CN115920026A - Heat-resistant polymer protein scaffold and application of heat-resistant polymer protein scaffold in vaccine - Google Patents

Heat-resistant polymer protein scaffold and application of heat-resistant polymer protein scaffold in vaccine Download PDF

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CN115920026A
CN115920026A CN202210345996.4A CN202210345996A CN115920026A CN 115920026 A CN115920026 A CN 115920026A CN 202210345996 A CN202210345996 A CN 202210345996A CN 115920026 A CN115920026 A CN 115920026A
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protein
tag
vaccine
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CN115920026B (en
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陈泽良
张劲松
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Sun Yat Sen University
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Sun Yat Sen University
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Abstract

The application belongs to the technical field of biomedicine, and particularly relates to a heat-resistant polymer protein scaffold and application of the heat-resistant polymer protein scaffold in a vaccine. The present application provides a heat-resistant multimeric protein scaffold comprising: a first tag for coupling and a thermostable multimeric protein; the first tag is connected to the N end of the heat-resistant polymer protein; the heat-resistant polymer protein has one or more of sequences shown in SeqIDNO.1-SeqIDNO.4. The application provides a heat-resistant polymer protein scaffold and application of the heat-resistant polymer protein scaffold in vaccines, and the novel heat-resistant polymer protein with higher immunogenicity is used for preparing heat-resistant polymer protein scaffolds and adjuvants of various vaccine antigens, so that a method for preparing vaccine heat-stable scaffolds based on the heat-resistant polymer protein and enhancing antigen immunogenicity is established.

Description

Heat-resistant polymer protein scaffold and application of heat-resistant polymer protein scaffold in vaccine
Technical Field
The application belongs to the technical field of biological medicines, and particularly relates to a heat-resistant polymer protein scaffold and application of the heat-resistant polymer protein scaffold in vaccines.
Background
Traditional vaccines are based primarily on attenuated or inactivated live pathogens. Although generally very effective in inducing a protective immune response in a host, this type of vaccine often presents safety concerns. For example, live attenuated vaccines may regain pathogenicity by reverting to wild-type, especially in immunodeficient populations. In addition, such vaccines often require stringent storage conditions to maintain their immunological activity, and accidental exposure to environmental stresses (e.g., light or temperature) may result in loss of immunogenicity. Batch-to-batch variability can also be a problem with such vaccines, especially during large-scale production of cultured pathogens. The improved subunit vaccines have higher safety and smaller lot-to-lot variation than attenuated live or inactivated vaccines, but suffer from the constant disadvantage of low immunogenicity, requiring high doses and adjuvant administration.
To address the problems of traditional vaccines, one strategy is to confer high stability or immunogenicity on antigens by combining them with a multimeric protein scaffold. The principle of operation is mainly that multimeric protein scaffolds have a well-defined parallel multimeric structure, have high stability, allow the introduction of target binding antigens and hinge regions, and achieve the desired multivalency by self-assembly without disrupting target antigen binding. Multimeric protein scaffolds can present multiple copies of an antigen or antigenic epitope on the surface, produce a high local density of structurally ordered epitopes, and are capable of high affinity interaction with B cell receptors, inducing B cell receptor signaling and strong immune stimulation (e.g., T helper cell independent B cell activation); second, due to the particulate nature and relatively large size of the multimeric protein scaffold, it can be efficiently taken up and processed by professional antigen presenting cells (e.g., dendritic cells and macrophages), thereby promoting a strong humoral and cellular immune response.
However, the existing multimeric protein antigen scaffolds have the problems of few types and much more candidate antigens than antigen scaffolds, so that the immune response of the organism to the antigen scaffold firstly can inhibit the immune response to the antigen-scaffold combination, and the development of a novel vaccine antigen scaffold is urgently needed.
Disclosure of Invention
In view of the above, the present application provides a heat-resistant polymer protein scaffold, an application of the heat-resistant polymer protein scaffold in vaccines, and a method for preparing vaccine heat-stable scaffolds and enhancing antigen immunogenicity based on the heat-resistant polymer protein by using the novel heat-resistant polymer protein as a heat-resistant polymer protein scaffold and an adjuvant of various vaccine antigens.
In a first aspect, the present application provides a heat-resistant multimeric protein scaffold comprising:
a first tag for coupling and a thermostable multimeric protein;
the first tag is attached to a terminal of the heat-resistant multimeric protein;
the heat-resistant polymer protein has one or more of sequences shown in Seq ID NO.1, seq ID NO.2, seq ID NO.3 and Seq ID NO. 4.
Specifically, the first label is connected to the N end or the C end of the heat-resistant polymer protein, preferably the N end; ensure that the N terminal or C terminal of the polymer protein is exposed on the surface of the particle protein.
In another embodiment, the first tag is selected from one or more of SpyCatcher, snoopCatcher, sdCatcher, and DogCatcher.
In another embodiment, the tag is linked to the end of the heat-resistant multimeric protein by a linker peptide, which may be N-terminal or C-terminal; the sequence of the connecting peptide is (GGGGS) n (1. Ltoreq. N. Ltoreq.4) or (GGGGA) n (1≤n≤4)。
Specifically, the connecting peptide is flexible connecting peptide, the flexible connecting peptide is soft and easy to bend and takes glycine Gly as a main component, the length can be properly adjusted according to different connecting proteins, and the connecting peptide has the best effect of selecting GGSGGGGSGGS.
The second aspect of the application provides the application of the heat-resistant polymer protein scaffold in the preparation of vaccines.
In a third aspect, the present application provides a vaccine comprising: the heat-resistant multimeric protein scaffold and an antigen to which a second tag is attached;
the second tag can be recombined with the first tag to form an isopeptide bond coupling; the antigen is a characteristic or protective antigen capable of presenting viruses or/and bacteria; the second tag is a tag corresponding to the first tag, and the second tag includes: one or more of SpyTag, snoeptag, sdTag and DogTag.
