Recombinant monkey pox virus antigen, monkey pox vaccine and preparation method and application of recombinant monkey pox virus antigen and monkey pox vaccine

Abstract

The invention relates to a recombinant monkey pox virus antigen, a monkey pox vaccine and a preparation method and application of the recombinant monkey pox virus antigen and the monkey pox vaccine. Truncated B6R-2 protein and helicobacter pylori ferritin are fused to form new recombinant protein B6R-Fer-2 which serves as the recombinant monkey pox virus antigen; the amino acid sequence of the truncated B6R-2 protein is as shown in SEQ ID NO: 3, especially the C end of helicobacter pylori ferritin is connected with a His tag through a flexible Linker, and the truncated B6R-2 protein and the His tag are connected through a hinge region flexible Linker to form a recombinant protein B6R-Fer-2. The recombinant protein B6R-Fer-2 can be self-assembled into a virus-like structure, so that the stability and the immunogenicity of the recombinant protein are improved, compared with the existing monkey pox virus single antigen protein, the number of antigens capable of being borne by single immunization can be obviously increased, the neutralizing antibody titer is greatly improved, and the recombinant protein B6R-Fer-2 can be applied to preparation of monkey pox vaccines and can be used for preparing monkey pox vaccines. Particularly, the monkey pox virus subunit vaccine is prepared. By adopting the preparation method, the recombinant protein B6R-Fer-2 can be expressed by using tobacco, the expression quantity of the recombinant protein B6R-Fer-2 is high, and compared with the existing method for expressing by using escherichia coli, the preparation cost of the method is lower.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a recombinant monkeypox virus antigen, a monkeypox vaccine, its preparation method, and its application. Background Technology

[0002] Monkeypox is a rare viral infectious disease similar to smallpox in humans, caused by the monkeypox virus (MPXV). It is also a zoonotic disease. MPXV is a double-stranded DNA virus belonging to the genus Orthopoxvirus in the family Poxviridae. It is round or oval in shape, with a diameter of 200-300 nm. Its nucleic acid core has a biconcave dumbbell-shaped structure. Other viruses in the same genus include smallpox virus, vaccinia virus, and cowpox virus. Currently, the FDA has approved two vaccines for pre-exposure prophylaxis against orthopox viruses, including monkeypox: the second-generation vaccine ACAM2000 and the third-generation vaccine Jynneos. However, ACAM2000 has the ability to replicate in humans and may cause side effects such as encephalitis and myocarditis after vaccination; therefore, it is not yet available to the public. Jynneos, administered in two doses at 28-day intervals, is used to prevent smallpox and monkeypox infection in high-risk adults over 18 years of age. It is currently unclear whether the first dose is sufficient for treatment. Therefore, researching a safe and effective new vaccine, especially one that is effective against monkeypox virus, is crucial for preventing monkeypox infection.

[0003] Screening suitable antigens from monkeypox virus surface antigens to prepare recombinant viral vaccines is an efficient and safe technique. However, due to the complex structure of monkeypox virus antigen proteins and potential immune escape mechanisms, directly expressing the complete protein presents some challenges. For example, the monkeypox virus B6R protein is an envelope protein, and its complete sequence typically includes a transmembrane domain (TM), an intracellular region, and an extracellular antigenic epitope region. Full-length proteins exhibit problems such as insufficient post-translational modification, poor stability, and antigenic epitope heterogeneity in heterogeneous expression systems (e.g., E. coli, mammalian cells). The immunogenicity of B6R depends on multiple discrete B-cell and T-cell epitopes, but these epitopes are relatively dispersed in sequence. In the full-length protein, some epitopes are masked or competitively inhibited, leading to different strengths of immune responses induced by different epitopes.

[0004] Currently, common expression systems include microbial bioreactors such as *E. coli* and yeast, and animal bioreactors such as mammalian cells. However, these systems still suffer from problems such as high production costs, low expression efficiency, and poor protein stability when producing high concentrations of soluble and functional proteins. Plant bioreactors utilize genetic engineering and endogenous metabolic reactions in plant cells or tissues to produce recombinant proteins and other target products on a large scale and at low cost, offering good scalability, safety, and cost-effectiveness. However, proteases are ubiquitous in living organisms, and whether plant bioreactors can be used to produce recombinant monkeypox virus antigen proteins remains unclear. Summary of the Invention

[0005] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a recombinant protein B6R-Fer, a monkeypox vaccine, its preparation method, and its applications. The aim is to discover that among the new recombinant proteins B6R-Fer, B6R-Fer-1, B6R-Fer-2, and B6R-Fer-3, formed by fusing the full-length B6R protein (which serves as a monkeypox virus antigen) and truncated versions of the B6R protein (B6R-1, B6R-2, and B6R-3) with Helicobacter pylori ferritin, only recombinant protein B6R-Fer-2 can self-assemble into virus-like particles, serving as a highly immunogenic monkeypox virus antigen. Furthermore, its expression level is high in tobacco leaves, thereby solving the technical problem of low immunogenicity of existing single monkeypox virus antigens.

[0006] To achieve the above objectives, according to one aspect of the present invention, a recombinant monkeypox virus antigen is provided, which comprises fusing a truncated B6R-2 protein with Helicobacter pylori ferritin to form a novel recombinant protein B6R-Fer-2, serving as the recombinant monkeypox virus antigen; the amino acid sequence of the truncated B6R-2 protein is as shown in SEQ ID NO:3; the fusion is performed by directly fusing the two proteins together or by linking them together with a flexible linker in the hinge region, wherein the amino acid sequence of the flexible linker includes GSGG, GSG, GGG, or SGGSG.

[0007] Preferably, the recombinant monkeypox virus antigen has the amino acid sequence of the Helicobacter pylori ferritin as shown in SEQ ID NO:9, and preferably the C-terminus of the Helicobacter pylori ferritin is linked to a His tag via a flexible linker.

[0008] Preferably, the recombinant monkeypox virus antigen has the amino acid sequence of the Helicobacter pylori ferritin as shown in SEQ ID NO:9 or SEQ ID NO:18; the amino acid sequence shown in SEQ ID NO:3 can be directly fused with the amino acid sequence shown in SEQ ID NO:9, or the two can be linked by a flexible linker in the hinge region to form a recombinant protein B6R-Fer-2; the amino acid sequence of the flexible linker includes GSGG, GSG, GGG or SGGSG.

[0009] Preferably, the recombinant monkeypox virus antigen has an amino acid sequence of the recombinant protein B6R-Fer-2 selected from those shown in SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:21 or SEQ ID NO:25.

[0010] According to another aspect of the present invention, a biomaterial for expressing recombinant monkeypox virus antigen is also provided, comprising a nucleic acid molecule encoding the recombinant protein B6R-Fer-2 as described in the present invention, and an expression cassette, recombinant expression vector, recombinant microorganism or plant bioreactor for expressing the nucleic acid molecule.

