A Monkeypox virus B6R antigen, its preparation method and application

Abstract

The present invention discloses a monkeypox virus B6R antigen, its preparation method and application. The present invention relates to the field of biotechnology, and specifically relates to a monkeypox virus B6R antigen, its preparation method and application. The fusion protein provided by the present invention is a protein obtained by fusing a polypeptide with the amino acid sequence of SEQ ID No.1 to the N-terminus of the monkeypox virus B6R antigen. The amino acid sequence of the fusion protein is SEQ ID No.5, the 1-275th position of SEQ ID No.5 or the 1-280th position of SEQ ID No.5. The present invention uses a signal peptide to guide the secretion and expression of the monkeypox virus B6R in eukaryotic cells. Among them, the luciferase signal peptide Ga guides the secretion and expression level of the monkeypox virus B6R antigen, which is significantly superior to the natural signal peptide Ns and the antibody light chain signal peptide Lc, and is more suitable for large-scale industrial production, reducing production costs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a monkeypox virus B6R antigen and a preparation method and application thereof. Background Art

[0002] Monkeypox is a zoonotic viral disease caused by the monkeypox virus (MPXV). Clinically, it presents with fever, rash, and swollen lymph nodes. It is predominantly found in Central and West Africa. Since May 2022, cases of monkeypox have been reported in some non-endemic countries, posing a risk of community transmission. Consequently, it has attracted the attention of health regulators in several countries.

[0003] Monkeypox virus particles are brick-shaped or oval, measuring 200 nm x 250 nm, and have an envelope. The virus particles contain structural proteins and a DNA-dependent RNA polymerase. The genome is a double-stranded DNA approximately 197 kb in length. The primary infectious form of monkeypox virus is the mature virion (MV). In addition, there is a form in which the mature virion is surrounded by an extracellular envelope (EV) derived from the endoplasmic reticulum. The B6R antigen is a membrane protein that encapsulates the lipid-encapsulated mature monkeypox virus virion (EV). It regulates the entry of virions into host cells and is also a key immune target. Therefore, the preparation of the B6R protein is a key step in the development of key products for monkeypox virus detection reagents or vaccines. Summary of the Invention

[0004] The main problem to be solved by the present invention is how to prepare monkeypox virus B6R antigen.

[0005] In order to solve the above problems, the present invention provides a luciferase signal peptide (signal peptide Ga, SEQ ID No. 1). The signal peptide Ga can guide the secretion of monkeypox virus B6R antigen into the culture supernatant. High-purity antigen can be obtained by nickel column affinity chromatography, and its secretory expression yield is significantly higher than that of the natural signal peptide (signal peptide Ns) and the antibody light chain signal peptide (signal peptide Lc).

[0006] The present invention first provides a fusion protein.

[0007] The fusion protein provided by the present invention is a protein obtained by fusing a polypeptide with an amino acid sequence of SEQ ID No. 1 to the N-terminus of the monkeypox virus B6R antigen.

[0008] Furthermore, the amino acid sequence of the fusion protein is SEQ ID No. 5, positions 1-275 of SEQ ID No. 5, or positions 1-280 of SEQ ID No. 5.

[0009] Positions 1-17 of SEQ ID No. 5 are the signal peptide Ga (i.e., SEQ ID No. 1), positions 18-275 are the monkeypox virus B6R antigen, positions 276-280 are the linker, and positions 281-286 are the histidine tag.

[0010] The present invention also provides a nucleic acid molecule encoding the above fusion protein.

[0011] Furthermore, the nucleic acid molecule is composed of the coding gene of the above polypeptide and the coding gene of monkeypox virus B6R antigen in sequence from the 5' end to the 3' end.

[0012] Furthermore, the polypeptide encoding gene may be any of the following:

[0013] (a1) a DNA molecule whose coding strand nucleotide sequence is SEQ ID No. 2;

[0014] (a2) a DNA molecule that hybridizes with the DNA molecule defined in (a1) under stringent conditions and encodes the polypeptide;

[0015] (a3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the DNA sequence defined in (a1) or (a2) and encodes the polypeptide.