In particular, the thermostable multimeric protein scaffold of the present application is used to present antigens characteristic of various viruses or bacteria to prepare vaccines of corresponding nanoparticles.
Specifically, the heat-resistant polymer protein has a sequence of Seq ID NO.1, wherein the sequence of Seq ID NO.1 is as follows:
MAFEFKLPAFEFKLIHEGEIVKWFVKPGDEVNEDDVLCEVQNDKAVVEIPSPVKGKVLEILVPEGTVATVGQTLITLDAPAFEFKLLKDKSKKKRKKRKKRKRCRKRKRLTLSLPMHRQLKRRLARTAASSPCRPCASMRAKKASIFGLSKERAAFEFKLKEDIDAFLAGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSGIRRAIAKAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCT。
specifically, the heat-resistant polymer protein has a sequence of Seq ID NO.1, and the nucleotide sequence is as follows:
ATGGCTTTTGAATTTAAGCTGCCGGCTTTTGAATTTAAGCTGATCCACGAAGGTGAAATTGTCAAATGGTTTGTGAAACCGGGCGATGAAGTGAACGAAGACGATGTATTGTGCGAAGTGCAAAATGACAAGGCGGTTGTCGAAATTCCCTCCCCGGTCAAAGGGAAAGTGCTTGAAATCCTCGTCCCGGAGGGAACAGTGGCAACGGTCGGGCAAACGCTCATCACGCTCGATGCGCCGGCTTTTGAATTTAAGCTGTTAAAGGACAAGAGCAAGAAGAAGCGAAAAAAGAGGAAAAAACGGAAACGGTGTCGAAAGAGGAAAAGGTTGACGCTGTCGCTCCCAATGCACCGGCAGCTGAAGCGGAGGCTGGCCCGAACCGCCGCGTCATCGCCATGCCGTCCGTGCGCAAGTATGCGCGCGAAAAAGGCGTCGATATTCGGCTTGTCCAAGGAACGGGCAGCTTTTGAATTTAAGCTGAAAGAAGATATTGACGCTTTCCTTGCCGGCGGCGCAAAACCGGCACCGGCCGCAGCGGAGGAAAAAGCGGCGCCGGCCGCAGCGAAACCGGCGACGACAGAAGGCGAATTCCCGGAAACGCGCGAGAAAATGAGCGGCATCCGTCGGGCGATTGCCAAAGCTCCGCACGTGACGCTGATGGACGAAGCCGATGTGACGAAGCTTGTTGCTCACCGAAAAAAATTCAAGGCTATTGCCGCGGAAAAAGGCATCAAGCTGACGTTTTTGCCGTACGTCGTCAAAGCGCTCGTTTCCGCGCTGCGTGAATACCCAGTGTTGAATACGTCCATTGATGATGAGACGGAAGAAATCATCCAGAAGCATTATTACAATATCGGCATCGCCGCTGATACGGACCGCGGCTTGCTTGTGCCGGTCATTAAACATGCCGATCGCAAGCCGATTTTCGCCTTGGCGCAAGAAATCAATGAGCTCGCCGAGAAAGCGCGCGACGGCAAACTGACGCCGGGAGAAATGAAAGGCGCTTCATGCACG。
in another embodiment, the antigen is a protective antigen of a pathogen, including, but not limited to, one or more of capsid protein VP1 of foot-and-mouth disease virus, receptor binding domain RBD of SARS-CoV-2S spike protein, brucella protective antigen, african swine fever protective antigen, matrix protein 2 extracellular domain M2e of avian influenza virus, GP glycoprotein of Ebola virus, RVGP glycoprotein of rabies virus, pre-membrane and envelope proteins of Japanese encephalitis virus, envelope glycoprotein and membrane proteins of dengue virus, glycoprotein precursor of Lassa virus, and envelope protein of West Nile virus.
Specifically, the thermostable multimeric protein scaffold is covalently coupled to the antigen to which the second tag is attached. The label coupling can effectively avoid the problems of complex antigen misfolding in gene fusion, coupling site heterogeneity in chemical combination and the like, and realizes the efficient and directional assembly of the antigen on the self-assembled polymer protein scaffold.
In another embodiment, the antigen is the capsid protein VP1 of foot and mouth disease virus.
The capsid protein VP1 of the foot-and-mouth disease virus is the capsid protein VP1 of the O-type foot-and-mouth disease virus, the GenBank: MZ634456.1, and the protein sequence is as follows:
MTTSTGESADPVTTTVENYGGETQVQRRQHTDVSFILDRFVKVTPKDQINVLDLMQTPAHTLVGALLRTATYYFADLEVAVKHEGNLTWVPNGAPEAALDNTTNPTAYHKAPLTRLALPYTAPHRVLATVYNGNCKYGEGAVTNVRGDLQVLAQKAARTLPTSFNYGAIKATRVTELLYRMKRAETYCPRPLLAIHPEQARHKQKIVAPVKQLL。
the nucleic acid sequence thereof:
ATGACCACCTCCACAGGTGAGTCCGCTGATCCCGTGACCACCACCGTTGAGAATTACGGTGGAGAAACACAGGTCCAGAGACGTCAGCACACCGACGTTTCTTTCATTTTGGACAGATTTGTGAAAGTAACACCAAAAGACCAAATCAATGTGTTGGACCTGATGCAAACCCCTGCTCACACTTTGGTAGGCGCGCTCCTCCGCACCGCCACTTACTACTTCGCAGATTTAGAAGTGGCAGTGAAGCACGAAGGCAACCTCACCTGGGTCCCAAACGGGGCGCCCGAGGCGGCGCTGGATAACACCACCAACCCGACGGCCTACCACAAGGCACCGCTCACCCGTCTTGCTTTGCCTTACACAGCACCACACCGTGTTCTGGCTACCGTCTACAACGGGAACTGCAAGTACGGCGAGGGCGCCGTGACCAACGTGAGGGGTGACCTGCAAGTCTTGGCCCAGAAAGCAGCAAGAACGCTGCCCACCTCCTTCAACTACGGTGCCATTAAGGCTACCCGGGTGACTGAACTGCTTTACCGCATGAAGAGGGCCGAAACATACTGCCCTCGGCCCCTGCTGGCCATTCACCCGGAACAAGCCAGACACAAGCAGAAGATTGTGGCACCTGTCAAACAGTTGTTG。
specifically, the amino acid sequence of the outer membrane protein BP26 of the Brucella:
MNTRASNFLAASFSTIMLVGAFSLPAFAQENQMTTQPARIAVTGEGMMTASPDMAILNLSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGINIQPIYVYPDDKNNLKEPTITGYSVSTSLTVRVRELANVGKILDESVTLGVNQGGDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPPMPMPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIK。
in another embodiment, the first tag is SpyCatcher; the second tag is SpyTag.