[0011] According to another aspect of the present invention, a method for preparing recombinant monkeypox virus antigen is also provided, which includes the step of expressing recombinant protein B6R-Fer-2 using biological materials as described in the present invention.

[0012] Preferably, the preparation method uses a plant expression vector to construct a recombinant expression vector containing a nucleic acid sequence encoding the recombinant protein B6R-Fer-2, expresses it by transfecting tobacco leaves, and obtains the recombinant protein B6R-Fer-2 after extraction and purification, which is used as a recombinant monkeypox virus antigen.

[0013] Preferably, in the preparation method, the amino acid sequence of the recombinant protein B6R-Fer-2 is as shown in SEQ ID NO:21 or SEQ ID NO:25, and the extraction and purification steps are as follows:

[0014] Transfected tobacco leaves were lysed using plant cell lysis buffer, and the supernatant was collected by centrifugation. The recombinant protein B6R-Fer-2 was purified by binding its His tag to a nickel column. The packing material was first rinsed with an imidazole-free buffer and a non-specific protein elution buffer to remove impurities, and then the target protein was eluted with a specific protein elution buffer. The eluent was collected, dialyzed, and concentrated to obtain the purified recombinant protein B6R-Fer-2.

[0015] According to another aspect of the present invention, a method for improving the immunogenicity of monkeypox virus antigen B6R is also provided, wherein a truncated B6R-2 protein, serving as the antigen, is fused with ferritin to form a recombinant protein B6R-Fer-2, which is then self-assembled into virus-like particles and used as the monkeypox virus antigen; the amino acid sequence of the truncated B6R-2 protein is as shown in SEQ ID NO:3. Preferably, the truncated B6R-2 protein and ferritin are fused together via a flexible linker, the amino acid sequence of which includes GSGG, GSG, GGG, or SGGSG.

[0016] Preferably, in the preparation method, the ferritin is Helicobacter pylori ferritin, whose amino acid sequence is shown in SEQ ID NO:9. The truncated B6R-2 protein is fused to the Helicobacter pylori ferritin via a flexible linker. Preferably, the C-terminus of the Helicobacter pylori ferritin is linked to a His tag via a flexible linker, as shown in SEQ ID NO:18. The truncated B6R-2 protein is fused to the Helicobacter pylori ferritin via a flexible linker, and the amino acid sequence of the flexible linker includes GSGG, GSG, GGG, or SGGSG.

[0017] According to another aspect of the present invention, the use of recombinant monkeypox virus antigen as described herein in the preparation of monkeypox virus vaccines or anti-monkeypox virus drugs is also provided.

[0018] According to another aspect of the present invention, a monkeypox virus vaccine is also provided, comprising the recombinant protein B6R-Fer-2 as described in the present invention, wherein the recombinant protein B6R-Fer-2 is self-assembled into virus-like particles and serves as a recombinant monkeypox virus antigen.

[0019] Preferably, the monkeypox virus vaccine is a monkeypox virus subunit vaccine, and the amino acid sequence of the recombinant protein B6R-Fer-2 is shown in SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:21 or SEQ ID NO:25.

[0020] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following advantages:

[0021] Beneficial effects:

[0022] This invention utilizes computer simulation prediction and a truncated expression strategy to segment the monkeypox virus antigen B6R protein and screen for three highly immunogenic truncated variants: B6R-1 (amino acid sequence shown in SEQ ID NO:2), B6R-2 (amino acid sequence shown in SEQ ID NO:3), and B6R-3 (amino acid sequence shown in SEQ ID NO:4). These three truncated variants, along with the antigen B6R protein, are fused with ferritin to form new recombinant proteins. The results show that only the recombinant protein B6R-Fer-2, formed by fusing antigen B6R-2 with ferritin, can self-assemble into a virus-like structure and can be used as a recombinant monkeypox virus antigen. Compared to existing single-antigen proteins of monkeypox virus, this recombinant protein B6R-Fer-2 can increase the number of antigens that can be carried in a single immunization, thereby improving the immunogenicity of the single antigen. Compared to existing single-antigen proteins of monkeypox virus, it significantly increases the neutralizing antibody titer and can be applied to the preparation of monkeypox virus vaccines or anti-monocholepenia virus drugs. In particular, using the recombinant protein B6R-Fer-2 with the amino acid sequence shown in SEQ ID NO:25 as the antigen, the antibody titer (log10) can reach more than 4 after one immunization 14 days later. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the full-length B6R protein and its various truncated expression variants;

[0024] Figure 2 This is a 3D model diagram of the predicted structure of the full-length B6R protein and its various truncated expression variants;

[0025] Figure 3 This is a diagram showing the phenotypic changes in tobacco leaves;

[0026] Figure 4 This is a Western blot analysis of recombinant protein extracts at different days (dpi) after transfection;

[0027] Figure 5 Transmission electron micrograph of recombinant protein;

[0028] Figure 6 This is a flowchart of the mouse immunization process;

[0029] Figure 7 This is a graph showing the titer of specific antibodies against B6R-Fer-2 antigen in mice. Detailed Implementation

[0030] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0031] While screening suitable antigens from monkeypox virus surface antigens to prepare recombinant viral vaccines is an efficient and safe technique, directly expressing the complete monkeypox virus antigen protein remains challenging due to its complex structure and potential immune escape mechanisms. Furthermore, single antigens often exhibit low immunogenicity. This invention utilizes computer simulation prediction and a truncated expression strategy to segment the monkeypox virus antigen B6R protein and screen for the most immunogenic regions while maintaining its immunogenicity and biological activity. By truncating the expression of the B6R protein, the conformational complexity of the protein is reduced, allowing for the design of shorter, smaller protein molecules. We truncated the full-length B6R protein (amino acid sequence as shown in SEQ ID NO:1) to express small proteins: B6R-1 protein (amino acid sequence as shown in SEQ ID NO:2), B6R-2 protein (amino acid sequence as shown in SEQ ID NO:3), and B6R-3 protein (amino acid sequence as shown in SEQ ID NO:4). These proteins were then fused with Helicobacter pylori ferritin to form new recombinant proteins B6R-Fer, B6R-Fer-1, B6R-Fer-2, and B6R-Fer-3, which were then used as monkeypox virus antigens.

[0032] Recombinant plasmids encoding recombinant proteins B6R-Fer, B6R-Fer-1, B6R-Fer-2, and B6R-Fer-3 were constructed using restriction endonucleases through genetic engineering. These plasmids were then transformed into Agrobacterium tumefaciens and used to infect tobacco leaves. The results showed that only recombinant proteins B6R-Fer and B6R-Fer-2 exhibited high expression levels in tobacco. However, only recombinant protein B6R-Fer-2, after expression in tobacco, was able to self-assemble into virus-like particles (VLPs), which can serve as a monkeypox virus antigen. Recombinant protein B6R-Fer, on the other hand, did not self-assemble into virus-like particles.

