rhizavidin-intein N-gb1。在2mM DTT溶液中，按摩尔比为1:1:1添加纳米颗粒、生物素化的CpG和蛋白rhizavidin-intein N-gb1，经蛋白剪接和抗生素蛋白与生物素化CpG高亲和相互作用得到CpG-HFT纳米颗粒，即为纳米佐剂给药系统。 Therefore, the biotinylated CpG can be covalently attached to the nanoparticle by the following method. As shown in Figure 18, ① the intein (intein) was introduced into the Streptococcus G protein B1 domain tag (gb1) to construct the linker gb1-intein C (shown as A in Figure 13). The N-terminal of human heavy chain ferritin HFT is genetically fused with the linker gb1-intein C to form the recombinant protein gb1-intein C- HFT. Twenty-four recombinant protein gb1-intein C- HFT molecules self-assembled into twenty-four polymer nanoparticles. ②Intein N- gb1 is introduced into the C-terminal of rhizavidin to form the protein rhizavidin-intein N- gb1. In 2mM DTT solution, the molar ratio is 1:1:1 by adding nanoparticles, biotinylated CpG and protein rhizavidin-intein N- gb1, obtained by protein splicing and the high affinity interaction between antibiotic protein and biotinylated CpG CpG-HFT nanoparticles are nano adjuvant drug delivery systems.
3.疫苗-佐剂共递送纳米制剂3. Vaccine-adjuvant co-delivery nanoformulation
3.1 CBLB与SP70基因融合成蛋白SP70-CBLB，CBLB与SP70的摩尔比为1:1。SP70-CBLB的CBLB的C端引入intein N构成蛋白SP70-CBLB-intein N，在2mM DTT溶液中，按HFT与SP70-CBLB摩尔比1:1添加纳米颗粒和SP70-CBLB-intein N，经蛋白质剪接获得SP70-CBLB-HFT，并自组装形成SP70-CBLB-HFT纳米颗粒。 3.1 The fusion of CBLB and SP70 genes into protein SP70-CBLB, the molar ratio of CBLB to SP70 is 1:1. Intein N is introduced into the C end of CBLB of SP70-CBLB to form the protein SP70-CBLB-intein N. In a 2mM DTT solution, add nanoparticles and SP70-CBLB-intein N at a molar ratio of HFT to SP70-CBLB 1:1. SP70-CBLB-HFT was spliced and self-assembled to form SP70-CBLB-HFT nanoparticles.
如图18a-b所示为Tricine-SDS-PAGE和UV曲线分析凝胶过滤分离纯化目的产物SP70-HFT的结果。结果显示：该方法可纯化较纯目的产物；before和after分别表示为：自剪切前后样品，数字8-19代表凝胶过滤纯化目的产物收集样品编号(1ml/管)。图18c：TEM分析目的产物SP70-HFT以纳米颗粒形式存在，箭头指示目的产物。图18d-f和图18g-i分别为目的产物SP70-CBLB-HFT和CBLB-HFT凝胶过滤法纯化及质量检测。图18a、18d和18g显示未被剪切的gb1-intein C-HFT条带几乎不可见，说明纳米颗粒的自剪切效率高。图18c、18f和18i显示三种目的产物均以纳米颗粒形式存在，表明本公开提供的纳米颗粒可通过自剪切与抗原、佐剂共价交联，从而赋予纳米颗粒不同的功能。 Figure 18a-b shows the results of Tricine-SDS-PAGE and UV curve analysis gel filtration separation and purification of the target product SP70-HFT. The results show that this method can purify a relatively pure target product; before and after are respectively denoted as: samples before and after shearing, and numbers 8-19 represent the sample number (1ml/tube) of the collected sample for gel filtration purification. Figure 18c: TEM analysis target product SP70-HFT exists in the form of nanoparticles, and the arrow indicates the target product. Figure 18d-f and Figure 18g-i are the purification and quality detection of the target product SP70-CBLB-HFT and CBLB-HFT by gel filtration, respectively. Figures 18a, 18d and 18g show that the unsheared gb1-intein C- HFT bands are almost invisible, indicating that the self-shearing efficiency of the nanoparticles is high. Figures 18c, 18f and 18i show that the three target products all exist in the form of nanoparticles, indicating that the nanoparticles provided in the present disclosure can be covalently cross-linked with antigens and adjuvants through self-shearing, thereby imparting different functions to the nanoparticles.
3.2如图18所示，①内含肽(intein)引入链球菌G蛋白B1结构域标签(gb1)构建接头gb1-intein C(图13中示为A)。人源重链铁蛋白HFT的N端与接头gb1-intein C进行基因融合构成重组蛋白gb1-intein C-HFT。24个重组蛋白gb1-intein C-HFT分子自组装成二十四聚体纳米颗粒。②SP70的C端引入intein N-gb1构成蛋白SP70-intein N-gb1。在2mM DTT溶液中，按摩尔比为1:1添加纳米颗粒和蛋白SP70-intein N-gb1，经蛋白剪接得到蛋白SP70-HFT。rhizavidin的C端引入intein N-gb1构成蛋白rhizavidin-intein N-gb1。在2mM DTT溶液中，添加纳米颗粒、生物素化的CpG、蛋白rhizavidin-intein N-gb1和蛋白SP70-intein N-gb1，经蛋白剪接得到SP70-CpG-HFT纳米颗粒，即为纳米共递送给药系统。其中，蛋白rhizavidin-intein N-gb1和蛋白SP70-intein N-gb1的摩尔比为1:5，纳米颗粒与蛋白rhizavidin-intein N-gb1和蛋白SP70-intein N-gb1的混合物的摩尔比为6:1：5，生物素化的CpG和蛋白rhizavidin-intein N-gb1的摩尔比为1:1。 3.2 As shown in Figure 18, ① the intein (intein) was introduced into the Streptococcus G protein B1 domain tag (gb1) to construct the linker gb1-intein C (shown as A in Figure 13). The N-terminal of human heavy chain ferritin HFT is genetically fused with the linker gb1-intein C to form the recombinant protein gb1-intein C- HFT. Twenty-four recombinant protein gb1-intein C- HFT molecules self-assembled into twenty-four polymer nanoparticles. ②Intein N- gb1 is introduced into the C-terminal of SP70 to form the protein SP70-intein N- gb1. Add nanoparticles and protein SP70-intein N- gb1 in a 2mM DTT solution at a molar ratio of 1:1, then protein SP70-HFT is obtained by protein splicing. Intein N- gb1 is introduced into the C-terminal of rhizavidin to form the protein rhizavidin-intein N- gb1. In 2mM DTT solution, add nanoparticles, biotinylated CpG, protein rhizavidin-intein N- gb1 and protein SP70-intein N- gb1, after protein splicing to obtain SP70-CpG-HFT nanoparticles, that is, nano co-delivery to Medicine system. Among them, the molar ratio of protein rhizavidin-intein N- gb1 and protein SP70-intein N- gb1 is 1:5, and the molar ratio of nanoparticles to the mixture of protein rhizavidin-intein N- gb1 and protein SP70-intein N- gb1 is 6. :1:5, the molar ratio of biotinylated CpG and protein rhizavidin-intein N- gb1 is 1:1.