Specifically, the Gene bank number of the SpyCatcher is: MN433887.1.
The amino acid sequence of SpyCatcher:
MVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT。
the Gene bank number of the SpyTag is as follows: MT945421.1.
The amino acid sequence of the SpyTag: RGVPHIVMVDAYKRYK.
In another embodiment, the heat-resistant multimeric protein scaffold has a His histidine tag attached to the C-terminus for later protein purification.
Specifically, a His histidine tag is linked to the C-terminus of the heat-resistant multimeric protein scaffold via a GS amino acid sequence.
In another embodiment, the C-terminus of the antigen to which the second tag is attached is a His histidine tag for later protein purification.
Specifically, a His histidine tag is linked to the C-terminus of the antigen to which the second tag is linked via the GS amino acid sequence.
Specifically, the amino acid sequence of SpyCatcher-E2 (SC-E2) connected with the His histidine tag is as follows:
MVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGGGGSGGGGSGGGGSMAFEFKLPAFEFKLIHEGEIVKWFVKPGDEVNEDDVLCEVQNDKAVVEIPSPVKGKVLEILVPEGTVATVGQTLITLDAPAFEFKLLKDKSKKKRKKRKKRKRCRKRKRLTLSLPMHRQLKRRLARTAASSPCRPCASMRAKKASIFGLSKERAAFEFKLKEDIDAFLAGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSGIRRAIAKAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTGSHHHHHH。
specifically, the nucleotide sequence of SpyCatcher-E2 (SC-E2) connected with the His histidine tag is as follows:
ATGGTAACCACCTTATCAGGTTTATCAGGTGAGCAAGGTCCGTCCGGTGATATGACAACTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAGGACGGCCGTGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGATTTCAGATGGACATGTGAAGGATTTCTACCTGTATCCAGGAAAATATACATTTGTCGAAACCGCAGCACCAGACGGTTATGAGGTAGCAACTCCAATTGAATTTACAGTTAATGAGGACGGTCAGGTTACTGTAGATGGTGAAGCAACTGAAGGTGACGCTCATACTGGGGGCGGAGGGTCAGGGGGAGGCGGGTCTGGCGGCGGAGGGTCTGGCGGTGGGGGATCAATGGCTTTTGAATTTAAGCTGCCGGCTTTTGAATTTAAGCTGATCCACGAAGGTGAAATTGTCAAATGGTTTGTGAAACCGGGCGATGAAGTGAACGAAGACGATGTATTGTGCGAAGTGCAAAATGACAAGGCGGTTGTCGAAATTCCCTCCCCGGTCAAAGGGAAAGTGCTTGAAATCCTCGTCCCGGAGGGAACAGTGGCAACGGTCGGGCAAACGCTCATCACGCTCGATGCGCCGGCTTTTGAATTTAAGCTGTTAAAGGACAAGAGCAAGAAGAAGCGAAAAAAGAGGAAAAAACGGAAACGGTGTCGAAAGAGGAAAAGGTTGACGCTGTCGCTCCCAATGCACCGGCAGCTGAAGCGGAGGCTGGCCCGAACCGCCGCGTCATCGCCATGCCGTCCGTGCGCAAGTATGCGCGCGAAAAAGGCGTCGATATTCGGCTTGTCCAAGGAACGGGCAGCTTTTGAATTTAAGCTGAAAGAAGATATTGACGCTTTCCTTGCCGGCGGCGCAAAACCGGCACCGGCCGCAGCGGAGGAAAAAGCGGCGCCGGCCGCAGCGAAACCGGCGACGACAGAAGGCGAATTCCCGGAAACGCGCGAGAAAATGAGCGGCATCCGTCGGGCGATTGCCAAAGCTCCGCACGTGACGCTGATGGACGAAGCCGATGTGACGAAGCTTGTTGCTCACCGAAAAAAATTCAAGGCTATTGCCGCGGAAAAAGGCATCAAGCTGACGTTTTTGCCGTACGTCGTCAAAGCGCTCGTTTCCGCGCTGCGTGAATACCCAGTGTTGAATACGTCCATTGATGATGAGACGGAAGAAATCATCCAGAAGCATTATTACAATATCGGCATCGCCGCTGATACGGACCGCGGCTTGCTTGTGCCGGTCATTAAACATGCCGATCGCAAGCCGATTTTCGCCTTGGCGCAAGAAATCAATGAGCTCGCCGAGAAAGCGCGCGACGGCAAACTGACGCCGGGAGAAATGAAAGGCGCTTCATGCACGGGATCCCATCACCATCACCATCAC。
specifically, the amino acid sequence of the SpyTag-VP1 (ST-VP 1) connected with the His histidine tag is as follows: <xnotran> MRGVPHIVMVDAYKRYKGGGGSGGGGSGGGGSMTTSTGESADPVTTTVENYGGETQVQRRQHTDVSFILDRFVKVTPKDQINVLDLMQTPAHTLVGALLRTATYYFADLEVAVKHEGNLTWVPNGAPEAALDNTTNPTAYHKAPLTRLALPYTAPHRVLATVYNGNCKYGEGAVTNVRGDLQVLAQKAARTLPTSFNYGAIKATRVTELLYRMKRAETYCPRPLLAIHPEQARHKQKIVAPVKQLLGSHHHHHH. </xnotran>
Specifically, the nucleotide sequence of the SpyTag-VP1 (ST-VP 1) connected with the His histidine tag is as follows: <xnotran> ATGCGTGGCGTGCCTCATATCGTGATGGTGGACGCCTACAAGCGTTACAAGGGGGGCGGAGGGTCAGGGGGAGGCGGGTCTGGCGGCGGAGGGTCTGGCGGTGGGGGATCAATGACCACCTCCACAGGTGAGTCCGCTGATCCCGTGACCACCACCGTTGAGAATTACGGTGGAGAAACACAGGTCCAGAGACGTCAGCACACCGACGTTTCTTTCATTTTGGACAGATTTGTGAAAGTAACACCAAAAGACCAAATCAATGTGTTGGACCTGATGCAAACCCCTGCTCACACTTTGGTAGGCGCGCTCCTCCGCACCGCCACTTACTACTTCGCAGATTTAGAAGTGGCAGTGAAGCACGAAGGCAACCTCACCTGGGTCCCAAACGGGGCGCCCGAGGCGGCGCTGGATAACACCACCAACCCGACGGCCTACCACAAGGCACCGCTCACCCGTCTTGCTTTGCCTTACACAGCACCACACCGTGTTCTGGCTACCGTCTACAACGGGAACTGCAAGTACGGCGAGGGCGCCGTGACCAACGTGAGGGGTGACCTGCAAGTCTTGGCCCAGAAAGCAGCAAGAACGCTGCCCACCTCCTTCAACTACGGTGCCATTAAGGCTACCCGGGTGACTGAACTGCTTTACCGCATGAAGAGGGCCGAAACATACTGCCCTCGGCCCCTGCTGGCCATTCACCCGGAACAAGCCAGACACAAGCAGAAGATTGTGGCACCTGTCAAACAGTTGTTGGGATCCCATCACCATCACCATCAC. </xnotran>