[0033] When the amino acid sequence of recombinant protein B6R-Fer-2 is as shown in SEQ ID NO:25, the diameter of the obtained recombinant protein B6R-Fer-2 is approximately 18–38 nm. Using it as a recombinant monkeypox virus antigen, it is mixed with an adjuvant to prepare a monkeypox subunit nanovaccine. After a single immunization, the antibody titer (log10) can reach over 4 14 days later. Compared to existing single-antigen proteins of monkeypox virus, the recombinant protein B6R-Fer-2 provided by this invention can significantly increase the number of antigens that can be carried in a single immunization. The fusion protein stably forms a virus-like structure (VLP), which can improve the stability and immunogenicity of the recombinant protein and greatly increase the neutralizing antibody titer.

[0034] Based on this, the present invention provides a recombinant monkeypox virus antigen, which fuses B6R-2 protein with Helicobacter pylori ferritin to form a new recombinant protein B6R-Fer-2, serving as the recombinant monkeypox virus antigen; the amino acid sequence of the B6R-2 protein is shown in SEQ ID NO:3, and the amino acid sequence shown in SEQ ID NO:3 is as follows:

[0035] WNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGS.

[0036] The amino acid sequence of the Helicobacter pylori ferritin is shown in SEQ ID NO:9, and the amino acid sequence shown in SEQ ID NO:9 is as follows:

[0037] DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS.

[0038] In some embodiments, the amino acid sequence shown in SEQ ID NO:3 is directly fused with the amino acid sequence shown in SEQ ID NO:9, or the two are linked by a flexible linker in the hinge region to form a recombinant protein B6R-Fer-2; the amino acid sequence of the flexible linker includes GSGG, GSG, GGG or SGGSG.

[0039] In this invention, the truncated B6R-2 protein as shown in SEQ ID NO:3, serving as an antigen, is directly fused with ferritin as shown in SEQ ID NO:9, serving as a fusion vector. The amino acid sequence of the resulting recombinant protein B6R-Fer-2 is shown in SEQ ID NO:12. As an alternative optimization, the two are linked together by a hinge region linker to form a recombinant protein. The amino acid sequence of the flexible linker includes GSGG, GSG, GGG, or SGGSG. When the flexible linker is GSGG, the amino acid sequence of the recombinant protein B6R-Fer-2 is shown in SEQ ID NO:16.

[0040] In some embodiments, the C-terminus of the ferritin is connected to a His tag via a flexible linker, wherein the flexible linker is GSG, and its amino acid sequence is shown in SEQ ID NO:18. The amino acid sequence of the recombinant protein B6R-Fer-2 obtained by directly fusing the truncated B6R-2 protein shown in SEQ ID NO:3 with the ferritin shown in SEQ ID NO:18 is shown in SEQ ID NO:21. As an alternative optimization, the two are linked by a hinge region linker to form a recombinant protein, wherein the amino acid sequence of the flexible linker includes GSGG, GSG, GGG, or SGGSG. When the flexible linker is GSGG, the amino acid sequence of the recombinant protein B6R-Fer-2 is shown in SEQ ID NO:25.

[0041] The recombinant protein B6R-Fer-2 can be used as a highly immunogenic recombinant monkeypox virus antigen in this invention for the preparation of monkeypox vaccines, especially for the preparation of monkeypox subunit vaccines. In some embodiments, the recombinant protein B6R-Fer-2 is self-assembled into virus-like particles (VLPs) and used as a monkeypox virus antigen with a diameter of 18-38 nm, which is then mixed with an adjuvant to prepare a monkeypox subunit nanovaccine.

[0042] The present invention also provides a method for improving the immunogenicity of a single monkeypox virus antigen B6R, comprising fusing a truncated B6R protein, B6R-2, with ferritin to form a recombinant protein, B6R-Fer-2, which is then self-assembled into virus-like particles and used as a monkeypox virus antigen; the amino acid sequence of the B6R-2 is shown in SEQ ID NO:3.

[0043] The preferred antigen B6R-2 is fused to ferritin via a flexible linker, the amino acid sequence of which includes GSGG, GSG, GGG or SGGSG.

[0044] In some embodiments, the ferritin is Helicobacter pylori ferritin, whose amino acid sequence is shown in SEQ ID NO:9; preferably, the C-terminus of Helicobacter pylori ferritin is linked to a His tag via a flexible linker, such as GSG, whose amino acid sequence is shown in SEQ ID NO:18.

[0045] In this invention, a recombinant expression vector can be constructed by inserting the target gene 1 encoding the protein B6R-2 as described in this invention and the target gene 2 encoding the ferritin as described in this invention into an expression vector. The recombinant protein B6R-Fer-2 is expressed by transfection into cells, microorganisms, or plants, and after self-assembly into virus-like particles (VLPs), it is extracted, purified, and used as a monkeypox virus antigen. For example, the amino acid sequence of the B6R-2 protein is shown in SEQ ID NO:3, and the nucleic acid sequence of the target gene 1 encoding the B6R-2 protein is shown in SEQ ID NO:7; the amino acid sequence of the ferritin is shown in SEQ ID NO:9, and the nucleic acid sequence of the target gene 2 encoding the ferritin is shown in SEQ ID NO:27.

[0046] In some embodiments, the expression vector is a plant expression vector. The constructed recombinant expression vector is transfected into tobacco leaves to express the recombinant protein B6R-Fer-2 as described in this invention. After self-assembly into virus-like particles (VLPs), it serves as a monkeypox virus antigen. Since this invention utilizes ferritin as a fusion vector, it can significantly increase the number of antigens that can be carried in a single immunization. The recombinant protein stably forms a virus-like structure (VLP), which can improve the stability and immunogenicity of the recombinant protein and greatly increase the neutralizing antibody titer.

[0047] The present invention also provides a biomaterial expressing the recombinant protein B6R-Fer-2 as described in the present invention, comprising the following biomaterials:

[0048] A1: A nucleic acid molecule encoding the recombinant protein B6R-Fer-2 as described in this invention;

[0049] A2: An expression cassette containing the nucleic acid molecules described in A1;

[0050] A3: Recombinant expression vectors, recombinant microorganisms, or plant bioreactors containing the nucleic acid molecules described in A1 or the expression cassettes described in A2;

[0051] In some embodiments, the recombinant expression vector is a recombinant plasmid prepared using a plant expression vector such as pFolia40108, and the recombinant microorganism is Agrobacterium tumefaciens containing the recombinant plasmid or Escherichia coli containing the recombinant plasmid; the plant bioreactor is a tobacco leaf transfected with the recombinant plasmid.

