Fusion protein and its application

Abstract

The present application relates to fusion proteins and their applications. A fusion protein is provided, which comprises a trimerization region and an immunogenic region connected by a linker, wherein the trimerization region comprises one or more repeating units shown in SEQ ID NO.1; the immunogenic region is an immunogenic protein of a pathogen, for example, selected from the coronavirus RBD region, HIV membrane protein or influenza virus hemagglutinin protein and their immunogenic fragments. The trimer obtained by the present invention can produce a higher level of neutralizing antibodies than the immunogenic monomer, will not induce strong antibodies against the trimerization region in the human body, and can promote the body's immune response to focus on the immunogenic region.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a fusion protein used as a vaccine. Background Art

[0002] The spike protein (S) of SARS-CoV-2 is responsible for recognizing and invading cells, among which the receptor binding domain (RBD) contained in the S protein is the most important, responsible for recognizing and binding to the angiotensin (ACE2) receptor to mediate the virus' entry into cells. The currently developed new coronavirus mRNA vaccines, protein subunit vaccines, and adenovirus vector vaccines are all developed for the S protein or RBD protein of SARS-CoV-2. The S protein of SARS-CoV-2 is a trimeric protein in its natural state. Many studies have shown that S protein trimers or RBD protein trimers can have higher immunogenicity than S protein monomers or RBD protein monomers. In order to achieve the expression of trimer proteins of RBD or S protein, some studies have added Foldon tags to RBD or S protein for fusion expression, and formed a trimer under the action of Foldon. However, Foldon has a large molecular weight and has very strong immunogenicity. Immunizing the human body with a trimer fused with Foldon and RBD / S protein will produce a strong immune response to the Foldon protein, weakening the antigenic response to SARS-CoV-2S or RBD. Likewise, trimers formed using other non-human tags or mediators still have similar disadvantages.

[0003] Therefore, it is still necessary to develop other strategies to induce immunogens, especially SARS-CoV-2 antigens, to form trimers. Summary of the invention

[0004] The inventors previously screened and optimized a type III collagen sequence, and the recombinant human collagen produced has very good hydrophilicity and stability. Its amino acid composition is 100% identical to the corresponding part of the natural collagen amino acid sequence. It will not produce an immune response when applied to the human body. At the same time, the recombinant human collagen is a trimer structure and can be widely used in industries such as biomedicine. Since the human type III collagen is an optimized human collagen peptide repeat, it may destroy the trimer structure of the collagen after fusion expression with the new coronavirus RBD protein or influenza virus hemagglutinin. At the same time, even if the new coronavirus RBD protein or influenza virus hemagglutinin does not destroy the trimer structure of collagen and forms a trimer protein of RBD, under the influence of the collagen, the trimer RBD may be different from the natural RBD trimer structure, resulting in a poor induction of the body's immune response. Therefore, the effect of fusion expression of type III collagen with the new coronavirus RBD protein or influenza virus hemagglutinin as a vaccine is unpredictable. In the present invention, the inventors introduced cysteine ​​at the C-terminus of the recombinant human type III collagen to form a disulfide bond to stabilize the collagen and fused it with the RBD protein of SARS-CoV-2 or the influenza virus hemagglutinin through a eukaryotic system or a prokaryotic system through the designed linker amino acid. Finally, the trimer fusion protein was successfully expressed. Immunizing mice with the recombinant human type III collagen-RBD trimer protein as a vaccine can produce more potent binding antibodies and neutralizing antibodies than the RBD protein of SARS-CoV-2 or the influenza virus hemagglutinin monomer. Therefore, the invention cleverly fused the human type III collagen peptide with the new coronavirus RBD protein or the influenza virus hemagglutinin in series, prompting the RBD protein or the influenza virus hemagglutinin to form the correct trimer conformation, enhancing the immunogenicity of the RBD or the influenza virus hemagglutinin, and at the same time, because the human type III collagen sequence comes from humans, it will not induce an immune response in the human body, prompting the immune response to focus on the RBD or influenza virus hemagglutinin trimer region.

[0005] In one aspect, the present invention provides a fusion protein comprising a trimerization region and an immunogenic region connected by a linker, wherein the trimerization region comprises one or more repeating units shown in SEQ ID NO.1 or repeating units having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity compared to the repeating units shown in SEQ ID NO.1, provided that the repeating units retain the trimerization function.

[0006] In one embodiment, the immunogenic region is an immunogenic protein of a pathogen.

[0007] In one embodiment, the pathogen is a virus, bacteria, fungus and / or parasite.