In a fourth aspect, the present application provides a method for preparing the vaccine, comprising the steps of: and mixing the heat-resistant polymer protein scaffold and the antigen connected with the second label in a buffer solution, and purifying to obtain the vaccine.
In another embodiment, the buffer is triethanolamine buffered saline solution (TBS buffer).
Specifically, the heat-resistant polymer protein scaffold and the antigen connected with the second label are mixed in a buffer solution, the heat-resistant polymer protein scaffold and the antigen connected with the second label form an amido bond for covalent binding, and the vaccine is obtained after purification.
Compared with the prior art, the beneficial effects of this application are:
the application provides a method for preparing a vaccine heat-resistant polymer protein scaffold based on heat-resistant polymer protein and serving as a vaccine adjuvant, and the stability and immunogenicity of a vaccine antigen are remarkably improved. 1. The heat-resistant polymer protein scaffold is constructed and used for delivering the vaccine antigen, so that the stability of the vaccine antigen is remarkably improved, and the antigen loss in the storage and transportation processes of the vaccine is reduced; 2. the heat-resistant polymer protein scaffold constructed by the application has higher immunogenicity, and can be used as an adjuvant to further improve the immune effect of a vaccine antigen; 3. most of the heat-resistant polymer proteins selected by the application are derived from archaea such as thermophilic bacteria, so that the probability of the organism generating autoantibody reaction is extremely low, the research and application of the heat-resistant polymer proteins as antigen supports are less, and the problem that the organism generates neutralizing antibodies and further affects the vaccine immunity effect due to repeated use of common supports as novel antigen supports can be avoided to a certain extent.
1. The heat-resistant polymer protein has heat resistance, and can endow vaccine antigen stability as a scaffold; 2. the heat-resistant polymer protein is polymer in nature and can endow antigen multivalency as a scaffold; 3. the heat-resistant polymer protein has immunogenicity, and can be used as an adjuvant to further improve the immunogenicity of vaccine antigens.
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In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a schematic diagram showing the construction of SC-E2 and ST-VP1 in this example;
FIG. 2 is a schematic diagram showing the conjugation of the heat-resistant multimeric protein scaffold of this example to a tag of an antigen;
FIG. 3 shows the purity of ST-VP1, SC-E2 and VP1-E2 by SDS-PAGE/Coomassie blue staining in this example;
FIG. 4 shows the results of electron microscopy of the 60-mer forms of SC-E2 and VP1-E2 of this example;
FIG. 5 shows the solubility results of SC-E2 and VP1-E2 of this example with temperature;
FIG. 6 shows the SC-E2 and VP1-E2 solubility assays before and after lyophilization for this example;
FIG. 7 shows the results of the MTT assay of this example for the cytotoxicity of SC-E2 and VP 1-E2;
FIG. 8 is a schematic diagram of the prime-boost immunization procedure of BALB/c mice of this example;
FIG. 9 is a graph showing the time course of the antibody titer against VP1 in serum of mice treated by the different methods of this example;
FIG. 10 shows the results of the antibody titer against E2 in the serum of mice treated by the different methods of this example;
FIG. 11 shows the results of measurement of the antibody titer of FRNT50 in mice treated by the different methods of this example;
FIG. 12 is a graph of the percentage of different types of T cells in mice treated with the different methods of this example;
FIG. 13 is a graph of the percentage of the RFP-marked DCs containing E2 and VP1-E2 of this example;
FIG. 14 is an RNA-Seq analysis chart of the thermostable multimeric protein scaffold E2 pulsed BMDC of this example.