[0052] In addition, this invention provides a method for preparing recombinant monkeypox virus antigen, which includes the step of expressing recombinant protein B6R-Fer-2 using biological materials as described in this invention. In some embodiments, a plant expression vector is used to construct a recombinant expression vector containing a nucleic acid sequence encoding the recombinant protein B6R-Fer-2. Expression is performed by transfecting tobacco leaves, and the recombinant protein B6R-Fer-2 is obtained after extraction and purification. After self-assembly, it is used as a recombinant monkeypox virus antigen. For example, a recombinant plasmid containing the target gene encoding the recombinant protein B6R-Fer-2 is prepared using plasmid pFolia40108 as a vector. This plasmid is then transformed into competent Escherichia coli cells for incubation, and resuspended cells are prepared. Positive E. coli are screened, and their recombinant plasmids are extracted. The recombinant plasmid is transformed into Agrobacterium tumefaciens strains for tobacco transfection. After 14 days of transfection, the accumulated recombinant protein B6R-Fer-2 is extracted, as detailed below:

[0053] Transfected tobacco leaves were lysed using plant cell lysis buffer, and the supernatant was collected by centrifugation. After filtration, the supernatant was purified using a His-tagged nickel column at low temperature. The packing material was rinsed with imidazole-free buffer and non-specific protein elution buffer to remove contaminating proteins. The target protein was then eluted with specific protein elution buffer, and the eluent containing the target protein was collected. After dialysis and concentration, the purified recombinant protein B6R-Fer-2 was obtained.

[0054] The recombinant protein B6R-Fer-2 constructed according to this method is expressed and then self-assembled into virus-like particles, which can be used as a monkeypox virus antigen to improve the immunogenicity of a single monkeypox virus antigen. It can be applied to the preparation of monkeypox virus vaccines or anti-monkeypox virus drugs.

[0055] This invention also provides the application of the recombinant protein B6R-Fer-2 as described herein in the preparation of monkeypox virus vaccines or anti-monocholevirus drugs, particularly in the preparation of monkeypox virus subunit vaccines. Subunit vaccines are vaccines prepared by expressing and purifying specific viral antigen proteins using recombinant technology. Compared to traditional inactivated or live attenuated vaccines, subunit vaccines do not contain the complete pathogen, thus offering higher safety and avoiding the infection risk in immunocompromised individuals that may be associated with live viruses.

[0056] On the other hand, the present invention also provides a monkeypox virus vaccine, which includes recombinant protein B6R-Fer-2 as described in the present invention as an antigen; the vaccine is a monkeypox virus subunit vaccine and further includes an adjuvant. In some embodiments, the amino acid sequence of recombinant protein B6R-Fer-2 in the monkeypox virus subunit vaccine is as shown in SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:21 or SEQ ID NO:25, and its mass ratio with aluminum hydroxide adjuvant is 1:1. Wherein, using recombinant protein B6R-Fer-2 with the amino acid sequence shown in SEQ ID NO:25 as the antigen, the antibody titer (log10) can reach 4 or higher after one immunization 14 days later.

[0057] The following are examples.

[0058] The main reagents and their manufacturers in the following examples are as follows:

[0059] E. coli DH5α strain: Qingke Biotechnology Co., Ltd.;

[0060] Agrobacterium tumefaciens C58C1 strain: Qingke Biotechnology Co., Ltd.;

[0061] T4 DNA ligase: New England Biolabs (NEB);

[0062] BsaI endonuclease: New England Biolabs (NEB);

[0063] BsmBI endonuclease: New England Biolabs (NEB);

[0064] Agarose Gel DNA Recovery Kit: Tiangen Biotech (Beijing) Co., Ltd.;

[0065] DNA Expression Vector Minimum Prep Kit: Tiangen Biotech (Beijing) Co., Ltd.;

[0066] 2-Morpholine ethanesulfonic acid (MES): Hefei Bomei Biotechnology Co., Ltd.;

[0067] Acetyleugenone: Hefei Bomei Biotechnology Co., Ltd.;

[0068] Dimethyl sulfoxide (DMSO): Biosharp Technologies Co., Ltd.

[0069] Tris(hydroxymethyl)aminomethane (Tris): Biosharp Technology Co., Ltd.

[0070] His-tagged protein agarose purification resin (Ni-NTA): Suzhou Lanxiao Biotechnology Co., Ltd.;

[0071] Triton X-100: Biosharp Technology Co., Ltd.

[0072] Benzyl sulfonyl fluoride (PMSF): Sangon Biotech (Shanghai) Co., Ltd.;

[0073] β-Mercaptoethanol (β-ME): Merck Sigma-Aldrich brand;

[0074] Imidazole: BBI Life Sciences, Inc.

[0075] NP-40 lysis buffer: Biosharp Technologies Co., Ltd.

[0076] Li Chunhong: Sangon Biotech (Shanghai) Co., Ltd.;

[0077] His antibody: Genscript Biotech Inc.;

[0078] Gradient PCR instrument: Tiangen Biotech (Beijing) Co., Ltd.;

[0079] Benchtop Refrigerated Centrifuge: Tianmei Instruments Laboratory Equipment (Shanghai) Co., Ltd.;

[0080] Ultraviolet spectrophotometer: Spyderco (Shanghai) Co., Ltd.;

[0081] Intelligent constant temperature incubator: Hefei Youke Instrument Equipment Co., Ltd.;

[0082] Thermostatic oscillator: Suzhou Jiemei Electronics Co., Ltd.;

[0083] General-purpose electrophoresis system: Wex Technology (Beijing) Co., Ltd.;

[0084] Chemical photoluminescence gel imaging system: Shanghai Jiapeng Technology Co., Ltd.

[0085] Example 1: Construction of four recombinant expression vectors: B6R-Fer, B6R-Fer-1, B6R-Fer-2, and B6R-Fer-3. This example uses the B6R protein (amino acid sequence as shown in SEQ ID NO:1), truncated versions of the B6R protein (B6R-1 protein, amino acid sequence as shown in SEQ ID NO:2), B6R-2 protein, and B6R-3 protein (amino acid sequence as shown in SEQ ID NO:3) of monkeypox virus antigen as the target antigen proteins. These proteins are fused with Helicobacter pylori ferritin (its amino acid sequence is shown in SEQ ID No. 9) to form new recombinant proteins B6R-Fer, B6R-Fer-1, B6R-Fer-2, and B6R-Fer-3. The antigen proteins and ferritin are directly fused together or linked using a flexible linker with a hinge region. The amino acid sequence of the flexible linker includes GSGG, GSG, GGG, or SGGSG. SEQ ID NO:1:

[0086] TCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSF GEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYH;

[0087] SEQ ID NO:2:

[0088] TCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNT;

[0089] SEQ ID NO:3:

[0090] WNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGS;

[0091] SEQ ID NO:4:

[0092] FSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYH;

[0093] SEQ ID NO:9:

[0094] DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS, preferably, the C-terminus of the Helicobacter pylori ferritin is linked to a His tag via a flexible linker.