[0008] In one embodiment, the immunogenic region is selected from the group consisting of a coronavirus RBD region, an HIV membrane protein, or an influenza virus hemagglutinin protein (eg, an H1N1 HA1 protein), and immunogenic fragments thereof.

[0009] In one embodiment, the trimerization region comprises 10-20 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, or 19) repeating units as shown in SEQ ID NO. 1 or repeating units with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity compared to the repeating units as shown in SEQ ID NO. 1. In one embodiment, the trimerization region comprises one or more regions as shown in SEQ ID NO. 2 at the C-terminus or sequences with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity compared to the sequence as shown in SEQ ID NO. 2.

[0010] In one embodiment, the trimerization region comprises the amino acid sequence shown in SEQ ID NO.3 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO.3.

[0011] In one embodiment, the immunogenic region comprises the amino acid sequence shown in SEQ ID NO.4 or SEQ ID NO.10, or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO.4 or SEQ ID NO.10.

[0012] In one embodiment, the linker is (GGGS)n or (GGGGS)n or (G)n. In one embodiment, n is an integer from 1 to 10. Preferably, the linker is (GGGS)n or (GGGGS)n.

[0013] In one embodiment, the trimerization region and the immunogenic region are at the N-terminus and C-terminus, respectively, of the fusion protein.

[0014] In one embodiment, the fusion protein comprises the amino acid sequence shown in SEQ ID NO.5 or 9 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO.5 or 9.

[0015] In one aspect, the present invention provides a nucleic acid comprising nucleotides encoding a fusion protein as described herein. In one embodiment, the nucleic acid comprises an amino acid sequence as shown in SEQ ID NO.7. In one embodiment, the nucleic acid further comprises a purification tag, such as a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag coding sequence. In one embodiment, the purification tag is at the C-terminus of the fusion protein. In one embodiment, the nucleic acid further comprises nucleotides encoding a leader sequence.

[0016] In one aspect, the invention provides a vector comprising a nucleic acid as described herein. In one embodiment, the vector comprises an expression control element operably linked to the nucleic acid. In one embodiment, the expression control element is a promoter, a terminator and / or an enhancer.

[0017] In one aspect, the invention provides a host cell comprising a nucleic acid or vector as described herein. In one embodiment, the host cell is a plant cell that cannot regenerate into a plant or is not a plant cell. In one embodiment, the host cell is a eukaryotic cell or a prokaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, an animal cell and / or an insect cell. In one embodiment, the prokaryotic cell is an Escherichia coli cell.

[0018] In one aspect, the present invention provides a trimeric fusion protein comprising the fusion protein described herein.

[0019] In one aspect, the present invention provides a method for producing a fusion protein or a trimer fusion protein according to the present invention, comprising:

[0020] (1) culturing the host cell described herein under appropriate culture conditions;

[0021] (2) harvesting host cells and / or culture medium containing the fusion protein or trimeric fusion protein; and

[0022] (3) Purify the fusion protein or trimer fusion protein.

[0023] In one embodiment, the fusion protein or trimeric fusion protein can be purified from the culture medium. In another embodiment, the fusion protein or trimeric fusion protein can be purified from the cell lysate by homogenizing the host cells.

[0024] In one aspect, the invention provides a composition comprising a fusion protein, a nucleic acid, a vector, a host cell and / or a trimeric fusion protein as described herein.

[0025] In one embodiment, the composition is a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a vaccine. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier, excipient, diluent and / or adjuvant. In one embodiment, the adjuvant is an aluminum adjuvant, a saponin adjuvant, lipid A, monophosphoryl lipid A, trehalose dimycolate, an oil emulsion adjuvant, a polysaccharide adjuvant, a Toll-like receptor activator and / or a STING agonist. For example, adjuvants are

[0026] In one aspect, the present invention provides the use of fusion proteins, nucleic acids, vectors, host cell trimeric fusion proteins, and / or compositions described herein for the preparation of a medicament for treating or preventing a condition or disease mediated by a pathogen. In one embodiment, the pathogen is a virus, a bacterium, a fungus, and / or a parasite. In one embodiment, the virus is selected from a coronavirus, an AIDS virus, and an influenza virus. In one embodiment, the coronavirus is SARS-CoV-2. In one embodiment, the influenza virus is an influenza A virus or an influenza B virus.

[0027] In one aspect, the present invention provides a method for vaccinating a subject, comprising administering the fusion protein, trimer or pharmaceutical composition of the present invention to the subject.

[0028] Compared with the prior art, the beneficial effects of the present invention include:

[0029] 1. The inventors discovered for the first time that by introducing disulfide bonds and linker molecules, the trimerization sequence of type III collagen can still perform the trimerization function after being fused with the SARS-CoV-2 RBD protein or influenza virus hemagglutinin, and the obtained RBD or influenza virus hemagglutinin trimer has the correct conformation and can produce higher neutralizing antibody levels than RBD or influenza virus hemagglutinin monomers.