[0016] Furthermore, the gene encoding the monkeypox virus B6R antigen may be any of the following:

[0017] (b1) the nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 4;

[0018] (b2) a DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in (b1) and encodes the same protein;

[0019] (b3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the DNA sequence defined in (b1) or (b2) and encodes the same protein.

[0020] More specifically, the nucleotide sequence of the coding strand of the nucleic acid molecule may be SEQ ID No. 7, positions 1 to 840 of SEQ ID No. 7, or positions 1 to 855 of SEQ ID No. 7.

[0021] Positions 1-6 of SEQ ID No. 7 are HindIII recognition sites, positions 7-15 are Kozak sequences, positions 16-66 are genes encoding luciferase signal peptide Ga, positions 67-840 are genes encoding B6R antigen, positions 841-855 are linker genes, positions 856-873 are histidine tag genes, positions 874-876 are stop codons, and positions 877-884 are PacI recognition sites. The recombinant expression plasmid pCGS3-Ga-B6R contains a gene encoding the recombinant protein Ga-B6R. The coding sequence of the gene encoding the recombinant protein Ga-B6R is positions 16-876 of SEQ ID No. 7. The amino acid sequence of the gene encoding the recombinant protein Ga-B6R is a protein of sequence 5.

[0022] In the above-mentioned nucleic acid molecules or coding genes, identity refers to nucleotide sequence identity. Nucleotide sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, Advanced BLAST 2.1 can be used by searching for the identity of a pair of nucleotide sequences using blastp as the program, setting the Expect value to 10, all filters to OFF, BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. Calculations are then performed to obtain the identity value (%).

[0023] In the above-mentioned nucleic acid molecules or encoding genes, the stringent conditions may be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 2×SSC, 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 1×SSC, 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 0.5×SSC, 0.1% SDS; hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and washing at 50°C in 0.1×SSC, 0.1% Rinse in SDS; alternatively: hybridize in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA at 50°C, rinse in 0.1×SSC, 0.1% SDS at 65°C; alternatively: hybridize in a solution of 6×SSC, 0.5% SDS at 65°C, then wash the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0024] The present invention also provides an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line of the nucleic acid molecule.

[0025] The expression cassette refers to a DNA capable of expressing the fusion protein in a host cell, which includes not only a promoter for initiating transcription of the target gene, but also a terminator for terminating transcription of the target gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0026] In a specific embodiment of the present invention, the recombinant vector is a recombinant plasmid obtained by inserting the DNA fragment represented by SEQ ID No. 7 (positions 16-66 of SEQ ID No. 5 are the gene encoding the signal peptide Ga, i.e., SEQ ID No. 2) into the multiple cloning sites (e.g., HindIII and PacI) of the pCGS3 vector. Accordingly, the transgenic cell line is obtained by introducing the recombinant plasmid into Expi293F cells.

[0027] The present invention also provides the use of the above-mentioned fusion protein or the nucleic acid molecule or the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line in any of the following:

[0028] P1. Application of increasing the secretory expression yield of monkeypox virus B6R antigen in host cells;

[0029] P2. Application of improving the secretory expression efficiency of monkeypox virus B6R antigen in host cells;

[0030] P3. Application in the preparation of monkeypox virus B6R antigen secretory protein products.

[0031] The amino acid sequence of the monkeypox virus B6R antigen secretory protein is SEQ ID No. 5.

[0032] Wherein, the host cell is a eukaryotic host cell.

[0033] Furthermore, the eukaryotic host cells can be HEK293 cells, CHO cells, yeast cells, insect cells, etc.

[0034] In a specific embodiment of the present invention, the host cell is specifically an Expi293F cell.

[0035] The method for preparing the monkeypox virus B6R antigen secretory protein also falls within the scope of protection claimed by the present invention.

[0036] The method for preparing the monkeypox virus B6R antigen secretory protein claimed in the present invention may include the following steps:

[0037] (A1) introducing the nucleic acid molecule described above into a host cell to obtain a recombinant cell;

[0038] (A2) Cultivating the recombinant cells and obtaining the monkeypox virus B6R antigen secretory protein from the culture supernatant.

[0039] The nucleic acid molecule can be introduced into the host cell via the recombinant vector mentioned above.