[0095] Taking the preparation of recombinant proteins B6R-Fer (the amino acid sequence of which is shown in SEQ ID No. 23), B6R-Fer-1 (the amino acid sequence of which is shown in SEQ ID No. 24), B6R-Fer-2 (the amino acid sequence of which is shown in SEQ ID No. 25), and B6R-Fer-3 (the amino acid sequence of which is shown in SEQ ID No. 26) as an example, the specific process is as follows:

[0096] (1) Computer simulation prediction

[0097] Using the B6R gene (its full-length nucleic acid sequence is shown in SEQ ID No. 5) as a template, AlphaFold2 was used to simulate the full-length three-dimensional structure of the B6R protein and predict antigenic epitopes. Based on the AlphaFold2 prediction results, the full-length B6R gene was truncated into three expression variants. The results are as follows: Figure 1 and Figure 2 As shown.

[0098] (2) Target gene amplification

[0099] Using the B6R gene (its nucleic acid sequence is shown in SEQ ID No. 5), B6R-1 gene (its nucleic acid sequence is shown in SEQ ID No. 6), B6R-2 gene (its nucleic acid sequence is shown in SEQ ID No. 7), and B6R-3 gene (its nucleic acid sequence is shown in SEQ ID No. 8) as templates, PCR amplification was performed using primers F-B6R, F-B6R-1, F-B6R-2, F-B6R-3 and R-B6R, R-B6R-1, R-B6R-2, R-B6R-3. The sequences of the upstream primer F and the downstream primer R are shown in Table 1.

[0100] Table 1. Sequences of each primer

[0101]

[0102]

[0103] The amplified products were identified by agarose gel electrophoresis (the synthesis was simulated in advance on the software before synthesis to obtain the size of the target band, and then compared).

[0104] The ferritin is custom-made and can be directly extracted from the expression vector by shaking bacteria. This ferritin is His-tagged ferritin, and the two are linked by a linker (GSG). Its amino acid sequence is shown in SEQ ID No. 18.

[0105] (3) Target gene enzyme digestion

[0106] The amplified B6R, B6R-1, B6R-2, and B6R-3 target genes and the ferritin target gene (SEQ ID No. 27) were added to the enzyme digestion system and digested simultaneously. The reaction was carried out at 37°C for 2 hours. The enzyme digestion system is shown in Tables 2 and 3. The enzyme digestion products were recovered by agarose gel electrophoresis.

[0107] Table 2 B6R / B6R-1 / B6R-2 / B6R-3 Enzyme Digestion Reaction System

[0108] name Volume (μL) Target gene (B6R / B6R-1 / B6R-2 / B6R-3) 40 Buffer (10×Tango) 5 Restriction endonuclease BsmBI 2 <![CDATA[ddH2O]]> 3 Total volume 40

[0109] Note: Reaction procedure: 37℃ for 2 hours. Enzyme digestion products were detected by agarose gel electrophoresis. BsmBI is a Type II restriction endonuclease that recognizes non-palindromic sequences and performs cleavage outside of the recognition sequence. The recognition sequences of BsmBI are 5'-CGTCTC(N)1-3' and 3'-GCAGAG(N)5-5', where N represents any nucleotide.

[0110] Table 3 Ferritin digestion reaction system

[0111] name Volume (μL) Target gene (Ferritin) 30 Buffer (10×Tango) 4 Restriction endonuclease BsmBI 2 <![CDATA[ddH2O]]> 4 Total volume 40

[0112] Note: Reaction procedure: 37℃ for 2 hours.

[0113] The enzyme digestion products were detected by agarose gel electrophoresis. The electrophoresis products were placed under UV light, and the target size bright band was excised along the edge and placed in a 1.5 mL centrifuge tube. Following the instructions of the Tiangen Agarose Gel DNA Recovery Kit, the fragment was recovered by excising it at the target band size. This recovered fragment is the enzyme-digested target band.

[0114] (4) Ligation and transformation of enzyme digestion products to prepare recombinant expression vectors

[0115] In this embodiment, a plant expression vector pFolia40108 (considered to be a special type of plasmid) was ligated with the enzyme digestion product and transformed to prepare a recombinant plasmid, which was used to improve the expression efficiency of the exogenous target gene in plants. Details are as follows:

[0116] The recovered enzyme digestion product B6R, ferritin, and vector pFolia40108 were mixed, and T4 DNA ligase (to achieve linker ligation) and 10× Buffer were added. The reaction system is shown in Table 4.

[0117] Table 4 B6R / B6R-1 / B6R-2 / B6R-3-Fer connection system

[0118] name Volume (μL) Vector (pFolia40108) 1 Target fragment (B6R / B6R-1 / B6R-2 / B6R-3) 1 Ferritin 1 T4 DNA ligase 1 10×T4TangoBuffer 1 <![CDATA[ddH2O]]> 5 Total volume 10

[0119] Note: Reaction procedure: Ligation at 16℃ to obtain recombinant plasmid pFolia40108-B6R / B6R-1 / B6R-2 / B6R-3-Fer with B6R-Fer inserted.

[0120] The four recombinant plasmids were transferred into E. coli. The competent DH5α cells were taken out of the -80℃ freezer and thawed on ice. 5 μL of recombinant plasmid was added to 70 μL of competent DH5α cells, and the cells were repeatedly pipetted 5-8 times. The cells were then incubated on ice for 30 min. The cells were then heat-shocked in a 42℃ water bath for 90 s and immediately placed on ice for 3 min. 800 μL of LB medium equilibrated to room temperature was added, and the cells were cultured at 200 rpm and 37℃ with shaking for 1 h. The culture of the competent cells was centrifuged at 6000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 100-120 μL of the remaining medium. 50 μL of the bacterial culture was evenly spread on a kanamycin LB agar plate and incubated upside down in a 37℃ incubator overnight.

[0121] (5) Extraction of recombinant plasmids

[0122] Single colonies were picked from LB agar plates and transferred to 5 mL of LB liquid medium (containing 50 mg / mL Amp). The culture was incubated overnight at 37°C with shaking. In a sterile environment under laminar flow hood, five single colonies were picked using a sterile toothpick. The end of the toothpick containing the colony was then touched to the bottom of a PCR tube to leave a small amount of colony for colony PCR detection, and the culture was expanded. The PCR reaction system is shown in Table 5.