[0030] 2. Since type III collagen is homologous to humans, it will not induce strong antibodies against this collagen, but can prompt the body's immune response to focus on the new coronavirus RBD protein or influenza virus hemagglutinin.

[0031] 3. The present invention also provides a strategy for other immunogens to form trimers. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Vector plasmid map used to construct collagen-RBD trimer protein.

[0033] Figure 2 This is the SDS-PAGE image of the expressed collagen-RBD trimer protein.

[0034] Figure 3To detect the molecular weight of collagen-RBD trimer protein using a particle size analyzer.

[0035] Figure 4 Immunization procedure for mice.

[0036] Figure 5 To detect the level of specific IgG binding antibodies against SARS-CoV-2 RBD in the serum of mice on the 21st day after immunization.

[0037] Figure 6 It is the specific IgG binding antibody titer against SARS-CoV-2 RBD in the serum of mice on the 21st day after immunization.

[0038] Figure 7 The neutralizing antibody titer against SARS-CoV-2 pseudovirus in the serum of mice on the 21st day after immunization with collagen-RBD trimer or RBD monomer with CF501 as adjuvant.

[0039] Figure 8 Neutralizing antibody titers against SARS-CoV-2 pseudovirus in the sera of mice immunized with aluminum-adjuvanted collagen-RBD trimer or RBD monomer on day 21.

[0040] Fig. 9 To detect the level of specific IgG binding antibodies against H1N1HA1 in the serum of mice immunized with influenza HA1-collagen / CF501 and HA1 / CF501 on the 17th day.

[0041] Fig.10 To detect the level of specific IgG binding antibodies against H1N1 HA1 in the serum of mice immunized with influenza HA1-collagen / aluminum adjuvant and HA1 / aluminum adjuvant on the 17th day. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] In this article, the inventor uses the previously discovered recombinant type III humanized collagen polypeptide as a part of the fusion protein, as a trimerization domain. In the field of proteins, the structure and / or function of proteins depend on the amino acid sequence to a certain extent. Before the present invention, the inventor envisioned that adding amino acid residues at one end of the recombinant type III humanized collagen polypeptide may affect the trimerization function of the recombinant type III humanized collagen polypeptide. Surprisingly, when the recombinant type III humanized collagen polypeptide is fused with an immunogenic peptide, it is completely capable of forming a trimerized fusion protein, and the trimerized fusion protein produces significantly improved immunogenicity. For example, in the embodiments, the inventors have demonstrated that when the recombinant type III humanized collagen polypeptide forms a fusion protein with SARS-CoV-2 RBD protein or influenza virus hemagglutinin, a trimer can be formed. Compared with SARS-CoV-2 RBD protein or SARS-CoV-2 RBD protein or influenza virus hemagglutinin and adjuvant, the trimer alone or in combination with an adjuvant can produce significantly improved immunogenicity and produce higher neutralizing antibody titers in vivo.

[0044] In this article, fusion protein refers to a protein formed by connecting two or more functional protein parts indirectly in a frame-matched manner through a joint. The fusion protein can include a trimerization region, an immunogenic region, and an optional joint. The position of each part is not particularly limited. For example, the trimerization region can be at the N-terminus or C-terminus of the fusion protein, and the immunogenic region can be at the C-terminus or N-terminus of the fusion protein.

[0045] In this article, the trimerization region refers to a functional region that can achieve the trimerization function. The trimerization region may include one or more repeating units shown in SEQ ID NO.1. Each repeating unit may be directly connected or separated by one or more amino acid residues. The number of repeating units may vary, for example, the repeating unit may be 2-50, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40. The trimerization region may include one or more stabilization regions shown in SEQ ID NO.2 at the C-terminus. The trimerization region may include the amino acid sequence shown in SEQ ID NO.3.

[0046] In this article, the immunogenic region refers to a protein or fragment thereof (e.g., a fragment in an antigen) derived from a pathogen that can cause an immune response in an organism, especially a mammal, such as a human body. The immunogenic protein may be selected from a coronavirus RBD region, an HIV membrane protein (e.g., gp140) or an influenza virus hemagglutinin protein and their immunogenic fragments. The immunogenic region may comprise an amino acid sequence shown in SEQ ID NO.4 or 10. Multiple documents of the prior art (e.g., CN202110676901.2 and CN202111337048.8) disclose coronavirus RBD regions and related immunogenic fragments.