Detailed Description
The application provides a heat-resistant polymer protein scaffold and application of the heat-resistant polymer protein scaffold in vaccines, and polymer proteins with higher immunogenicity are used for preparing heat-resistant polymer protein scaffolds and adjuvants of various antigens.
The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without making any creative effort belong to the protection scope of the present application.
The raw materials and reagents used in the following examples are commercially available or self-made.
Example 1
The embodiment of the application provides the expression and purification of a heat-resistant polymer protein scaffold SC-E2 and a vaccine VP1-E2, which comprises the following steps:
1. expression of thermostable multimeric protein scaffolds and antigens:
1) Carrying out gene synthesis on the constructed protein sequence, subcloning the DNA sequence into a pET28a expression plasmid, and respectively constructing pET28a-Spycatcher-E2 and pET28a-SpyTag-VP1; wherein E2 is a heat-resistant polymer protein E2 (E2 has the sequence of Seq ID NO. 1), VP1 is a capsid protein VP1 of foot-and-mouth disease virus (GenBank: MZ 634456.1), and Gene bank number of Spycatcher (marker SC) is: MN433887.1, gene bank number for SpyTag (marker ST): MT945421.1.
2) The pET28a expression plasmids of SpyCatcher-E2 and SpyTag-VP1 were transformed into E.coli BL21 (DE 3) -RIPL (Agilent), and the cells were grown for 16h at 37 ℃ on LB-agar plates containing 50. Mu.g/mL kanamycin.
3) Individual colonies were picked into 5mL LB starting culture containing 50. Mu.g/mL kanamycin and incubated at 37 ℃ for 16h with shaking at 220 rpm.
4) 5mL of the culture was diluted into 500mL of LB containing 50. Mu.g/mL of kanamycin and incubated at 37 ℃ with shaking at 220 rpm. When A is 600 =0.8, 0.5mM IPTG was added to induce culture and the culture was cultured at 22 ℃ for 16 to 20 hours with shaking at 200 rpm.
2. Purification of thermostable multimeric protein scaffolds and antigens:
1) 500mL of the cell culture pellet was resuspended with 20mL PBS, 0.1mg/mL lysozyme and 1mM protease inhibitor (PMSF) were added and lysed on ice for 15min.
2) The lysate was sonicated on ice for 5min, the sonicated lysate was centrifuged at 15000rpm/min for 15min, and the supernatant was collected.
3) Referring to FIG. 1, the supernatant was incubated with Ni-NTA agarose resin (GE Healthcare) to enrich for His-tagged Spycatcher-E2 and SpyTag-VP1, yielding two proteins, respectively: SC-E2-HIS and ST-VP1-HIS. And eluting the target protein by using Tris buffer solution containing imidazole to obtain purified proteins of the heat-resistant polymer protein scaffold SpyCatcher-E2 and SpyTag-VP 1.
4) The purified protein described above was concentrated by ultrafiltration, replacing the imidazole-containing Tris buffer with a conventional Tris buffer.
5) The protein of interest is further purified by Size Exclusion Chromatography (SEC).
6) Endotoxin was removed from the sample by TritonX-114, and the BCA method was used to determine the endotoxin-removing granule protein concentration.
7) Purity was assessed by SDS-PAGE/Coomassie brilliant blue staining and SEC.
3. Coupling and purifying the heat-resistant polymer protein scaffold Spycatcher-E2 and SpyTag-VP 1:
referring to FIG. 2, purified SpyCatcher-E2 and purified SpyTag-VP1 were covalently bound in TBS buffer by amide bond formation.
1) Purified SpyCatcher-E2 was incubated with 1.5-fold molar excess (amount of monomer VP1 relative to E2) of purified SpyTag-VP1 in TBS buffer overnight at room temperature;
2) The VP 1-coupled E2 nanoparticle scaffold was collected by separation of conjugated VP1-E2 from free VP1 by SEC using a Superose 610/300 (GE Healthcare) column equilibrated with PBS, followed by concentration by an ultrafiltration device.
3) The purity of the target protein was verified by SDS-PAGE separation.
4) The BCA method was used to determine the target protein concentration for subsequent analysis.
4. General characterization of ST-VP1, thermostable multimeric protein scaffold SC-E2 and vaccine VP 1-E2:
1) The purity of the vaccine VP1-E2 nanoparticle scaffold was successfully prepared by SDS-PAGE/Coomassie blue staining, as shown in FIG. 3, as can be seen from FIG. 3;
2) The 60-mer morphology of the heat-resistant polymer protein scaffold SC-E2 and the vaccine VP1-E2 is observed by a Transmission Electron Microscope (TEM), and the result is shown in FIG. 4, the left image of FIG. 4 is the transmission electron microscope image of the SC-E2 scaffold, the right image of FIG. 4 is the transmission electron microscope image of the VP1-E2 vaccine, the diameter of the SC-E2 scaffold is about 23nm in the left image of FIG. 4, and the diameter of the VP1-E2 vaccine is about 26nm in the right image of FIG. 4. The results show that the ST-VP1, the heat-resistant polymer protein scaffold SC-E2 and the vaccine VP1-E2 have high purity, the scaffold and the vaccine show uniform 60-polymer morphology under an electron microscope, and particle size measurement data show that the E2 particle size is increased after ST-VP1 coupling, but the 60-polymer morphology is not influenced.
Example 2
The embodiment of the application provides the stability analysis of the heat-resistant polymer protein scaffold SC-E2 and the vaccine VP1-E2, which specifically comprises the following steps:
1) Analysis of the thermostability of the Heat-resistant multimeric protein scaffold SC-E2 and vaccine VP 1-E2:
the purified heat-resistant polymer protein scaffold SC-E2 and the vaccine VP1-E2 are incubated for 1h in a neutral buffer solution at the temperature ranging from 25 ℃ to 95 ℃. Aggregates were removed by centrifugation and the proportion of protein in the soluble fraction was measured as a function of temperature.