实验例4不同的免疫制剂的免疫效果Experimental example 4 The immune effect of different immune preparations
1.建立小鼠试验组：SP70-CpG-HFT组使用摩尔比为1:1的0.4μg/只生物素化CpG和10μg/只SP70-rhizavidin-HFT冰浴混合30分钟后，免疫小鼠。SP70-HFT+CpG-HFT组使用纳米疫苗SP70-HFT与纳米佐剂CpG-HFT共同免疫小鼠，使用剂量分别为10μg/只和1.6μg/只。纳米疫苗SP70-HFT组：SP70-HFT免疫小鼠，使用剂量分别为10μg/只。SP70-HFT+CpG组：SP70-HFT和0.4μg/只生物素化CpG共同免疫小鼠。各组小鼠5只，3次接种，每次接种间隔2周。第三次免疫2周后眼眶采血开展免疫分析。1. Establish a mouse test group: SP70-CpG-HFT group used 0.4μg/bottle of biotinylated CpG and 10μg/bottle SP70-rhizavidin-HFT at a molar ratio of 1:1 to mix for 30 minutes in an ice bath to immunize mice. SP70-HFT+CpG-HFT group used nano vaccine SP70-HFT and nano adjuvant CpG-HFT to immunize mice together, using doses of 10μg/mouse and 1.6μg/mouse respectively. Nano-vaccine SP70-HFT group: SP70-HFT immunized mice with a dose of 10 μg/mouse. SP70-HFT+CpG group: SP70-HFT and 0.4μg/mouse of biotinylated CpG co-immunized mice. Five mice in each group were vaccinated 3 times with an interval of 2 weeks between each inoculation. Two weeks after the third immunization, blood was collected from the orbit for immune analysis.
表2：Table 2:
Figure PCTCN2020091636-appb-000003
Figure PCTCN2020091636-appb-000003
注：SP70-HFT：纳米疫苗；SP70-HFT+CpG：纳米疫苗和游离CpG混合剂；SP70-CpG-HFT：SP70和CpG共递送纳米制剂；SP70-HFT+CpG-HFT：纳米疫苗和纳米佐剂联合制剂。Note: SP70-HFT: Nano vaccine; SP70-HFT+CpG: Nano vaccine and free CpG mixture; SP70-CpG-HFT: SP70 and CpG co-delivery nano formulation; SP70-HFT+CpG-HFT: Nano vaccine and nano adjuvant Agent combination preparation.
不同剂型疫苗接种小鼠后，接种3次，每次间隔2周，第三次免疫2周后眼眶取血进行ELISA分析。After inoculating mice with different dosage forms, the mice were vaccinated 3 times with an interval of 2 weeks. Blood was taken from the orbit for ELISA analysis after 2 weeks of the third immunization.
如图20a所示，ELISA分析不同方式递送CpG佐剂诱导IgG滴度。低剂量游离的0.4μg/只CpG无明显佐剂效果，纳米佐剂CpG-HFT的助力效果明显，SP70-CpG-HFT共递送纳米制剂的佐剂效果最佳。可见，低剂量的0.4μg/只游离CpG佐剂效果不明显。但是，用纳米颗粒递送的纳米佐剂CpG-HFT及抗原-佐剂共递送纳米颗粒SP70-CpG-HFT佐剂效果显著，且共递送纳米制剂诱导B细胞免疫应答最高效。这是因为共递送纳米制剂最大程度保证抗原与佐剂定位至同一个抗原呈递细胞(antigen presenting cell，简称APC)，充分发挥佐剂的助力效应。使用ELISA检测不同亚型的IgG抗体滴度(1:1000)，IgG亚型分析结果显示，共递送纳米制剂SP70-CpG-HFT同时明显增强Th1和Th2型的IgG滴度，见表1，其中Th2型的IgG1占比例最高。As shown in Figure 20a, ELISA analysis of different ways to deliver CpG adjuvant induces IgG titers. The low-dose free 0.4μg/CpG has no obvious adjuvant effect, the nano adjuvant CpG-HFT has obvious boosting effect, and the adjuvant effect of SP70-CpG-HFT co-delivery nano formulation is the best. It can be seen that the low-dose 0.4μg/free CpG adjuvant has no obvious effect. However, the nano-adjuvant CpG-HFT delivered with nanoparticles and the antigen-adjuvant co-delivered nano-particle SP70-CpG-HFT adjuvant effect is significant, and the co-delivery of nano-formulations induces the most efficient B cell immune response. This is because the co-delivery of nanoformulations ensures that the antigen and the adjuvant are localized to the same antigen presenting cell (APC) to the fullest extent possible to fully exert the boosting effect of the adjuvant. ELISA was used to detect IgG antibody titers of different subtypes (1:1000). The IgG subtype analysis results showed that the co-delivery of the nanoformulation SP70-CpG-HFT also significantly enhanced the IgG titers of Th1 and Th2 types, as shown in Table 1. Th2 type IgG1 accounted for the highest proportion.