[0040] In step (A1), the host cell is a eukaryotic host cell, such as HEK293 cells, CHO cells, yeast cells, and insect cells.

[0041] In a specific embodiment of the present invention, the host cell is specifically an Expi293F cell.

[0042] In step (A2), the culturing is performed until the cell viability drops to 65%-75%, and the culturing is terminated.

[0043] In step (A2), the monkeypox virus B6R antigen secretory protein is obtained from the culture supernatant according to a method comprising the following steps: collecting the culture and centrifuging at 3500g for 30 minutes, collecting the supernatant for ultrafiltration concentration and nickel column purification.

[0044] The present invention also provides the use of a polypeptide having an amino acid sequence of SEQ ID No. 1 or a related biological material thereof in any of the following:

[0045] P1. Application of increasing the secretory expression yield of monkeypox virus B6R antigen in host cells;

[0046] P2. Application of improving the secretory expression efficiency of monkeypox virus B6R antigen in host cells;

[0047] P3, application in the preparation of monkeypox virus B6R antigen secretory protein products;

[0048] The relevant biological material is the coding gene of the polypeptide described in SEQ ID No. 1 or an expression cassette or recombinant vector or recombinant bacteria or transgenic cell line containing the coding gene.

[0049] In a specific embodiment of the present invention, the recombinant vector is a recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No. 7 (positions 16-66 of SEQ ID No. 5 are the gene encoding the signal peptide Ga, i.e., SEQ ID No. 2) into the multiple cloning sites (such as Hind III and Pac I) of the pCGS3 vector.

[0050] Furthermore, the host cell may be a eukaryotic host cell, such as HEK293 cells, CHO cells, yeast cells, and insect cells.

[0051] In a specific embodiment of the present invention, the host cell is specifically an Expi293F cell.

[0052] Furthermore, the coding gene may be any of the following:

[0053] (a1) a DNA molecule whose coding strand nucleotide sequence is SEQ ID No. 2;

[0054] (a2) a DNA molecule that hybridizes with the DNA molecule defined in (a1) under stringent conditions and encodes the polypeptide shown in SEQ ID No. 1;

[0055] (a3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the DNA sequence defined in (a1) or (a2) and encodes the polypeptide.

[0056] In the above-mentioned proteins, homology refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, blastp can be used as the program, with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated and the identity value (%) can be obtained.

[0057] In the above proteins, the 95% or greater homology may be at least 96%, 97%, or 98% identity. The 90% or greater homology may be at least 91%, 92%, 93%, or 94% identity. The 85% or greater homology may be at least 86%, 87%, 88%, or 89% identity. The 80% or greater homology may be at least 81%, 82%, 83%, or 84% identity.

[0058] The present invention uses natural signal peptide Ns, luciferase signal peptide Ga (SEQ ID No. 1) and antibody signal peptide Lc to guide the eukaryotic cell secretory expression of monkeypox virus B6R antigen. Studies have shown that the secretory expression of monkeypox virus B6R antigen in the luciferase signal peptide Ga experimental group is significantly better than that in the natural signal peptide Ns and antibody light chain signal peptide Lc experimental groups, and is more suitable for large-scale industrial production and reduces production costs. The present invention uses eukaryotic cell secretory expression to prepare the extracellular region of B6R protein, which has the following advantages: 1) low background of secreted supernatant protein and easy purification; 2) soluble expression to avoid the formation of inclusion bodies; 3) precise cleavage by signal peptidase, no redundant Met residues at the N-terminus, and the production of expected protein sequences. The present invention is suitable for applications such as antigen preparation in vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The enzyme digestion identification diagram for the recombinant expression plasmid construction is shown in Figure 1. 1 is pCGS3-Ns-B6R (Ns is the natural signal peptide), 2 is pCGS3-Ga-B6R (Ga is the luciferase signal peptide), and 3 is pCGS3-Lc-B6R (Lc is the antibody light chain signal peptide).