[0123] Table 5 PCR detection reaction system

[0124] name Volume (μL) Primers F-B6R, F-B6R-1, F-B6R-2 or F-B6R-3 1 Primers R-B6R, R-B6R-1, R-B6R-2 or R-B6R-3 1 2×TaqBuffer 5 <![CDATA[ddH2O]]> 3

[0125] Note: A 10 μL system was used, and the reaction program was 2×Taq: 94℃ deformation, 30 cycles, 72℃ extension;

[0126] PCR products were detected by agarose gel electrophoresis. Colonies with bands consistent with the expected gene fragment and in the correct position were selected for recombinant plasmid extraction by shaking culture. The recombinant plasmid was extracted according to the instructions provided by Tiangen Company's plasmid mini-prep kit.

[0127] (6) Sequencing

[0128] The recombinant plasmid was identified by PCR and sequencing. Sequencing was performed by Changsha Qingke Biotechnology Co., Ltd., and the obtained sequence was compared with the target fragment using Snapgene software. The results showed that the sequence of the synthesized plasmid was consistent with the designed sequence, indicating that the recombinant plasmid was successfully synthesized.

[0129] Example 2: Transient expression experiment of recombinant proteins B6R-Fer, B6R-Fer-1, B6R-Fer-2, and B6R-Fer-3

[0130] (1) Transformation of Agrobacterium tumefaciens with recombinant plasmid

[0131] Recombinant plasmids pFolia40108-B6R-Fer, pFolia40108-B6R-1-Fer, pFolia40108-B6R-2-Fer, and pFolia40108-B6R-3-Fer were transformed into Agrobacterium tumefaciens C58C1 strain via heat shock transformation. Competent Agrobacterium cells were collected and thawed on ice for 30 min. 5 μl of the recombinant plasmid (approximately 1-2 μg) was added to 100 μl of competent Agrobacterium cells and mixed thoroughly. The cells were then incubated on ice for 30 min, flash-frozen in liquid nitrogen for 1 min, then incubated in a 37°C water bath for 5 min, followed by an ice bath for 2 min. 800 μl of liquid LB medium was added, and the cells were incubated at 28°C and 200 rpm for 2 h using a shaker. Centrifuge the bacterial suspension at 6000 rpm for 3 min, remove the culture medium to a final volume of 100 μL, gently pipette to mix the suspension, and spread the suspension onto solid LB agar plates containing Rif and Carb. Incubate at 28°C for 48 h. After plaque growth, select suitable single colonies in a sterile environment under laminar flow hood. Confirm successful transformation with positive PCR testing of the colonies, and then propagate the bacterial suspension. Aliquot the suspension into 1.5 mL centrifuge tubes, add 60% glycerol, and store at -80°C for subsequent tobacco transfection.

[0132] (2) Injection transfection of tobacco and sampling

[0133] ① Solution preparation:

[0134] LB medium (1L): 10g tryptone, 5g yeast extract, 10g NaCl, and ddH2O to make up the volume.

[0135] 0.1M MES (pH 5.6) (1L): 19.524g MES, pH adjusted to 5.6 with NaOH, volume made up with ddH2O, filtered through a 0.22μm filter for sterilization, and stored at room temperature.

[0136] 100mM acetylsyl syringone (AS): 0.036g acetylsyl syringone dissolved in 1mL dimethyl sulfoxide (DMSO).

[0137] 1M MgCl2 (400mL): 95.2g MgCl2·H2O, add ddH2O to make up the volume, and store at room temperature.

[0138] Transfection solution: 10mM MES, 100μM AS, 10mM MgCl2, ddH2O to make up the volume.

[0139] ② Injecting tobacco:

[0140] Agrobacterium containing the target recombinant plasmid was cultured by shaking, and approximately 5 mL of bacterial suspension was collected. The bacterial pellet was collected by gentle centrifugation (room temperature, 6000 rpm, 5 min). The colonies were resuspended in transfection buffer and activated by shaking at 28°C and 100 rpm for 1 h. The OD values ​​of the three activated bacterial suspensions were adjusted to 0.1. The bacterial suspension was injected into the leaves using a sterile syringe for transfection. The transfected tobacco leaves were photographed and sampled for two consecutive weeks.

[0141] ③ Sampling of tobacco leaves:

[0142] Take 0.2g of tobacco leaves with expression at corresponding days after transfection (6dpi, 8dpi, 10dpi, 12dpi, 14dpi) and place them in a 1.5mL centrifuge tube. After quick freezing in liquid nitrogen, grind them into powder with a grinder. Add 500μL of NP-40 lysis buffer to the centrifuge tube, resuspend the leaf powder, let it stand on ice for 10min, and then centrifuge at 14000rpm for 15min at 4℃. Collect the supernatant.

[0143] (3) Phenotypic changes in tobacco leaves

[0144] The transfected tobacco leaves were observed daily, and the results were as follows: Figure 3 As shown, the surface of tobacco leaves expressing B6R-Fer showed no obvious changes on day 6, but black necrotic spots appeared on the leaf surface on day 8 after transfection. On day 10, the infected parts remained green, while the surrounding normal leaves turned yellow. On day 12, some wilting began to appear, and the surrounding normal leaves turned increasingly yellow and were accompanied by leaf deformation. On day 14 after transfection, the uninfected parts of the leaves continued to wilt and turn yellow, while the infected parts remained green.

[0145] On day 6, injection marks became more visible on the surface of tobacco leaves expressing B6R-Fer-1. On day 8, a few black necrotic spots began to appear on the parts infected with Agrobacterium, but at this time, the infected parts of the tobacco leaves were not significantly different from the surrounding normal leaves. On day 12 post-transfection, the Agrobacterium-infected parts of the leaves became lighter in color than the surrounding normal parts, and the leaves became increasingly uneven. On day 14 post-transfection, the tobacco leaves expressing B6R-Fer-2 turned yellow overall but showed no obvious signs of wilting.

[0146] On day 6 post-transfection, small black necrotic spots began to appear on the surface of tobacco leaves expressing B6R-Fer-2. By day 8, these spots had increased in size and number. On day 12, the uninfected parts of the tobacco leaves began to yellow, and the black necrotic spots became more pronounced. On day 14, the yellowing of the surrounding uninfected normal leaves became more pronounced, and the leaves expressing B6R-Fer also turned slightly yellow, exhibiting a yellowish-green hue, with the black necrotic spots becoming larger and more noticeable. However, on day 14 post-transfection, the leaves did not show obvious wilting; the infected areas, however, bulged significantly, leading to overall leaf deformation.

[0147] On days 6 and 8, the surface of tobacco leaves expressing B6R-Fer-3 showed no significant changes; the leaves remained green overall, and the parts infected with Agrobacterium were indistinguishable from normal leaves. However, on day 10, black necrotic spots appeared on the infected parts, and the Agrobacterium-infected leaves remained green, while the surrounding normal leaves began to turn yellow. On day 14 post-transfection, the uninfected leaves began to wilt completely and turn gray, while the Agrobacterium-infected leaves remained green.