2.根据本实验例的第1点建立小鼠试验组(每组5只小鼠，接种3次，每次间隔2周)，分别为SP70-CBLB-HFT(10μg/只)组以及SP70-HFT(10μg/只)+CBLB-HFT(10μg/只)组，然后取血进行ELISA分析(第3次免疫两周后眼眶取血)。如图20b所示，SP70-CBLB-HFT诱导的抗体滴度低于SP70-HFT+CBLB-HFT。2. According to the first point of this experimental example, establish a mouse test group (5 mice in each group, vaccinated 3 times, 2 weeks apart), respectively SP70-CBLB-HFT (10μg/mouse) group and SP70- In the HFT (10μg/mouse) + CBLB-HFT (10μg/mouse) group, blood was taken for ELISA analysis (two weeks after the third immunization, blood was taken from the orbit). As shown in Figure 20b, the antibody titer induced by SP70-CBLB-HFT was lower than SP70-HFT+CBLB-HFT.
3.CpG作为佐剂，对B细胞免疫应答的助力效果优于鞭毛蛋白；对比共递送的CpG和鞭毛蛋白，发现CpG诱导抗体滴度更高；详见图20c。3. As an adjuvant, CpG has a better boosting effect on B cell immune response than flagellin; comparing co-delivered CpG and flagellin, it is found that CpG induces higher antibody titers; see Figure 20c for details.
4.分别取本实验例的第1～2点的各试验组小鼠的血清，以2倍为梯度系列稀释血清(50μl)，与100TCID50的EV71G082(50μl)在37℃的CO2培养箱中放置1h后，加入15000个人胚胎横纹肌肉瘤细胞(human embryo rhabdomyosarcoma cells，RD cells)。感染3天后观察CPE，统计中和抗体效价。如图20d所示，对抗体中和效价分析发现，SP70-CpG-HFT、SP70-HFT+CpG-HFT、SP70-CBLB-HFT以及SP70-HFT+CBLB-HFT的抗体效价均显著高于SP70-HFT，其中以SP70-CpG-HFT诱导的抗体效价最高。4. Take the sera of mice in each test group from points 1 to 2 of this experimental example, serially dilute the sera (50μl) in a 2-fold gradient, and place them in a CO2 incubator at 37°C with 100TCID50 EV71G082 (50μl) 1h later, 15,000 human embryo rhabdomyosarcoma cells (RD cells) were added. CPE was observed 3 days after infection, and the neutralizing antibody titer was counted. As shown in Figure 20d, the antibody neutralization titer analysis showed that the antibody titer of SP70-CpG-HFT, SP70-HFT+CpG-HFT, SP70-CBLB-HFT and SP70-HFT+CBLB-HFT were significantly higher than SP70-HFT, among them, the antibody titer induced by SP70-CpG-HFT is the highest.
综上，以SP70表位为模型分析不同疫苗剂型对免疫效果的影响分析图详见图20：其中，图20a-c为ELISA分析不同方式递送CpG佐剂以及CBLB诱导IgG滴度；图20a为不同方式递送CpG对SP70IgG抗体滴度的影响，与纳米疫苗SP70-HFT相比，纳米疫苗混合游离的CpG(SP70-HFT+CpG)无明显佐剂效果。但是纳米疫苗与纳米佐剂CpG-HFT混合制剂及SP70与CpG的共递送纳米制剂SP70-CpG-HFT可显著提高特异性抗体滴度。且共递送纳米制剂效果比纳米疫苗与纳米佐剂混合制剂诱导的抗体滴度更高。图20b揭示当CBLB为佐剂，共递送纳米制剂诱导特异抗体滴度高于纳米疫苗SP70-HFT和纳米佐剂CBLB-HFT混合制剂。图20c表明CpG的佐剂效果高于CBLB。图20d体外细胞滴定分析不同疫苗制剂诱导的抗体中和效价。抗体的中和效价结果显示，共递送纳米制剂诱导的抗体中和能力最高，纳米疫苗和纳米佐剂混合制剂刺激产生的抗体中和能力次之，均明显高于纳米疫苗本身。且CpG为佐剂诱导的抗体中和滴度优于CBLB佐剂。图20e：体内致死保护分析揭示共递送纳米制剂可提供最为高效的免疫保护(75％)，纳米疫苗和纳米佐剂提供的保护效果次之(50％)，两者均高于纳米疫苗(25％)。免疫分析结果显示，共递送纳米制剂诱导的免疫应答最有效，纳米佐剂与纳米疫苗混合剂亦有显著佐剂效果。In summary, using SP70 epitope as a model to analyze the impact of different vaccine formulations on the immune effect is shown in Figure 20: Figure 20a-c shows the ELISA analysis of different ways of delivering CpG adjuvant and CBLB to induce IgG titers; Figure 20a shows The effect of different ways of delivering CpG on SP70IgG antibody titer. Compared with the nano-vaccine SP70-HFT, the nano-vaccine mixed with free CpG (SP70-HFT+CpG) has no obvious adjuvant effect. But the nano-vaccine and nano-adjuvant CpG-HFT mixed preparation and SP70 and CpG co-delivery nano-preparation SP70-CpG-HFT can significantly increase the specific antibody titer. And the effect of co-delivery of nano preparations is higher than the antibody titer induced by the mixed preparation of nano vaccine and nano adjuvant. Figure 20b reveals that when CBLB is used as an adjuvant, the co-delivery of the nanoformulation induces a higher specific antibody titre than the nanovaccine SP70-HFT and nanoadjuvant CBLB-HFT mixed formulation. Figure 20c shows that the adjuvant effect of CpG is higher than that of CBLB. Figure 20d In vitro cell titration analysis of antibody neutralization titers induced by different vaccine preparations. The results of antibody neutralization titers showed that the co-delivery of nanoformulations induced the highest antibody neutralization ability, and the neutralization ability of the antibodies stimulated by the nanovaccine and nanoadjuvant mixed formulation was the second, which was significantly higher than the nanovaccine itself. And CpG adjuvant induced antibody neutralization titer is better than CBLB adjuvant. Figure 20e: In vivo lethal protection analysis reveals that co-delivery of nanoformulations can provide the most effective immune protection (75%), followed by nanovaccine and nanoadjuvant (50%), both of which are higher than nanovaccine (25 %). The results of immunological analysis showed that the immune response induced by co-delivery of nano-formulations was the most effective, and the mixture of nano-adjuvant and nano-vaccine also had significant adjuvant effects.