The results are shown in FIG. 5. The soluble protein determination result shows that the ratio of the SC-E2 and VP1-E2 soluble protein is not obviously changed when the temperature is 80 ℃, which indicates that the SC-E2 and VP1-E2 have higher thermal stability (figure 5).
2) Analysis of lyophilization stability of the Heat-resistant multimeric protein scaffold SC-E2 and vaccine VP 1-E2:
the purified thermostable multimeric protein scaffold SC-E2 and vaccine VP1-E2 were lyophilized and reconstituted in the same buffer, and the particle solubility before and after lyophilization was examined.
The results are shown in FIG. 6. SC-E2 and VP1-E2 both maintain higher solubility before and after lyophilization, indicating that SC-E2 and VP1-E2 have higher lyophilization stability (FIG. 6).
Example 3
The present embodiments provide for the safety assessment of thermotolerant multimeric protein scaffolds SC-E2 and vaccines VP1-E2, comprising:
the heat-resistant polymer protein scaffold SC-E2 and the constructed vaccine VP1-E2 are subjected to safety evaluation by adopting an in vitro cytotoxicity experiment. The specific scheme is that Chinese hamster ovary Cells (CHO) 48h are treated by heat-resistant polymer protein scaffold SC-E2 and vaccine VP1-E2 with different concentrations (0, 0.05, 0.1, 0.15, 1, 2 and 5 mu M) respectively, and MTT kit is used for detecting cell activity to evaluate the toxicity of the scaffold and the vaccine.
The results are shown in FIG. 7. MTT toxicity results show that when heat-resistant polymer protein scaffold SC-E2 and vaccine VP1-E2 with different concentrations are used for treating CHO cells for 48 hours, the proliferation capacity of the cells is not obviously changed, which shows that the SC-E2 and the VP1-E2 have low toxicity and good safety, and can be used for subsequent animal in-vivo immunological evaluation.
Example 4
The embodiment of the application provides a vaccine VP1-E2 inoculated in a mouse body to induce a humoral immune response, which comprises the following steps:
1. animal experiments were set up to perform immunogenicity assessment of heat resistant multimeric protein scaffolds and vaccines:
the method is specifically divided into 4 groups: blank group, SC-E2 group, ST-VP1 group, VP1-E2 group, each group of 6 BALB/c mice. The VP1-E2 groups were immunized subcutaneously with a 5. Mu.g dose of VP1-E2 vaccine, the ST-VP1 group was immunized with VP1 in equimolar amounts to the VP1-E2 groups, the SC-E2 group was immunized with E2 in equimolar amounts to the VP1-E2 groups, and the blank group was not treated at all. Referring to FIG. 8, all mice were vaccinated in a prime-boost fashion, i.e., at week 0 (W0) and week 4 (W4). Sera were collected every two weeks and mice were euthanized at week 10 (W10). ELISA detected varying levels of antibodies in serum against VP1 and E2.
The time-dependent VP1 antibody levels in the sera of the groups of mice are shown in FIG. 9. ELISA results show that compared with ST-VP1 monomer group, vaccine VP1-E2 can rapidly induce mice to generate higher antibody level which can last for a longer time; compared with the blank group, the later period of the heat-resistant polymer protein scaffold SC-E2 can also induce the mice to generate certain antibody level, but the duration is shorter, which indicates that the heat-resistant polymer protein scaffold SC-E2 has certain immunogenicity but is not enough to induce higher and lasting immune response.
The antibody titer levels against the thermostable multimeric protein scaffold E2 in the sera of the groups of mice are shown in fig. 10. ELISA results show that high-titer heat-resistant polymer protein scaffold E2 antibodies are detected in the heat-resistant polymer protein scaffold SC-E2 and vaccine VP1-E2 groups, but do not influence the antibody level of VP1, and the heat-resistant polymer protein E2 scaffold is proved to have good safety.
2. Plaque reduction neutralization assay (FRNT), comprising:
this example evaluates the effectiveness of VP1-E2 vaccine constructed from heat-resistant polymer protein E2 scaffold in inducing neutralizing antibody in animal body, and uses plaque reduction neutralization test (FRNT) to test whether it can inhibit real foot-and-mouth disease virus infection. Due to the limitation of experimental conditions, the example adopts a pseudotyped foot-and-mouth disease virus to carry out Vero cell infection.
The test was divided into 4 groups: blank group, SC-E2 group, ST-VP1 group, VP1-E2 group, 6 BALB/c mice per group. The plaque reduction neutralization test was performed, and as a result, as shown in FIG. 11, the neutralizing antibody induced by the VP1-E2 vaccine was able to strongly inhibit the infection of the pseudotyped foot-and-mouth disease virus. VP1-E2 vaccinated mice with a 50% plaque reduction neutralization assay (FRNT 50) titer in excess of 3.2X 10 4 About 100-fold higher than monomer-vaccinated VP1 mice; the FRNT50 titer of the mice in the heat-resistant polymer protein scaffold E2 inoculated group is slightly higher than that in the blank group, which shows that the E2 scaffold alone can also trigger certain immune response.