[0060] Figure 2The following is an SDS-PAGE analysis of the supernatants secreted from cells expressing the monkeypox virus B6R antigen. Figure 1 shows the supernatant containing the natural signal peptide Ns (culture supernatant of cells transfected with pCGS3-Ns-B6R), 2 shows the supernatant containing the luciferase signal peptide Ga (culture supernatant of cells transfected with pCGS3-Ga-B6R), 3 shows the supernatant containing the antibody light chain signal peptide Lc (culture supernatant of cells transfected with pCGS3-Lc-B6R), and 4 shows the negative control group without the transfected expression plasmid (culture supernatant of cells transfected with the pCGS3 vector).

[0061] Figure 3 Grayscale analysis of the supernatant secreted by monkeypox virus B6R antigen cells. Figure 1 shows the supernatant secreted by the natural signal peptide Ns (culture supernatant of cells transfected with pCGS3-Ns-B6R), 2 shows the supernatant secreted by the luciferase signal peptide Ga (culture supernatant of cells transfected with pCGS3-Ga-B6R), 3 shows the supernatant secreted by the antibody light chain signal peptide Lc (culture supernatant of cells transfected with pCGS3-Lc-B6R), and 4 shows the negative control group without transfected expression plasmid (culture supernatant of cells transfected with the pCGS3 vector).

[0062] Figure 4 The monkeypox virus B6R antigen protein was purified and identified by SDS-PAGE. Sample 1 is a sample of secretory expression and purification of the natural signal peptide Ns, sample 2 is a sample of secretory expression and purification of the luciferase signal peptide Ga, and sample 3 is a sample of secretory expression and purification of the antibody light chain signal peptide Lc. DETAILED DESCRIPTION

[0063] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0064] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0065] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0066] The main reagents and their manufacturers in the following examples are as follows:

[0067] pCGS3 vector: Merck;

[0068] HindIII endonuclease: NEB;

[0069] PacI endonuclease: NEB;

[0070] GXL Premix: TAKARA;

[0071] DNA Ligation Kit Ver.2.1: TAKARA;

[0072] Expi293F TM Cells: Thermo Fisher Scientific;

[0073] Expi293 TM Expression Medium: Thermo Fisher Scientific;

[0074] ExpiFectamine TM 293 Transfection Kit: Thermo Fisher;

[0075] Opti-MEM TM I Reduced Serum Medium: Thermo Fisher Company;

[0076] Ni-NTA protein purification kit: Sangon Biotech (Shanghai) Co., Ltd.;

[0077] Amicon Ultra-15 centrifugal filter device: Millipore;

[0078] Amicon Ultra-0.5 centrifugal filter device: Millipore;

[0079] PBS pH 7.4 (1×): Gibco;

[0080] Gel imaging system: Protein Simple;

[0081] Cell counter: Roche;

[0082] Clean bench: Suzhou Antai Air Technology Co., Ltd.

[0083] Electric constant temperature water bath: Fisher Scientific;

[0084] CO2 constant temperature shaker: CRYSTAL;

[0085] HYG-A fully constant temperature shaker cabinet: Taicang Experimental Equipment Factory;

[0086] DYY-6C electrophoresis apparatus: Beijing Liuyi Instrument Factory;

[0087] DYCP-31DN horizontal electrophoresis tank: Beijing Liuyi Instrument Factory;

[0088] Micropipette: Eppendorf.

[0089] Example 1: Construction of recombinant expression plasmid

[0090] The monkeypox virus was selected from the latest monkeypox virus in 2022 (NCBI genome accession number is ON563414.3), among which the B6R antigen NCBI accession number is URK20605.1. The present invention selected the extracellular region of the B6R antigen sequence, that is, the amino acid sequence at positions 20-277.

[0091] The genes encoding the natural signal peptide Ns, luciferase signal peptide Ga, and antibody light chain signal peptide Lc were fused to the 5' end of the gene encoding the monkeypox virus B6R antigen protein (the amino acid sequence of the B6R antigen protein is SEQ ID No. 3, and the coding sequence of the gene encoding it is SEQ ID No. 4). The corresponding fragments are named Ns-B6R, Ga-B6R, and Lc-B6R, respectively, and cloned into the pCGS3 vector to construct eukaryotic recombinant expression plasmids. The specific operation process is described below. Ns-B6R gene synthesis: The carbon terminus of the natural signal peptide Ns was fused to the monkeypox antigen B6R. Sangon Biotechnology was commissioned to perform codon optimization and synthesize the Ns-B6R gene (nucleotide sequence is SEQ ID No. 6). Sangon Biotechnology delivered the synthesis plasmid pUC57-Ns-B6R (containing the Ns-B6R gene).