实验例5纳米载体模块平台的稳定性Experimental example 5 The stability of the nanocarrier module platform
流感病毒血凝素茎部区是通用型流感疫苗的重要靶标，其中大肠杆菌表达的H1HA10三聚体诱导的抗体可中和不同亚型的毒株。采用现有的基因融合方法进行H1HA10(简写为HA)与ferrtin融合获得重组蛋白HA-HFT。Influenza virus hemagglutinin stem region is an important target of universal influenza vaccine, in which antibodies induced by H1HA10 trimer expressed by E. coli can neutralize different subtypes of strains. H1HA10 (abbreviated as HA) and ferrtin were fused using the existing gene fusion method to obtain the recombinant protein HA-HFT.
结果详见图21：即蛋白编辑剪切技术可在HFT/PFT载体上高效负载HA抗原；其中，图21a为重组基因HA-HFT表达分析电泳图；图中，control：pET28a载体空白对照菌裂解液；all：细菌裂解液；up：细菌裂解液上清；HA-HFT：HA(H1HA10，简写为HA)与HFT直接基因融合形成重组蛋白。图21b为凝胶过滤纯化基于蛋白编辑剪切技术制备的HA-HFT的电泳分析图。图21c为凝胶过滤纯化的HA-HFT的UV曲线图。图21d为HA-HFT的透射电镜图(负染制样)。图21e为凝胶过滤纯化基于蛋白编辑剪切技术制备的HA-PFT的电泳分析图。图21f为凝胶过滤纯化的HA-PFT的UV曲线图。图21g为HA-PFT的透射电镜图(负染制样)。before：内含肽自剪切前，after：内含肽剪切后，图21b及21e中的8-19编号：分管收集凝胶过滤纯化的目的产物样品的编号(1ml/管)。The results are shown in Figure 21: the protein editing and shearing technology can efficiently load HA antigen on HFT/PFT vector; Figure 21a is the electrophoresis diagram of recombinant gene HA-HFT expression analysis; in the figure, control: pET28a vector blank control bacteria lysis All: bacterial lysate; up: bacterial lysate supernatant; HA-HFT: direct gene fusion of HA (H1HA10, abbreviated as HA) with HFT to form a recombinant protein. Figure 21b is an electrophoretic analysis diagram of gel filtration purification of HA-HFT prepared based on protein editing and shearing technology. Figure 21c shows the UV curve of HA-HFT purified by gel filtration. Figure 21d is a transmission electron microscope image of HA-HFT (negative staining). Figure 21e is an electrophoretic analysis diagram of gel filtration purification of HA-PFT prepared based on protein editing and shearing technology. Figure 21f shows the UV curve of HA-PFT purified by gel filtration. Figure 21g is a transmission electron microscope image of HA-PFT (negative staining). Before: before the intein self-cleavage, after: after the intein is cut, the numbers 8-19 in Figure 21b and 21e: the numbers (1ml/tube) of the target product samples collected by the gel filtration purification.
如图21a所示，菌裂解液的上清液中不含有重组蛋白HA-HFT，可见重组蛋白形成包涵体沉淀。As shown in Figure 21a, the supernatant of the bacterial lysate does not contain the recombinant protein HA-HFT, and it can be seen that the recombinant protein forms an inclusion body precipitate.
采用本公开的方法将HA共价偶联至HFT形成HA-HFT，如图21b-d所示基于蛋白编辑剪切制备的HA-HFT以纳米颗粒形式存在。The method of the present disclosure is used to covalently couple HA to HFT to form HA-HFT. As shown in Figure 21b-d, HA-HFT prepared based on protein editing and shearing exists in the form of nanoparticles.
采用本公开的方法将HA共价偶联至PFT形成HA-PFT，如图21e-g所示基于蛋白编辑剪切制备的HA-PFT同样以纳米颗粒形式存在，也就是说PFT亦可负载HA。The method of the present disclosure is used to covalently couple HA to PFT to form HA-PFT. As shown in Figure 21e-g, HA-PFT prepared based on protein editing and shearing also exists in the form of nanoparticles, which means that PFT can also carry HA .
由于H1HA10需要维持天然三聚体构象，因此本公开中仅研究纳米佐剂CpG-HFT对纳米疫苗HA-HFT/PFT免疫原性的影响。Since H1HA10 needs to maintain the natural trimer conformation, this disclosure only studies the effect of the nano-adjuvant CpG-HFT on the immunogenicity of the nano-vaccine HA-HFT/PFT.
实验例6 CpG-HFT增强针对HA的Th1型IgG应答Experimental Example 6 CpG-HFT enhances Th1 type IgG response to HA
建立小鼠试验组：每组小鼠5只，免疫三次，每次接种间隔2周，免疫3周后眼眶采血用于免疫分析。HA组使用H1HA10-intein N免疫小鼠，使用剂量10μg/只。HA-HFT+CpG-HFT组使用纳米疫苗HA-HFT与纳米佐剂CpG-HFT共同免疫小鼠，使用剂量分别为10μg/只和1.6μg/只。纳米疫苗HA-HFT组：HA-HFT免疫小鼠，使用剂量分别为10μg/只。纳米疫苗HA-PFT组：HA-PFT免疫小鼠，使用剂量分别为10μg/只。HA-PFT+CpG-HFT组使用纳米疫苗HA-PFT与纳米佐剂CpG-HFT共同免疫小鼠，使用剂量分别为10μg/只和1.6μg/只。 Establish a mouse test group: 5 mice in each group, immunized three times, with an interval of 2 weeks between each inoculation, and blood was collected from the orbit for immunoassay after 3 weeks of immunization. The HA group used H1HA10-intein N to immunize mice with a dose of 10 μg/mouse. The HA-HFT+CpG-HFT group used the nano-vaccine HA-HFT and the nano-adjuvant CpG-HFT to immunize mice together, and the dosages were 10μg/mouse and 1.6μg/mouse respectively. Nano-vaccine HA-HFT group: HA-HFT immunized mice with a dose of 10μg/mouse. Nano-vaccine HA-PFT group: HA-PFT immunized mice with a dose of 10 μg/mouse. In the HA-PFT+CpG-HFT group, mice were immunized with the nano-vaccine HA-PFT and the nano-adjuvant CpG-HFT at the doses of 10μg/mouse and 1.6μg/mouse respectively.