[0148] Experimental results showed that the leaf phenotypes of *Nicotiana benthamiana* containing B6R-Fer, B6R-Fer-1, B6R-Fer-2, and B6R-Fer-3 recombinant proteins exhibited significant pathological changes with the number of days of growth after transfection.

[0149] (4) Western blot analysis

[0150] Given the variations in leaf phenotype, proteins extracted from leaves at corresponding days (6 dpi, 8 dpi, 10 dpi, 12 dpi, 14 dpi) were analyzed by Western blot using His antibody as the primary antibody. The consistency of sample loading in each lane was verified by Ponceau S staining of the PVDF membrane. The results are as follows: Figure 4 As shown.

[0151] Tobacco plants expressing B6R-Fer-2 showed slight expression on day 6, with a significant increase in protein expression on day 12. Expression gradually increased with the number of post-transfection growth days, reaching its highest level on day 14. The B6R-Fer-2 protein is approximately 25 kDa, indicating a relatively high overall expression level. Tobacco plants expressing B6R-Fer-1 showed slight expression on day 6, with a slight increase on day 10, reaching its highest level on day 14, but the overall expression level remained low. The B6R-Fer-1 protein is approximately 20 kDa. Tobacco plants expressing B6R-Fer showed significant expression on day 6, with the highest protein expression on day 12, decreasing on day 14 after transfection. The B6R-Fer protein is approximately 35 kDa, indicating a relatively high protein expression level. Tobacco expressing B6R-Fer-3 showed slight expression on days 6 and 8, but protein expression increased significantly on day 14 post-transfection. The B6R-Fer-3 protein is approximately 25 kDa, and its overall expression level is low. This indicates that the expression of different recombinant proteins in tobacco varies significantly and follows no discernible pattern. Among the four recombinant proteins, B6R-Fer-2 was found to have the highest expression level in tobacco.

[0152] Combination Figure 3 Phenotypic changes in tobacco leaves: The phenotype of leaves expressing B6R-Fer changed with the number of days of expression. B6R-Fer protein expression was highest on day 12 post-transfection, and may have degraded as leaves withered on day 14. Combining the phenotypic changes in tobacco leaves expressing B6R-Fer-1, the Agrobacterium-infected parts of the leaves showed no significant color change compared to the surrounding healthy leaves. Combining the phenotypic changes in tobacco leaves expressing B6R-Fer-2, the most significant phenotypic changes occurred on day 14 post-transfection. Combining the phenotypic changes in tobacco leaves expressing B6R-Fer-3, the leaves began to wither significantly on day 14, but protein expression was at its highest. Recombinant protein expression levels were analyzed by Western blotting. After entering the host cell, the recombinant gene sequence underwent transcription and translation independently of the host genome, accumulating and expressing recombinant proteins within the host cell. However, different recombinant gene sequences exhibited different levels of transcription and translation in the host cell, indicating significant differences in the expression levels of different recombinant proteins in tobacco.

[0153] Example 3: Purification experiments of recombinant proteins B6R-Fer, B6R-Fer-1, B6R-Fer-2, and B6R-Fer-3

[0154] (1) Harvesting tobacco

[0155] Tobacco leaves transfected with Agrobacterium and exhibiting a distinct phenotype were harvested, and the main veins of the leaves were removed. Two volumes of plant cell lysis buffer, primarily composed of PBS, were used as the lysis buffer. The leaf tissues were homogenized using a high-speed homogenizer, and the crude homogenate was collected. The homogenate was centrifuged at 9000 rpm for 40 min at 4°C, and the supernatant was collected. This process was repeated several times until the precipitate was significantly reduced. The resulting supernatant was filtered through a 0.45 μm filter to obtain a crude extract free of significant plant tissue residue, which was used for subsequent purification experiments.

[0156] (2) Protein purification

[0157] (1) Solution preparation:

[0158] 10mM Phosphate Buffered Sodium (PBS): 137mM NaCl, 2.67mM KCl, 10mM Na₂HPO₄, 2mM KH₂PO₄

[0159] Plant cell lysis buffer: 10 mM PBS, 4.8 mM mercaptoethanol (β-ME), 1 mM benzosulfonyl fluoride (PMSF)

[0160] Equilibrium solution: 250mM NaCl, 50mM Tris-HCl, pH 8.0

[0161] Nonspecific protein elution buffer (WB): 500mM NaCl, 50mM Tris-HCl, pH 8.0, 20mM imidazole

[0162] Protein elution buffer EB: 500mM NaCl, 50mM Tris-HCl, pH 8.0, 500mM imidazole

[0163] Dialysis buffer: 500mM NaCl, 50mM Tris-HCl, pH 8.0

[0164] (2) Nickel column affinity chromatography:

[0165] Purification was performed using a His tag bound to a nickel column at low temperatures. 5 mL of Ni-NTA agarose gel column packing (nickel column) was added to 500 mL of crude extract. An appropriate amount of packing was pipetted into an empty column, allowing the medium to settle freely and the stock solution to drain. The Ni-NTA agarose gel column packing was equilibrated with 10 column volumes of equilibration buffer. The packed column was fixed at 4°C. The crude extract of the target recombinant protein containing the His tag was loaded into the column, allowing the liquid to flow out under gravity. The His tag is attached to the ferritin gene sequence.

[0166] (3) Rinse:

[0167] Elute the packing material with a dialysis buffer that does not contain imidazole for approximately 10 column volumes; then rinse the packing material with nonspecific protein elution buffer (WB) containing 20 mM imidazole for approximately 10 column volumes.

[0168] (4) Washing:

[0169] The target protein was eluted using a specific protein elution buffer EB (containing 500mM imidazole) for approximately 5 column volumes. The elution buffer was then collected in centrifuge tubes.

[0170] (5) Dialysis:

[0171] After cleaning and checking for leaks in the dialysis bag, add the eluent to the pre-cooled dialysis solution and perform dialysis overnight in a 4°C refrigerator.

[0172] (5) Concentration:

[0173] Select an appropriate ultrafiltration tube based on the protein size. Pre-cool the ultrafiltration tube on ice for a few minutes, and add the purified sample after dialysis to the upper part of the ultrafiltration tube. Balance the ultrafiltration tube to ensure that both the mass and center of gravity are balanced. Centrifuge at 4°C and 5000×g for 15 minutes using a high-speed refrigerated centrifuge. Repeat the centrifugation process several times until the purified sample is concentrated to approximately 1 mL.

[0174] (3) Transmission electron microscopy and particle size analysis

[0175] Transmission electron microscopy analysis was performed on the four purified recombinant proteins B6R-Fer, B6R-Fer-1, B6R-Fer-2, and B6R-Fer-3. The results are as follows: Figure 5 As shown, B6R-Fer-2 clearly shows protein particles and successfully assembles into virus-like particles (VLPs), while B6R-Fer-1 shows very few protein particles, and B6R-Fer and B6R-Fer-3 show no protein particles. The purified B6R-Fer-2 recombinant protein exhibits a spherical structure, corresponding to virus-like particles (VLPs) in both size and morphology, with a diameter of approximately 18–38 nm.