3. VP1-E2 vaccination induces an effective cellular immune response in mice, including:
this example demonstrates that VP1-E2 vaccination enhances T cell activation in animals, that 10d after vaccination in mice, spleens were harvested by sacrifice and the percentage of different types of lymphocytes, including CD4, was assessed by flow cytometry + T and CD8 + T cells, wherein Th1 cell surface markers: CD4 + CCR5 + CCR1 + (ii) a Th2 cell surface markers: CD4 + CCR8 + CRTH2 + (ii) a Tfh cell surface markers: CD4 + CXCR5 + PD-1 + ；CD8 + T cell surface markers: CD62L + CD44 + Tcm。
The experiments were divided into 4 groups: blank group, SC-E2 group, ST-VP1 group, VP1-E2 group, each group of 6 BALB/c mice. Cell immunoreaction tests are carried out, the results are shown in figure 12, and flow detection results show that compared with a monomer inoculation ST-VP1 group, VP1-E2 vaccination can activate a larger number of different types of T cells in a mouse body, and the cell immunoreaction is obviously enhanced; vaccination with SC-E2 thermotolerant multimeric protein scaffolds alone also activated a small number of T cells compared to the blank group, although the difference was not statistically significant.
4. VP1-E2 vaccination induces potent antigen presentation effects in mice, including:
the self-assembled multimeric protein of the present example, due to its large particle size, can be preferentially taken up and processed by professional antigen presenting cells (e.g., dendritic cells, DCs), and further presented to T cells after processing to enhance immune response. This example demonstrates how self-assembled multimeric proteins are recognized and processed by the host immune system, and DC cell antigen presentation experiments were performed. RFP-labeled VP1, RFP-labeled E2, and RFP-labeled VP1-E2 were constructed for antigen tracking.
The experiments were divided into 3 groups: SC-E2 group, ST-VP1 group, VP1-E2 group, each group of 6 BALB/c mice. Equimolar amounts of 3 antigens were injected subcutaneously into 6 BALB/c mice. After 4h, inguinal lymph nodes were isolated and DCs (B220-CD 11 c) were harvested + MHC-II + ) And calculating the percentage of the fluorescence-labeled DC, and further reflecting the antigen uptake capacity of the DC.
Results as in fig. 13, flow results show that the percentage of RFP-labeled E2 scaffold and VP1-E2 containing DCs is significantly higher than RFP-labeled VP1 monomer containing cells, indicating that DCs preferentially capture multimeric protein particles.
5. Analysis of transcriptome profiles of thermostable multimeric protein scaffolds E2 pulsed BMDCs:
the gene expression characteristics of the E2 heat-resistant polymer protein scaffold induced in antigen presenting cells are identified by using a whole genome transcription method, and the molecular mechanism of the immune response triggered by the E2 scaffold is clarified. The specific scheme is that RNA sequencing analysis is carried out on immature mouse BMDC treated by an E2 scaffold, and the influence of the E2 scaffold on a BMDC transcription profile is analyzed, wherein the specific method comprises the following steps:
grouping: mice were randomly divided into E2 thermostable multimeric protein scaffold pulsed and unpulsed groups.
Treatment of mice:
1. collecting bone marrow derived dendritic cells (BMDC) of the mice, culturing with complete medium (90% RPMI 1640 and 10% FBS);
2. the cells were inoculated in complete medium supplemented with 200U/mL recombinant murine granulocyte-macrophage colony stimulating factor (GM-CSF) and cultured for 7d.
3. Immature cells were harvested and co-incubated with 50. Mu.g/mL of E2 thermotolerant multimeric protein scaffoldIncubate for 1d, final concentration 2.5X 10 6 Individual cells/mL.
4. Total RNA was extracted from untreated or E2-treated BMDCs using Tri Reagent according to the manufacturer's protocol.
5. The double-ended library (100 × 2 bp) prepared using the TruSeq RNA sample preparation kit was sequenced on the Illumina HiSeq2000 platform.
6. Cuffdiff and CummeRBund were used to identify differentially expressed genes using normalized expression values (FPKM: reads per million mapping per kilobase of transcription).
7. Any threshold of 0.05FDR (false discovery rate) and 1FPKM was used under at least one condition to filter out differentially expressed genes.
8. Gene ontology and pathway analysis was performed using DAVID (database for annotation, visualization and integrated discovery). The results are shown in FIG. 14.
Significant deregulation of gene expression after E2 scaffold treatment (more than 5300 genes, FDR < 0.05) compared to transcriptome of unpulsed BMDCs with E2 treated cells; a significant upregulation of 2984 genes was measured under E2 scaffold stimulation, including those associated with immune responses, such as the "chemokine signaling" and "Jak-STAT signaling" pathways.
Furthermore, in E2 scaffold pulsed BMDCs, il12b, il6 and other genes encoding innate immune system receptors, such as the Nlrp3 (NOD-like receptor family, pyrin domain 3, tlr2, toll-like receptor 2) and Il7r (interleukin 7 receptor) genes, are up-regulated. When BMDCs were stimulated with E2 scaffolds, the expression levels of genes encoding the co-stimulatory molecules CD40 and Notch2 signaling components (Notch 2, rbpj and Mib 1) were increased, with the Notch2-Rbpj signaling receptor having been described as a unique signaling molecule required to control DC functional differentiation and initiate T cell activation.
The above results indicate that E2 scaffold can activate DC through innate immune receptor (PRR, pattern recognition receptor) or related immune signaling molecular pathway, and further activate T cells, inducing strong cellular immune response.
Example 5
The embodiment of the application provides antigenic characterization of heat-resistant polymer protein, which specifically comprises the following steps:
the above-mentioned Seq ID No.2 to Seq ID No.4 sequences were subjected to antigenicity analysis using a software VaxiJen v 2.0Server, TARGET ORGANISM selected bacteria, and the antigenicity scores of Seq ID No.2 to Seq ID No.4 were measured. The results were: seq ID No.2 (antigenicity score: 0.6461); seq ID No.3 (antigenicity score: 0.6953); seq ID No.4 (antigenicity score: 0.6197).