不同剂型疫苗接种小鼠后，第3次免疫2周后眼眶取血进行ELISA分析。用纳米颗粒递送HA，比HA诱导抗体滴度提高10倍。CpG-HFT的联合使用，特异性抗体梯度再次提高10倍。以PFT为载体诱导抗体滴度高于HFT。PFT与小鼠的ferrtin相似度低，可能具有一定的佐剂作用。After inoculating mice with different dosage forms of vaccines, blood was taken from the orbit for ELISA analysis 2 weeks after the third immunization. Using nanoparticles to deliver HA can induce a 10-fold increase in antibody titer than HA. The combined use of CpG-HFT increases the specific antibody gradient by 10 times again. PFT as a carrier induces higher antibody titers than HFT. PFT has low similarity with mouse ferrtin and may have certain adjuvant effects.
纳米佐剂CpG-HFT增强HA特异性体液免疫应答并调控IgG类型的分析图详见图22；其中，图22a为ELISA分析揭示纳米载体和纳米佐剂均可增强HA特异性体液免疫应答。与HA抗原相比，纳米疫苗HA-HFT可将HA特异性IgG抗体滴度提高10倍左右，纳米佐剂CpG-HFT与纳米疫苗混合制剂可进一步提高特异性抗体滴度(10倍左右)。另外，PFT为载体效果高于HFT，可能是由PFT与小鼠内铁蛋白ferritin同源性低，有一定的佐剂作用引起。人源HFT与小鼠铁蛋白高度同源(氨基酸序列99％相同)，HFT蛋白自身无佐剂效果。图22b-d ELISA分析揭示，疫苗诱导抗体可特异性识别H1N1(P)、H3N2和H7H9毒株，且纳米佐剂与纳米疫苗混合制剂诱导的抗体识别能力高于纳米疫苗本身。(一抗是接种3次剂量疫苗小鼠的血清，二抗是辣根过氧化物酶HRP标记的鼠二抗)。The analysis diagram of nano adjuvant CpG-HFT enhancing HA specific humoral immune response and regulating IgG type is shown in Fig. 22; among them, Fig. 22a is an ELISA analysis revealing that both nanocarrier and nano adjuvant can enhance HA specific humoral immune response. Compared with HA antigen, nano-vaccine HA-HFT can increase HA-specific IgG antibody titer by about 10 times, and the mixed preparation of nano-adjuvant CpG-HFT and nano-vaccine can further increase specific antibody titer (about 10 times). In addition, the effect of PFT as a carrier is higher than that of HFT, which may be caused by the low homology of PFT and mouse ferritin, which has a certain adjuvant effect. Human HFT is highly homologous to mouse ferritin (99% identical in amino acid sequence), and HFT protein itself has no adjuvant effect. Figure 22b-d ELISA analysis revealed that vaccine-induced antibodies can specifically recognize H1N1(P), H3N2, and H7H9 strains, and the antibody recognition ability induced by the nano-adjuvant and nano-vaccine mixture is higher than the nano-vaccine itself. (The primary antibody is the serum of mice vaccinated with 3 doses of vaccine, and the secondary antibody is a mouse secondary antibody labeled with horseradish peroxidase HRP).
图22a显示，纳米颗粒递送HA显著改善机体响应HA的B细胞免疫应答，比HA诱导抗体滴度提高10倍，与纳米佐剂CpG-HFT联合使用，特异性抗体梯度再次提高10倍，进一步提高应答水平。以PFT为载体诱导的抗体滴度高于HFT载体，提示PFT与小鼠的ferrtin相似度低，PFT载体可能具有一定的佐剂功能。图22b-d显示，HA特异性相关抗体可识别不同亚型的流感毒株，识别亚型涵盖：H1NI的(简写P)、H3N2和H7N9(安徽毒株，简写Ahpri)。ELISA分析IgG亚型(1:10 5稀释接种不同剂型疫苗小鼠的血清)发现，CpG-HFT主要增强Th2型IgG2和IgG3免疫应答，对Th1型的IgG有很强的佐剂效果，见表3。用纳米模块负载CpG制备纳米佐剂，与负载HA的纳米疫苗联合使用，显著增强HA特异性抗体免疫应答，诱导抗体识别不同亚型的流感病毒毒株能力亦增强。 Figure 22a shows that the delivery of HA by nanoparticles significantly improves the body's B cell immune response in response to HA, which is 10 times higher than HA-induced antibody titer, and when used in combination with the nano adjuvant CpG-HFT, the specific antibody gradient is increased again by 10 times, further increasing Response level. The antibody titer induced by PFT carrier is higher than that of HFT carrier, suggesting that PFT has low similarity with mouse ferrtin, and PFT carrier may have certain adjuvant function. Figure 22b-d shows that HA-specific related antibodies can recognize influenza strains of different subtypes, including: H1NI (abbreviated P), H3N2 and H7N9 (Anhui strain, abbreviated Ahpri). ELISA analysis of IgG subclass (1:10 dilution of 5 mice inoculated with different vaccine formulations serum) found, CpG-HFT primary enhanced IgG2 and IgG3 Th2 type immune response, a strong adjuvant effect on Th1-type IgG, Table 3. Nano-modules loaded with CpG to prepare nano-adjuvants, combined with HA-loaded nano-vaccine, significantly enhance the HA-specific antibody immune response, and the ability of inducing antibodies to recognize influenza virus strains of different subtypes is also enhanced.