[0176] The experimental results above show that among the four purified recombinant proteins B6R-Fer, B6R-Fer-1, B6R-Fer-2, and B6R-Fer-3, recombinant protein B6R-Fer-2 had the highest expression level and purity, and only recombinant protein B6R-Fer-2 successfully assembled into virus-like particles (VLPs). Therefore, recombinant protein B6R-Fer-2 was selected as the subject of subsequent immunogenicity experiments.

[0177] Example 4: Immunogenicity test of recombinant protein B6R-Fer-2

[0178] (1) Mouse immunization

[0179] Take 10 μg of the recombinant protein B6R-Fer prepared as described above and mix it thoroughly with 10 μg of aluminum hydroxide adjuvant to prepare a monkeypox virus subunit vaccine.

[0180] Five healthy 9-week-old BALB / c mice (approximately 20g in weight) were selected. The mice were immunized intramuscularly via injection according to the following method: an immunization cycle of 14 days, a immunization program of 3 doses, and administration of the immunization via intramuscular injection in the leg. Serum samples were collected from the mice before immunization and 14 days after the first, second, and third immunizations. The specific immunization procedure is as follows: Figure 6 As shown.

[0181] (2) Enzyme-linked immunosorbent assay (ELISA) analysis

[0182] The titer of B6R-Fer-2 antigen-specific antibodies was determined by indirect ELISA. Recombinant monkeypox virus protein B6R-Fer-2 was diluted to 1 μg / mL with 50 mM carbonate buffer for coating in microplates. 100 μL was added to each well, and the plates were incubated overnight at 4°C. The next day, the coating buffer containing the antigen was discarded, and each well was washed three times with 300 μL of PBST and patted dry. 200 μL of blocking buffer (20% BSA + 80% PBS) was added to each well, and the plates were incubated at room temperature for 2–4 hours. The blocking buffer was discarded, and the plates were washed three times with PBST and patted dry. Mouse serum collected 14 days after immunization and before immunization was diluted 1000-fold with blocking buffer. 5-fold serial dilutions were performed starting from 1000 ng / mL, with 100 μL of diluted mouse serum added to each well and incubated at 37°C for 1 hour. The primary antibody was discarded, and the plates were washed three times with PBST and patted dry. The secondary antibody (Anti-Mouse antibody) was diluted with blocking buffer. IgG (Goat, HRP-Labeled) 6000-fold, 100 μL added to each well, incubated at room temperature for 1 h; discard the secondary antibody, wash 3 times with PBST and blot dry; add 200 μL of OPD substrate to each well, incubate at room temperature in the dark for 15 min; stop the reaction by adding 50 μL of 3M HCl to each well; measure the OD value at 450 nm using a microplate reader. Results are as follows. Figure 7 As shown, p < 0.05 indicates a statistically significant difference. Figure 7 ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0183] Depend on Figure 7 The results showed that the subunit vaccine prepared from the recombinant monkeypox virus protein B6R-Fer-2 could stimulate the body to produce antibodies, and the antibodies maintained for a long time. This indicates that the prepared recombinant monkeypox virus protein B6R-Fer-2 has good immunogenicity and can stimulate the body to produce antibodies with strong binding ability and long maintenance time.

[0184] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A recombinant monkeypox virus antigen, characterized in that The truncated B6R-2 protein was fused with Helicobacter pylori ferritin to form a new recombinant protein B6R-Fer-2, which was used as a recombinant monkeypox virus antigen; the amino acid sequence of the truncated B6R-2 protein is shown in SEQ ID NO: 3; The fusion is to directly fuse the truncated B6R-2 protein and the Helicobacter pylori ferritin or to connect the two with a flexible linker in the hinge region, wherein the amino acid sequence of the flexible linker includes GSGG, GSG, GGG or SGGSG.

2. The recombinant monkeypox virus antigen according to claim 1, wherein The amino acid sequence of the Helicobacter pylori ferritin is shown in SEQ ID NO:

9. Preferably, the C-terminus of the Helicobacter pylori ferritin is connected to a His tag via a flexible linker.

3. The recombinant monkeypox virus antigen according to claim 2, wherein The amino acid sequence of the recombinant protein B6R-Fer-2 is selected from SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 21 or SEQ ID NO:

25.

4. A biomaterial expressing a recombinant monkeypox virus antigen, characterized in that: The invention comprises a nucleic acid molecule encoding the recombinant monkeypox virus antigen according to any one of claims 1 to 3 and an expression cassette, a recombinant expression vector, a recombinant microorganism or a plant bioreactor for expressing the nucleic acid molecule.

5. A method for preparing a recombinant monkeypox virus antigen, characterized in that: A plant expression vector is used to construct a recombinant expression vector containing a nucleic acid sequence encoding the recombinant protein B6R-Fer-2, which is expressed by transfecting tobacco leaves. The recombinant protein B6R-Fer-2 is obtained after extraction and purification as a recombinant monkeypox virus antigen.

6. The preparation method according to claim 5, wherein The amino acid sequence of the recombinant protein B6R-Fer-2 is shown in SEQ ID NO: 21 or SEQ ID NO: 25, and the extraction and purification steps are as follows: The transfected tobacco leaves were taken and lysed with plant cell lysate. The supernatant was collected by centrifugation and purified by binding the His tag of the recombinant protein B6R-Fer-2 to a nickel column. The filler was first rinsed with an imidazole-free buffer and a non-specific protein eluent to remove impurities. The target protein was then eluted with a specific protein eluent. The eluate was collected, dialyzed, and concentrated to obtain the purified recombinant protein B6R-Fer-2.

7. A method for improving the immunogenicity of monkeypox virus antigen B6R, characterized in that: The truncated B6R-2 protein is fused with ferritin to form a recombinant protein B6R-Fer-2, which is self-assembled into virus-like particles and used as a monkeypox virus antigen; the amino acid sequence of the truncated B6R-2 protein is shown in SEQ ID NO:

3.

8. The method according to claim 7, wherein The ferritin is Helicobacter pylori ferritin, and its amino acid sequence is shown in SEQ ID NO:

9. The truncated B6R-2 protein is fused with the Helicobacter pylori ferritin via a flexible linker; preferably, the C-terminus of the Helicobacter pylori ferritin is connected to a His tag via a flexible linker.

9. Use of the recombinant monkeypox virus antigen according to any one of claims 1 to 3 in the preparation of a monkeypox virus vaccine or an anti-monkeypox virus drug.

10. A monkeypox virus vaccine, characterized in that Comprising the recombinant monkeypox virus antigen according to any one of claims 1 to 3.

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