The heat-resistant polymer protein with high immunogenicity is used for constructing a stent and a vaccine, the brucella outer membrane protein BP26 is selected as an antigen, the stability and the immunogenicity of the antigen endowed by the stent to the vaccine are evaluated, and the specific scheme is the same as that of the E2-VP1 vaccine of the embodiment 2-5:
(1) Constructing and purifying heat-resistant polymer protein scaffold and vaccine expression; (2) Thermostable multimeric protein scaffolds and vaccine stability assessment; (3) Heat resistant multimeric protein scaffolds and vaccine safety assessments; (4) Evaluation of heat-resistant multimeric protein scaffolds and vaccine immunogenicity; (5) The heat-resistant polymer protein with higher immunogenicity is determined to be used for preparing vaccine heat-stable scaffolds and adjuvants.
As a result, the 3 heat-resistant polymer protein scaffolds (Seq ID No.2 to Seq ID No. 4) all can endow the vaccine antigen with high stability (heat, freeze-thaw, freeze-drying and the like) and immunogenicity, and can be used as a novel scaffold and an adjuvant for vaccine antigen delivery.
As described above, the heat-resistant multimeric protein E2 based on Seq ID No.1 was selected as an antigen scaffold, and the capsid protein VP1 of foot-and-mouth disease virus (GenBank: MZ 634456.1) was selected as a vaccine antigen in the examples of the present application. The antigen scaffold with higher immunogenicity is used for preparing a novel heat-stable scaffold and an adjuvant for delivering various antigens so as to construct a universal vaccine antigen scaffold. The VP1-E2 vaccine constructed by the heat-resistant polymer protein E2 scaffold has good stability and can induce strong humoral and cellular immunity, and the vaccine is probably based on (1) the special self-assembly of the E2 scaffold and the easy uptake of a polymer structure by antigen presenting cells, and presents a plurality of antigen copies on the surface, so that the affinity of the antigen with B and T cell receptors is increased; (2) The E2 scaffold has higher immunogenicity, and can be used as an adjuvant to further enhance the immune effect of the vaccine antigen.
The foregoing is only a preferred embodiment of the present application and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.
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Claims (10)

1. A heat-resistant multimeric protein scaffold, comprising:
a first tag for coupling and a thermostable multimeric protein;
the first tag is attached to a terminal of the heat-resistant multimeric protein;
the heat-resistant polymer protein has one or more of sequences shown in Seq ID NO.1, seq ID NO.2, seq ID NO.3 and Seq ID NO. 4.
2. The thermotolerant multimeric protein scaffold according to claim 1, wherein the first tag is selected from one or more of SpyCatcher, snoopatcher, sdCatcher and DogCatcher.
3. The thermotolerant multimeric protein scaffold according to claim 1, wherein the tag is linked to a terminus of the thermotolerant multimeric protein by a linking peptide; the sequence of the connecting peptide is (GGGGS) n Or (GGGGA) n Wherein n is more than or equal to 1 and less than or equal to 4.
4. Use of a heat-resistant multimeric protein scaffold according to any one of claims 1 to 3 in the manufacture of a vaccine.
5. A vaccine, comprising: a heat-resistant multimeric protein scaffold according to any one of claims 1 to 3 and an antigen to which a second tag is attached;
the second tag can be recombined with the first tag to form an isopeptide bond coupling; the antigen is a characteristic or protective antigen capable of presenting viruses or/and bacteria; the second tag is a tag corresponding to the first tag, and the second tag includes: one or more of SpyTag, snooppag, sdTag and DogTag.
6. The vaccine of claim 5, wherein the antigen is a protective antigen of a pathogen, including but not limited to one or more of capsid protein VP1 of foot and mouth disease virus, protective antigen of African swine fever, receptor binding domain RBD of SARS-CoV-2S spike protein, protective antigen of Brucella, matrix protein 2 extracellular domain M2e of avian influenza virus, GP glycoprotein of Ebola virus, RVGP glycoprotein of rabies virus, pre-membrane protein and envelope protein of Japanese encephalitis virus, envelope glycoprotein and membrane protein of dengue virus, glycoprotein precursor of Lassa virus and envelope protein of West Nile virus.
7. The vaccine of claim 5, wherein the first tag is a SpyCatcher; the second tag is SpyTag.
8. The vaccine of claim 7, wherein the SpyCatcher has the amino acid sequence:
<xnotran> MVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT; </xnotran> The amino acid sequence of the SpyTag: RGVPHIVMVDAYKRYK.
9. A method for preparing a vaccine according to any one of claims 5 to 8, comprising the steps of: the heat-resistant polymer protein scaffold of any one of claims 1 to 3 and the antigen to which the second tag is attached are mixed in a buffer solution and purified to obtain a vaccine.
10. The method of claim 9, wherein the buffer is a triethanolamine-buffered saline solution.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112010984A (en) * 2020-08-04 2020-12-01 广州千扬生物医药技术有限公司 Novel coronavirus S protein polymer nano vaccine based on helicobacter pylori ferritin
CN112638411A (en) * 2018-05-04 2021-04-09 斯拜生物技术有限公司 Vaccine composition
WO2021084282A1 (en) * 2019-11-01 2021-05-06 SpyBiotech Limited Viruses with modified capsid proteins
CN113621031A (en) * 2021-04-23 2021-11-09 中山大学 Combination of peptide linkers for covalent self-assembly of proteins using spontaneous isopeptide bonds

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112638411A (en) * 2018-05-04 2021-04-09 斯拜生物技术有限公司 Vaccine composition
WO2021084282A1 (en) * 2019-11-01 2021-05-06 SpyBiotech Limited Viruses with modified capsid proteins
CN112010984A (en) * 2020-08-04 2020-12-01 广州千扬生物医药技术有限公司 Novel coronavirus S protein polymer nano vaccine based on helicobacter pylori ferritin
CN113621031A (en) * 2021-04-23 2021-11-09 中山大学 Combination of peptide linkers for covalent self-assembly of proteins using spontaneous isopeptide bonds

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