[0047] The trimerization region and the immunogenic region can be indirectly connected via a linker. The type of linker may not be particularly limited. The linker may be (GGGS)n or (GGGGS)n or (G)n. For example, n may be an integer of 1-10, such as 2, 3, 4, 5, 6, 7, 8 or 9. Preferably, the linker may be (GGGS)n or (GGGGS)n, thereby introducing a disulfide bond.

[0048] In order to facilitate the production and purification of the fusion protein herein, the fusion protein of the present invention can add additional parts at the N-terminus or C-terminus. For example, the fusion protein of the present invention can include a secretion signal, a leader region at the N-terminus, so that the fusion protein is secreted into the culture medium. The fusion protein of the present invention can also include a purification tag at the C-terminus, such as a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag to facilitate the purification of the fusion protein. For example, the fusion protein can include the amino acid sequence shown in SEQ ID NO.6.

[0049] In this context, nucleic acid comprises a polynucleotide encoding a protein. The nucleic acid may be a natural nucleic acid of each part of the fusion protein, or a codon-optimized nucleic acid for expression in a target host. The nucleic acid may comprise the sequence of SEQ ID NO.7.

[0050] As used herein, the term "vector" is a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc. A vector may contain a variety of elements for controlling expression, including but not limited to promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector may also contain a replication initiation site. A vector may contain a nucleic acid of the present invention to facilitate introduction into a cell for expression. A vector may contain expression control elements, such as promoters, terminators and / or enhancers, operably linked to the nucleic acid.

[0051] The term "host cell" refers to a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. The host cell can be a eukaryotic cell or a prokaryotic cell. For example, a eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. A prokaryotic cell can be an E. coli cell.

[0052] The term "subject" refers to any person or other animal, particularly other mammal, who is receiving prevention, treatment, or diagnosis. Other mammals may include, for example, dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, guinea pigs, mice, and the like.

[0053] The term "vaccine" in this article refers to a suspension or solution of an antigenic portion, usually containing an infectious agent or some portion of an infectious agent, which is injected into the body to produce immunity. The antigenic portion of the vaccine can be a microorganism, or a natural product purified from a microorganism, a synthetic product, or a genetically engineered protein, peptide, polysaccharide or similar product. The vaccine herein can be an oral or parenteral vaccine, such as a vaccine administered subcutaneously, intradermally, intravenously, intramuscularly, intraarterially or by infusion.

[0054] The term "adjuvant" refers to any substance that is mixed with the injected immunogen and can increase or modify the immune response. The adjuvant can be an aluminum adjuvant, a saponin adjuvant, lipid A, monophosphoryl lipid A, trehalose dimycolate, an oil emulsion adjuvant, a polysaccharide adjuvant, a Toll-like receptor activator and / or a STING agonist.

[0055] The fusion protein of the present invention can be assembled into a trimeric fusion protein due to the presence of the trimerization region. The trimeric fusion protein can be a homotrimer or a heterotrimer. Preferably, the trimeric fusion protein is a homotrimer. The trimeric fusion protein of the present invention can produce higher neutralizing antibodies than a single immunogen without immunogenicity to the trimerization region. The fusion protein or trimeric fusion protein of the present invention can be prepared by the following method, which comprises: (1) culturing a host cell according to the present invention under suitable culture conditions; (2) harvesting host cells and / or culture medium containing the fusion protein or trimeric fusion protein; and (3) purifying the fusion protein or trimeric fusion protein. There is no particular limitation on the culture and purification conditions, and those skilled in the art can select suitable culture and purification conditions to separate the fusion protein or trimeric protein of the present invention.

[0056] The fusion protein, nucleic acid, vector, host cell and / or trimeric fusion protein of the present invention can be prepared into a composition. The composition can include the fusion protein, nucleic acid, vector, host cell and / or trimeric fusion protein of the present invention. For example, the composition can be a pharmaceutical composition, in particular a vaccine. In this case, the composition can further include a pharmaceutically acceptable carrier, excipient, diluent and / or adjuvant. For example, in the case of a vaccine, the vaccine can further include an aluminum adjuvant, a saponin adjuvant, lipid A, monophosphoryl lipid A, trehalose dimycolate, an oil emulsion adjuvant, a polysaccharide adjuvant, a Toll-like receptor activator and / or a STING agonist to enhance the subject's immune response to the immunogen. Such fusion proteins, nucleic acids, vectors, host cells and / or trimeric fusion proteins or compositions of the present invention can be used to treat or prevent conditions or diseases mediated by pathogens. Pathogens are viruses, bacteria, fungi and / or parasites. For example, the virus can be selected from various types of coronaviruses, HIV and influenza viruses. For example, the fusion protein of the present invention can be prepared into a vaccine for preparing a vaccine for the currently popular SARS-CoV-2 virus or influenza virus.