表3：table 3:
Figure PCTCN2020091636-appb-000004
Figure PCTCN2020091636-appb-000004
注：HA：H1HA10-intein N；HA-HFT：HFT纳米颗粒递送H1HA10；HA-HFT+CpG-HFT：HA-HFT与CpG-HFT联合制剂；HA-PFT：PFT纳米颗粒递送H1HA10；HA-PFT+CpG-HFT：HA-PFT与CpG-HFT联合制剂(备注：由于gb1特异性地结合所有类型的IgG，因此在检测HA特异性抗体滴度时使用H1HA10-intein N蛋白。在利用蛋白编辑剪切时，基于gb1可显著改善蛋白的可溶性，因此使用H1HA10-intein N-gb1)。 Note: HA: H1HA10-intein N ; HA-HFT: HFT nanoparticles deliver H1HA10; HA-HFT+CpG-HFT: HA-HFT and CpG-HFT combined preparation; HA-PFT: PFT nanoparticles deliver H1HA10; HA-PFT +CpG-HFT: HA-PFT and CpG-HFT combined preparation (Note: Since gb1 specifically binds to all types of IgG, H1HA10-intein N protein is used when detecting HA-specific antibody titer. Use protein editing scissors When cutting, gb1-based can significantly improve the solubility of the protein, so use H1HA10-intein N -gb1).
综上所述，本公开的纳米载体模块平台可在纳米颗粒表面高效展示抗原蛋白、免疫增强剂、多肽表位，用于增强型的预防性亚单位疫苗和个性化多肽表位肿瘤疫苗研发等领域。解决了负载蛋白对蛋白自装配纳米颗粒稳定性影响以及纳米颗粒难以共递送佐剂和抗原分子的难题。In summary, the nanocarrier module platform of the present disclosure can efficiently display antigen proteins, immune enhancers, and polypeptide epitopes on the surface of nanoparticles, and is used for the development of enhanced preventive subunit vaccines and personalized polypeptide epitope tumor vaccines. field. The problem of the influence of loaded protein on the stability of protein self-assembled nanoparticles and the difficulty of co-delivering adjuvant and antigen molecules with nanoparticles is solved.
以上所述仅为本公开的优选实施方式而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。The above are only the preferred embodiments of the present disclosure and are not used to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.
工业实用性Industrial applicability
本公开提供了一种基于内含肽介导纳米载体及其应用，旨在提供一种综合使用基因工程技术和内含肽介导的蛋白编辑剪切技术，完美解决encapsulin的C端亚稳定性问题，提供一种通用的纳米载体。本公开提供的技术方案通过分子设计，成功将内含肽介导蛋白编辑技术引入encapsulin纳米载体的表面修饰；制备的纳米颗粒与N端带有互补的断裂内含肽C片段(split intein C)货物分子混合，intein自我切除，货物分子共价偶联至纳米颗粒表面，改纳米颗粒表面修饰技术有效解决encapsulin的C端亚稳定性的限制，可在encapsulin的表面特异、高效递送不同的货物分子。本公开提供的技术方案以GFP蛋白和鼠巨细胞病毒M44蛋白的C端氨基酸315-411为模型，利用新开发的纳米递送技术递送上述货物蛋白，接种小鼠诱导高滴度的特异性抗体；利用halo共交联技术和抗原-抗体高亲和相互作用，成功纯化出GFP和M44相关IgG抗体，可用于western blotting检测和免疫荧光(immunofluorescence，IF)分析。本公开还提供了一种可同时递送抗原和免疫增强剂的纳米制剂，该纳米制剂可高效同时递送抗原和免疫增强剂，且具有良好特异性。本公开的纳米制剂可在纳米颗粒表面高效展示抗原蛋白、免疫增强剂、多肽表位和抗体，用于增强型的预防性亚单位疫苗和个性化多肽表位肿瘤疫苗研发及抗体偶联药物领域。本公开解决了负载蛋白对蛋白自装配纳米颗粒稳定性影响，实现了纳米制剂高效地同时递送抗原和免疫增强剂，同时且具有良好特异性。The present disclosure provides an intein-mediated nanocarrier and its application, and aims to provide a comprehensive use of genetic engineering technology and intein-mediated protein editing and shearing technology to perfectly solve the C-terminal metastability of encapsulin The problem is to provide a universal nanocarrier. The technical solution provided by the present disclosure successfully introduces the intein-mediated protein editing technology into the surface modification of the encapsulin nanocarrier through molecular design; the prepared nanoparticle and the N-terminus have a complementary split intein C fragment (split intein C) Cargo molecules are mixed, intein is self-removed, and cargo molecules are covalently coupled to the surface of nanoparticles. The nanoparticle surface modification technology effectively solves the limitation of C-terminal metastability of encapsulin, and can deliver different cargo molecules specifically and efficiently on the surface of encapsulin. . The technical solution provided by the present disclosure uses the GFP protein and the C-terminal amino acids 315-411 of the murine cytomegalovirus M44 protein as models, and uses the newly developed nano-delivery technology to deliver the aforementioned cargo protein, and inoculate mice to induce high-titer specific antibodies; Using halo co-cross-linking technology and antigen-antibody high-affinity interaction, GFP and M44-related IgG antibodies were successfully purified, which can be used for western blotting detection and immunofluorescence (IF) analysis. The present disclosure also provides a nanoformulation capable of simultaneously delivering antigen and immune enhancer. The nanoformulation can efficiently deliver antigen and immune enhancer at the same time, and has good specificity. The nano-formulations of the present disclosure can efficiently display antigen proteins, immune enhancers, polypeptide epitopes and antibodies on the surface of nano particles, and are used in the field of enhanced preventive subunit vaccines and personalized polypeptide epitope tumor vaccines and antibody-conjugated drugs. . The present disclosure solves the effect of the loaded protein on the stability of the protein self-assembled nanoparticle, and realizes that the nano preparation can efficiently deliver the antigen and the immune enhancer at the same time, and has good specificity.