[0057] Example

[0058] The purpose, technical features and beneficial effects of the present invention are further described in detail below in conjunction with the embodiments, but the embodiments of the present invention are not limited thereto.

[0059] Example 1: Construction and expression of recombinant human type III collagen-RBD trimer protein.

[0060] The amino acid sequence of the human recombinant type III collagen designed by the inventor is shown in SEQ ID NO.3. The amino acid sequence of the SARS-CoV-2 RBD protein is shown in SEQ ID NO.4. The amino acid sequence of the intermediate connecting arm is shown in SEQ ID NO.8. The amino acid sequence of the fusion protein of collagen-RBD protein is shown in SEQ ID NO.6. The optimized nucleotide sequence encoding the fusion protein of collagen-RBD protein is shown in SEQ ID NO.7, in which 8 histidine coding nucleotides and a stop codon are added.

[0061] According to the nucleotide sequence of collagen-RBD fusion protein, Nanjing GenScript Biotech was commissioned to perform gene synthesis. The synthesized gene fragment was connected to the pFUSE-hIgG1-Fc2 vector through the EcoR I and Nco I restriction sites ( Figure 1 ) to obtain the pFUSE-hIgG1-Fc2 vector containing the nucleotide sequence of collagen-RBD fusion protein. After the sequencing is correct, the plasmid is transformed into DH5α competent cells. The specific steps are as follows:

[0062] 1) Take a tube of DH5α competent cells from -80℃ and place it on ice.

[0063] 2) Add 1 μl of the constructed plasmid to DH5α competent cells and place on ice for 30 min.

[0064] 3) After heat shock at 42°C for 90 seconds, place the cells on ice immediately.

[0065] 4) After standing for 2 minutes, add 600 μl of LB without resistance and place in a shaker at 37°C for 1 hour.

[0066] 5) Take 100ul of bacterial solution and spread it on a LAB plate containing bleomycin.

[0067] 6) After the colonies have grown, pick out the single clones and add them to 5 ml of LB containing bleomycin. After 12 hours, transfer the 5 ml of LB to 500 ml of LB.

[0068] 7) After 8 hours, use Tiangen's plasmid extraction kit to extract the plasmid.

[0069] Then, the collagen-RBD protein fusion protein is expressed, and the specific steps are as follows:

[0070] 1) Subculture the well-growing EXPi293F cells to a density of 1x10 6 / ml.

[0071] 2) After 12 h, the plasmid containing collagen-RBD encoding nucleotide was transfected according to the instructions of EZ Trans (Liji Biotechnology) transfection reagent.

[0072] 3) After 6 days of continuous cultivation, the cells were centrifuged at 2000 rpm and the cell supernatant was collected.

[0073] Collagen-RBD protein was purified using NI-NTA. The specific steps are as follows:

[0074] 1) Filter the collected cell supernatant through a 0.45 μm filter membrane.

[0075] 2) Add 1 ml of NI-NTA (Qiagen) to the protein purification column.

[0076] 3) Add 5 ml of ddH2O to remove the alcohol in NI-NTA.

[0077] 4) Add 10 ml of binding buffer (40 mM NaH2PO3, 500 mM NaCl, pH:

[0078] 7.8) Equilibrate NI-NTA.

[0079] 5) Add the treated NI-NTA to the protein supernatant and incubate at 4°C for 3 h.

[0080] 6) Then use the binding buffer containing 5mM imidazole to wash the impurities for 30 minutes.

[0081] ml volume.

[0082] 7) Elute the target protein using binding buffer containing 40 mM imidazole.

[0083] 8) Run SDS-PAGE on the eluted target protein.

[0084] The results are as follows Figure 2 As shown, collagen-RBD can be successfully expressed and high-purity collagen-RBD protein is obtained. After denaturation treatment, the molecular weight is about 80kDa, which is consistent with the expected size.

[0085] In order to verify whether the protein forms a trimer, we used a particle size analyzer to detect the molecular weight of the protein. The results showed that the predicted molecular weight of the collagen-RBD protein was 255 kDa, which is about 3 times the size of the monomer ( Figure 3 ). Therefore, the collagen-RBD is a trimeric protein.

[0086] Example 2: Comparison of the levels of RBD-specific binding antibodies produced after immunization of mice with collagen-RBD trimer protein and RBD monomer protein

[0087] Materials: Balb / c was purchased from Beijing Weitonglihua Biotechnology Co., Ltd.; aluminum adjuvant was purchased from Thermo Scientic; RBD-His monomer protein was purchased from Shanghai Kaika Biotechnology Co., Ltd. CF501 adjuvant has the following structure and is owned by the applicant's laboratory.

[0088]

[0089] Twenty-four Balb / c mice aged 6 weeks were divided into 4 groups, 6 mice in each group. The first group was injected intramuscularly with 5 μg collagen-RBD trimer protein and 20 μg CF501 adjuvant; the second group was injected intramuscularly with 5 μg RBD monomer protein and 20 μg CF501 adjuvant; the third group was injected intramuscularly with 5 μg collagen-RBD trimer protein and an equal volume of aluminum adjuvant; the fourth group was injected intramuscularly with 5 μg RBD monomer protein and an equal volume of aluminum adjuvant. The immunization procedure of mice is as follows Figure 4 As shown. The mice were immunized twice on days 0 and 14, and the mouse serum was collected on day 21. The mouse serum was inactivated at 56°C for 30 min.

[0090] The ELISA method was used to detect the IgG antibody titer specific to SARS-CoV-2 RBD in mouse serum. The specific steps are as follows:

[0091] 1) 1 μg / ml RBD-His monomer protein (Kaika Biotechnology) was coated on the ELISA plate and placed at 4°C overnight for coating.

[0092] 2) Block the ELISA plate, add 200 μl of PBS containing 5% BSA to each well, and incubate at 37° C. for 2 h.

[0093] 3) Dilute the mouse serum with PBST, starting from 100-fold dilution and then performing 3-fold dilution.

[0094] 4) Add the diluted mouse serum to the ELISA plate and incubate at 37°C for 45 min.

[0095] 5) Wash the ELISA plate 5 times with PBST.

[0096] 6) Add HRP-labeled rabbit anti-mouse IgG secondary antibody (Dako) and incubate at 37°C for 45 min.

[0097] 7) Wash the ELISA plate 5 times with PBST.

[0098] 8) Add TMB substrate for color development and allow to react for 15 minutes.

[0099] 9) Add H2SO4 to stop color development.

[0100] 10) Use an ELISA reader to read OD450 and calculate the antibody titer in the serum.

[0101] The results are as follows Figure 5 , Figure 6 As shown in Table 1. After mice were immunized with CF501 / collagen-RBD trimer, the average titer of IgG antibodies against SARS-CoV-2 RBD in the serum reached 656100, which was 28 times higher than the RBD-specific IgG antibody titer in the serum of mice immunized with CF501 / RBD monomer. At the same time, the IgG antibody titer in the serum of mice immunized with aluminum adjuvant / collagen-RBD trimer was also nearly 5 times higher than that in the serum of mice immunized with aluminum adjuvant / RBD monomer. These data indicate that collagen-RBD trimer protein can effectively enhance the immunogenicity of RBD as an immunogen.

[0102] Table 1: SARS-CoV-2 RBD-specific IgG antibody titers in mouse sera 21 days after immunization

[0103]

[0104]

[0105] Example 3: Collagen-RBD trimer protein can induce high-strength neutralizing antibody titers against SARS-CoV-2 pseudovirus in mice.

[0106] To compare the titers of neutralizing antibodies against SARS-CoV-2 induced by collagen-RBD trimers and RBD monomers, we tested the neutralizing effect of sera from immunized mice on SARS-CoV-2 pseudoviruses. The packaging steps of SARS-CoV-2 pseudoviruses are as follows:

[0107] 1) Subculture the HEK-293T cells that have grown well.

[0108] 2) After 24 hours, the backbone plasmid PNL-4-3 and the SARS-CoV-2 envelope plasmid PcDNA3.1-SARS-CoV-2-S (Liu, Z., Zhou, J., Xu, W. et al. A novel STING agonist-adjuvanted pan-sarbecovirus vaccine elicits potent and durable neutralizingantibody and T cell responses in mice, rabbits and NHPs. Cell Res 32, 269–287 (2022). https: / / doi.org / 10.1038 / s41422-022-00612-2) were co-transfected into HEK-293T cells using Vigofect transfection reagent.

[0109] 3) After 12 hours, replace with new DMEM medium containing 10% FBS.

[0110] 4) After another 48 hours, the cell supernatant is collected to be the SARS-CoV-2 pseudovirus.

[0111] The specific steps of the SARS-CoV-2 pseudovirus neutralization experiment are as follows:

[0112] 1) Well-growing Huh-7 cells were plated in a 96-well plate, with 10,000 cells per well.

[0113] 2) After 8 hours, mouse serum was diluted with DMEM.