Claims (20)

  1. 一种基于内含肽介导的纳米载体，其特征在于，所述的纳米载体是通过下述步骤构建的：An intein-mediated nanocarrier is characterized in that the nanocarrier is constructed through the following steps:
    1)encapsulin蛋白的C端通过基因融合的方法引入int N、gb1和halo共3个串联蛋白形成重组蛋白encapsulin-int N-gb1-halo； 1) The C-terminus of the encapsulin protein is introduced by gene fusion into three tandem proteins of int N , gb1 and halo to form a recombinant protein encapsulin-int N -gb1-halo;
    2)60个重组蛋白单体自装配成纳米颗粒，其暴露的int N-gb1-halo作为通用“适配器”，与货物重组蛋白gb1-int C和cargo混合后，在DTT的帮助下发生分子重组； 2) 60 recombinant protein monomers self-assemble into nanoparticles. The exposed int N- gb1-halo serves as a universal "adapter". After mixing with the cargo recombinant protein gb1-int C and cargo, molecular recombination occurs with the help of DTT ；
    3)内含肽intein自我切除，形成副产物int N-gb1-halo和gb1-int C，而encapsulin与cargo共价偶联，生产目的产物encapsulin-cargo。 3) The intein intein is self-excised, forming by-products int N -gb1-halo and gb1-int C , and encapsulin is covalently coupled with cargo to produce the target product encapsulin-cargo.
  2. 根据权利要求1所述的纳米载体，其特征在于，所述encapsulin蛋白的核酸序列如SEQ ID No.1所示，所述int N的核酸序列如SEQ ID No.2所示，所述gb1的核酸序列如SEQ ID No.3所示，所述halo的核酸序列如SEQ ID No.4所示，所述int C的核酸序列如SEQ ID No.16所示。 The nanocarrier according to claim 1, wherein the nucleic acid sequence of the encapsulin protein is shown in SEQ ID No. 1, the nucleic acid sequence of int N is shown in SEQ ID No. 2, and the nucleic acid sequence of the gb1 The nucleic acid sequence is shown in SEQ ID No. 3, the nucleic acid sequence of halo is shown in SEQ ID No. 4, and the nucleic acid sequence of int C is shown in SEQ ID No. 16.
  3. 根据权利要求1或2所述的纳米载体，其特征在于，所述的cargo为GFP或M44或者strep2。The nanocarrier according to claim 1 or 2, wherein the cargo is GFP or M44 or strep2.
  4. 根据权利要求3所述的纳米载体，其特征在于，所述GFP的核酸序列如SEQ ID No.9所示。The nanocarrier according to claim 3, wherein the nucleic acid sequence of the GFP is shown in SEQ ID No.9.
  5. 根据权利要求3所述的纳米载体，其特征在于，所述M44的核酸序列如SEQ ID No.10所示。The nanocarrier according to claim 3, wherein the nucleic acid sequence of M44 is shown in SEQ ID No. 10.
  6. 根据权利要求3所述的纳米载体，其特征在于，所述strep2的核酸序列如SEQ ID No.11所示。The nanocarrier according to claim 3, wherein the nucleic acid sequence of strep2 is shown in SEQ ID No. 11.
  7. 根据权利要求3所述的纳米载体，其特征在于，步骤3)为encapsulin-int N-gb1-halo和gb1-int C-GFP按照浓度比例为1:1-2，两者在室温条件下孵育2小时后，用superose 6B increase分子排阻法分离目的产物encapsulin-GFP。 The nanocarrier according to claim 3, wherein step 3) is that encapsulin-int N -gb1-halo and gb1-int C- GFP are in a concentration ratio of 1:1-2, and the two are incubated at room temperature After 2 hours, the target product encapsulin-GFP was separated by superose 6B increase molecular exclusion method.
  8. 根据权利要求3所述的纳米载体，其特征在于，步骤3)为encapsulin-int N-gb1-halo和gb1-int C-strep2按照浓度比例为1:1-3，两者在室温条件下孵育2小时后，用superose 6B increase分子排阻法分离目的产物encapsulin-strep2。 The nanocarrier according to claim 3, wherein step 3) is encapsulin-int N -gb1-halo and gb1-int C- strep2 in a concentration ratio of 1:1-3, and the two are incubated at room temperature After 2 hours, the target product encapsulin-strep2 was separated by superose 6B increase molecular exclusion method.
  9. 根据权利要求3所述的纳米载体，其特征在于，步骤2)为encapsulin-int N-gb1-halo和gb1-int C-M44按照浓度比例为1:1-2，两者在室温条件下孵育2小时后，用superose 6B increase分子排阻法分离目的产物encapsulin-M44。 The nanocarrier according to claim 3, wherein step 2) is encapsulin-int N -gb1-halo and gb1-int C -M44 in a concentration ratio of 1:1-2, and the two are incubated at room temperature After 2 hours, the target product encapsulin-M44 was separated by superose 6B increase molecular exclusion method.
  10. 权利要求1所述的纳米载体作为递送货物蛋白，接种小鼠诱导高滴度特异性抗体的应用。The nanocarrier of claim 1 is used as the delivery cargo protein, and the application of inoculating mice to induce high titer specific antibodies.
  11. 根据权利要求10所述的应用，其特征在于，所述的抗体的纯化方法为：The application according to claim 10, wherein the antibody purification method is:
    1)使用gb1-int C-GFP或gb1-intein C-M44与halo-int N-gb1蛋白进行蛋白编辑剪切，生成重组蛋白halo-cargo，以及副产物gb1-int C和int N-gb1； 1) Use gb1-int C -GFP or gb1-intein C -M44 and halo-int N -gb1 protein for protein editing and cutting to generate recombinant protein halo-cargo, and by-products gb1-int C and int N -gb1;
    2)反应结束后，混合物与Promega halo TM beads混合，目的产物halo-cargo共价结合至halo TM beads，离心弃上清后，加入免疫后的血清，货物分子相关抗体利用抗原-抗体之间高亲和相互作用与halo TM-halo-cargo结合，其它抗体和蛋白通过离心、洗涤后分离，最后用200mM glycine pH2.8洗脱目的抗体。 2) After the reaction, the mixture is mixed with Promega halo TM beads, and the target product halo-cargo is covalently bound to halo TM beads. After centrifugation to discard the supernatant, the immunized serum is added. Affinity interaction is combined with halo TM -halo-cargo, other antibodies and proteins are separated after centrifugation and washing, and finally the target antibody is eluted with 200mM glycine pH2.8.