[0114] 3) The diluted mouse serum was mixed with the SARS-CoV-2 pseudovirus and incubated for 30 minutes.

[0115] 4) Add pseudovirus and mouse serum into Huh-7 cells.

[0116] 5) After 12 hours, replace with fresh DMEM medium.

[0117] 6) After 48 h, the luciferase value of each well was detected using Promega's luciferase kit.

[0118] 7) Calculate the neutralizing antibody titer in the serum.

[0119] The results are as follows Figure 7 , Figure 8As shown in Table 2. The average neutralizing antibody titer against SARS-CoV-2 pseudovirus produced by collagen-RBD trimer protein with CF501 as adjuvant reached 3620, while the average neutralizing antibody titer against SARS-CoV-2 pseudovirus produced by RBD monomer with CF501 as adjuvant was only 191. Similarly, the neutralizing antibody titer of mice in the aluminum adjuvant-collagen-RBD trimer protein group was also higher than that of aluminum adjuvant-RBD monomer protein.

[0120] Table 2: Neutralizing antibody titers against SARS-CoV-2 pseudovirus in sera of mice immunized 21 days ago

[0121]

[0122] Example 4: Comparison of HA1-specific binding antibody levels produced after immunization of mice with collagen-H1N1 HA1 trimer protein and H1N1 HA1 monomer protein

[0123] Construction and expression of recombinant type III collagen-H1N1 HA1 trimer protein

[0124] According to the nucleotide sequence of collagen-H1N1 HA1 fusion protein, Nanjing GenScript Biotech Co., Ltd. was commissioned to perform gene synthesis. The synthesized gene fragment was connected to the pFUSE-hIgG1-Fc2 vector through the EcoR I and Nco I restriction sites to obtain the pFUSE-hIgG1-Fc2 vector containing the nucleotide sequence of collagen-H1N1 HA1 fusion protein. The collagen-HA1 fusion protein was expressed according to the protein expression steps of Example 1.

[0125] Twenty-four Balb / c mice aged 6 weeks were divided into 4 groups, 6 mice in each group. The first group was injected intramuscularly with 5 μg collagen-H1N1 HA1 trimer protein and 20 μg CF501 adjuvant; the second group was injected intramuscularly with 5 μg H1N1 HA1 monomer protein and 20 μg CF501 adjuvant; the third group was injected intramuscularly with 5 μg collagen-H1N1 HA1 trimer protein and an equal volume of aluminum adjuvant; the fourth group was injected intramuscularly with 5 μg H1N1 HA1 monomer protein and an equal volume of aluminum adjuvant. The immunization procedure of mice is as follows Figure 4 As shown. The mice were immunized twice on days 0 and 14, and the mouse serum was collected on day 21. The mouse serum was inactivated at 56°C for 30 min.

[0126] The ELISA method was used to detect the IgG antibody titer specific to H1N1 HA1 in mouse serum. The specific steps are as follows:

[0127] 1) 1 μg / ml H1N1 HA1-His monomer protein was coated on an ELISA plate and placed at 4°C overnight for coating.

[0128] 2) Block the ELISA plate, add 200 μl of PBS containing 5% BSA to each well, and incubate at 37° C. for 2 h.

[0129] 3) Dilute the mouse serum with PBST, starting from 100-fold dilution and then performing 3-fold dilution.

[0130] 4) Add the diluted mouse serum to the ELISA plate and incubate at 37°C for 45 min.

[0131] 5) Wash the ELISA plate 5 times with PBST.

[0132] 6) Add HRP-labeled rabbit anti-mouse IgG secondary antibody (Dako) and incubate at 37°C for 45 min.

[0133] 7) Wash the ELISA plate 5 times with PBST.

[0134] 8) Add TMB substrate for color development and allow to react for 15 minutes.

[0135] 9) Add H2SO4 to stop color development.

[0136] 10) Use an ELISA reader to read OD450 and calculate the antibody titer in the serum.

[0137] The results are as follows Fig. 9 and Fig.10 As shown in the figure, whether using CF501 adjuvant or aluminum adjuvant, H1N1 HA1 protein with collagen tag can produce higher levels of IgG antibodies against HA1 protein than H1N1 HA1 protein alone. Therefore, whether it is for new coronavirus antigen or influenza virus antigen, collagen trimer tag can significantly improve the immunogenicity of antigen.

[0138] sequence

[0139] Repeating unit in the trimerization region: GERGAPGFRGPAGPNGIPGEKGPAGERGAP (SEQ ID NO. 1). The region at the C-terminus of the trimerization region: GAPGPCCGG (SEQ ID NO. 2).

[0140] Exemplary trimerization regions:

[0141]

[0142] Exemplary immunogenic regions (SARS-CoV-2 RBD protein amino acid sequence):

[0143]

[0144] Exemplary fusion proteins (without His tag):

[0145]

[0146]

[0147] (SEQ ID NO. 5, the trimerization region is underlined and the immunogenic region is in bold).

[0148] Exemplary fusion protein (containing His tag):

[0149] (SEQ ID NO. 6, His tag is in bold).

[0150] Exemplary fusion protein coding sequences:

[0151]

[0152]

[0153] Exemplary linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO.8). Collagen-H1N1 HA1 fusion protein (with His tag):

[0154]

[0155] Influenza virus H1N1 (A / WSN / 1933) HA1 protein amino acid sequence:

[0156]

Claims

1. A fusion protein, which consists of a trimerization region and an immunogenic region connected by a linker, wherein the trimerization region consists of the amino acid sequence shown in SEQ ID NO. 3; the immunogenic region is an immunogenic protein of a pathogen, and the immunogenic protein is selected from the coronavirus RBD region, HIV membrane protein or influenza virus hemagglutinin protein, and the fusion protein is a trimer fusion protein.

2. The fusion protein according to claim 1, wherein the influenza virus hemagglutinin protein is H1N1 HA1 protein.

3. The fusion protein according to claim 1, wherein the immunogenic region comprises the amino acid sequence shown in SEQ ID NO. 4 or SEQ ID NO.

10. The fusion protein according to claim 1 , wherein the linker is (GGGS)n or (GGGGS)n or (G)n. The fusion protein according to claim 4 , wherein n is an integer of 1-10.

6. The fusion protein of claim 5, wherein n is 2, 3, 4, 5, 6, 7, 8 or 9.

7. The fusion protein according to any one of claims 1 to 6, wherein the trimerization region and the immunogenic region are at the N-terminus and C-terminus of the fusion protein, respectively.

8. The fusion protein according to claim 7, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO. 5 or 9.

9. A nucleic acid comprising nucleotides encoding the fusion protein according to any one of claims 1 to 8.

10. The nucleic acid of claim 9, further comprising nucleotides encoding a purification tag. The nucleic acid according to claim 10 , wherein the purification tag is a His tag, a GST tag, a MBP tag, a SUMO tag or a NusA tag.

12. The nucleic acid according to any one of claims 9 to 11, further comprising nucleotides encoding a leader sequence.

13. The nucleic acid according to claim 12, comprising the sequence shown in SEQ ID NO.

7.

14. A vector comprising the nucleic acid according to any one of claims 9 to 13.

15. The vector of claim 14, comprising an expression control element operably linked to the nucleic acid. The vector according to claim 15 , wherein the expression control element comprises a promoter, a terminator and / or an enhancer.

17. A host cell comprising a nucleic acid according to any one of claims 9 to 13 or a vector according to any one of claims 14 to 16. The host cell according to claim 17 , which is a eukaryotic cell or a prokaryotic cell. The host cell according to claim 18 , wherein the eukaryotic cell is a yeast cell, an animal cell and / or an insect cell.

20. The host cell according to claim 18, wherein the prokaryotic cell is an Escherichia coli cell.

21. A method for producing a fusion protein according to any one of claims 1 to 8, comprising: (1) culturing the host cell according to any one of claims 17 to 20 under suitable culture conditions; (2) harvesting host cells and / or culture medium containing the fusion protein; and (3) Purify the fusion protein.

22. A composition comprising a fusion protein according to any one of claims 1-8, a nucleic acid according to any one of claims 9-13, a vector according to any one of claims 14-16 and / or a host cell according to any one of claims 17-20.

23. The composition of claim 22, wherein the composition is a pharmaceutical composition.

24. The composition of claim 23, wherein the pharmaceutical composition is a vaccine.

25. The composition according to claim 24, further comprising a pharmaceutically acceptable carrier, excipient, diluent and / or adjuvant.

26. The composition according to claim 25, wherein the adjuvant is an aluminum adjuvant, a saponin adjuvant, lipid A, monophosphoryl lipid A, trehalose dimycolate, an oil emulsion adjuvant, a polysaccharide adjuvant, a Toll-like receptor activator and / or a STING agonist.

27. The composition according to claim 26, The adjuvant is .

28. Use of the fusion protein according to any one of claims 1 to 8 for the preparation of a medicament for preventing a condition or disease mediated by a pathogen selected from the group consisting of coronavirus, HIV and influenza virus.

29. The use according to claim 28, wherein the influenza virus is influenza A virus or influenza B virus.

30. The use according to claim 28, wherein the coronavirus is SARS-CoV-2.

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