  12. 根据权利要求11所述的应用，其特征在于，所述的halo TM-halo-GFP的制备与纯化方法为：将halo-int N-gb1和gb1-int C-GFP按照浓度比1：1-2在室温条件下孵育4小时后，将250μL反应液与50μL平衡好的Promega halo TM室温摇床混合半个小时后离心，用500μL PBS洗涤beads，除去非特异性结合蛋白；免疫2次后的血清；血清与halo TM-halo-GFP孵育后的流出；先用500μL PBST洗涤halo TM-halo-GFP；再90μL的200mM glycine pH2.8洗脱目的抗体。 The application according to claim 11, wherein the preparation and purification method of halo TM -halo-GFP is: halo-int N- gb1 and gb1-int C- GFP are in a concentration ratio of 1:1- 2 After incubating for 4 hours at room temperature, mix 250 μL of the reaction solution with 50 μL of the balanced Promega halo TM room temperature shaker for half an hour, then centrifuge, wash the beads with 500 μL of PBS to remove non-specific binding proteins; serum after 2 immunizations The flow of serum after incubation with halo TM -halo-GFP; first wash halo TM -halo-GFP with 500 μL PBST; then 90 μL of 200mM glycine pH2.8 to elute the target antibody.
  13. 根据权利要求10所述的应用，其特征在于，所述的halo TM-halo-M44的制备及M44相关抗体的纯化方法为：将halo-int N-gb1和gb1-int C-M44按照浓度比1：1-2在室温条件下孵育2-4小时后，250μL反应液与50μL平衡好的Promega halo TM室温摇床混合半个小时，而后离心，用500μL PBS洗涤beads，除去非特异性结合蛋白，反应液与Promega halo TM混合后离心上清；PBS洗涤液洗涤3次，使得目的蛋白halo-GFP特异性结合至Promega halo TM beads；免疫2次后的血清；血清与halo TM-halo-M44孵育后的流出；先用500μL PBST洗涤halo TM-halo-M44；再90μL的200mM glycine pH2.8洗脱目的抗体。 The application according to claim 10, wherein the preparation method of halo TM -halo-M44 and the purification method of M44-related antibodies are: halo-int N- gb1 and gb1-int C- M44 according to the concentration ratio 1:1-2 After incubating for 2-4 hours at room temperature, 250μL of the reaction solution was mixed with 50μL of the balanced Promega halo TM room temperature shaker for half an hour, and then centrifuged, and the beads were washed with 500μL of PBS to remove non-specific binding proteins. The reaction solution was mixed with Promega halo TM and the supernatant was centrifuged; the PBS washing solution was washed 3 times to make the target protein halo-GFP specifically bind to Promega halo TM beads; the serum after immunization twice; the serum was incubated with halo TM -halo-M44 After the flow out; first wash halo TM -halo-M44 with 500 μL PBST; then eluate the target antibody with 90 μL 200 mM glycine pH2.8.
  14. 根据权利要求10所述的应用，其特征在于，诱导后的抗体用于WB和/或IF分析。The application according to claim 10, wherein the induced antibody is used for WB and/or IF analysis.
  15. 一种可同时递送抗原和免疫增强剂的纳米制剂，其特征在于，由铁蛋白的N端与接头gbl-intein 的C端融合形成重组蛋白gbl-intein c-ferrtin后自装配成24聚体构成纳米颗粒；外源蛋白的C端融合内含肽的N端形成重组蛋白，内含肽的N端特异性识别并切除暴露在所述纳米颗粒表面的铁蛋白；外源蛋白与铁蛋白共价交联而得； A nano preparation capable of delivering antigen and immune enhancer at the same time, which is characterized in that the N-terminal of ferritin and the C-terminal of the linker gbl-intein are fused to form a recombinant protein gbl-intein c- ferrtin and then self-assembled into a 24-mer. Nanoparticles; the C-terminus of the foreign protein is fused with the N-terminus of the intein to form a recombinant protein, and the N-terminus of the intein specifically recognizes and excises the ferritin exposed on the surface of the nanoparticle; the foreign protein and ferritin are covalently Cross-linked;
    其中，所述的外源蛋白为免疫增强剂或抗原或二者组合。Wherein, the foreign protein is an immune enhancer or an antigen or a combination of the two.
  16. 根据权利要求1所述的纳米制剂，其特征在于，所述gbl的核酸序列如SEQ ID No.22所示，所述intein c的核酸序列如SEQ ID No.19所示。 The nanoformulation of claim 1, wherein the nucleic acid sequence of the gbl is shown in SEQ ID No. 22, and the nucleic acid sequence of the intein c is shown in SEQ ID No. 19.
  17. 根据权利要求15或16所述的纳米制剂，其特征在于，所述的免疫增强剂为鞭毛蛋白或抗生物素蛋白单体类似物rhizavidin。The nano-formulation according to claim 15 or 16, wherein the immune enhancer is flagellin or rhizavidin, a monomer analog of avidin.
  18. 根据权利要求15至17中任一项所述的纳米制剂，其特征在于，所述的抗原为单一多肽表位的蛋白抗原或不同多肽表位的蛋白抗原。The nanoformulation according to any one of claims 15 to 17, wherein the antigen is a protein antigen of a single polypeptide epitope or a protein antigen of different polypeptide epitopes.
  19. 根据权利要求15至18中任一项所述的纳米制剂，其特征在于，所述的外源蛋白为免疫增强剂与抗原二者组合时，其摩尔比为1～3:5。The nanoformulation according to any one of claims 15 to 18, wherein when the exogenous protein is a combination of an immune enhancer and an antigen, the molar ratio is 1 to 3:5.
  20. 根据权利要求15至19中任一项所述的纳米制剂，其特征在于，所述的铁蛋白为人源的重链铁蛋白或强烈火球菌的重链铁蛋白或其他物种来源的铁蛋白。The nanoformulation according to any one of claims 15 to 19, wherein the ferritin is human-derived heavy-chain ferritin or Pyrococcus furiosus or other species-derived ferritin.
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