[0092] Using pUC57-Ns-B6R as template, polymerase GXL Premix (TAKARA) was used to amplify the target fragments of Ga-B6R and Lc-B6R. The details are as follows:

[0093] 1) Ga-B6R gene fragment amplification: Primers 1 and 5 were used to amplify fragment A. Fragment A was used as the second round template. Primers 2 and 5 were used for the second round of amplification to obtain fragment B, which was the Ga-B6R target fragment.

[0094] 2) Lc-B6R gene fragment amplification: Primers 3 and 5 amplified fragment C, which was used as the second round template. Primers 4 and 5 were used for the second round of amplification to obtain fragment D, which was the Lc-B6R target fragment.

[0095] Primer 1: 5'-TGTTTGCTCTGATTTGTATTGCCGTGGCTGAGGCCACCTGCACCGTGCCTACCAT-3';

[0096] Primer 2: 5'-CCCAAGCTTGCCGCCACCATGGGGGTGAAGGTGTTGTTGCTCTGATTTGTATTG-3';

[0097] Primer 3: 5'-CTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAGATGCACCTGCACCGTGCCTACCAT-3';

[0098] Primer 4: 5'-CCCAAGCTTGCCGCCACCATGAGCGTGCCAACCCAGGTGCTGGGCCTGCTGCTGCTGTG-3';

[0099] Primer 5: 5'-CCTTAATTAATCAGTGGTGGTGATGATGGTGAGAG-3'.

[0100] The pCGS3 vector was double-digested with HindIII (NEB) and PacI (NEB) to obtain the vector fragment and the fully synthesized gene pUC57-Ns-B6R. The amplified fragments Ga-B6R and Lc-B6R were double-digested with HindIII and PacI to obtain the target fragments. Ligation, transformation, plasmid extraction, and identification were performed using the DNA Ligation Kit Ver. 2.1 (TAKARA). Three recombinant expression plasmids, pCGS3-Ns-B6R, pCGS3-Ga-B6R, and pCGS3-Ga-B6R, were obtained.

[0101] The enzyme digestion identification results of the three recombinant expression plasmids are as follows Figure 1 As shown, lane 1 shows double-enzyme digestion analysis of the pCGS3-Ns-B6R expression plasmid, lane 2 shows double-enzyme digestion analysis of the pCGS3-Ga-B6R expression plasmid, and lane 3 shows double-enzyme digestion analysis of the pCGS3-Lc-B6R expression plasmid. The vector fragment after digestion is approximately 7100 bp, and the target gene is approximately 890 bp. The sizes of the digested bands are consistent with expectations.

[0102] The structure of the recombinant expression plasmid pCGS3-Ns-B6R is described as follows: The recombinant expression vector is obtained by replacing the region between the HindIII and PacI recognition sites of the pCGS3 vector with the DNA molecule having the nucleotide sequence of SEQ ID No. 6, while keeping the other nucleotides of the pCGS3 vector unchanged. Positions 1-6 of SEQ ID No. 6 represent a HindIII recognition site, positions 7-15 represent a Kozak sequence, positions 16-72 represent the gene encoding the native signal peptide Ns, positions 73-846 represent the gene encoding the B6R antigen, positions 847-861 represent a linker gene, positions 862-879 represent a histidine tag gene, positions 880-882 represent a stop codon, and positions 883-890 represent a PacI recognition site. The recombinant expression plasmid pCGS3-Ns-B6R contains the gene encoding the recombinant protein Ns-B6R. The coding sequence of the gene encoding the recombinant protein Ns-B6R is positions 16 to 882 of SEQ ID No. 6. The encoded recombinant protein Ns-B6R is a protein fused with the Ns signal peptide and positions 18 to 286 of sequence 5, and consists of 288 amino acids.

[0103] The structure of the recombinant expression plasmid pCGS3-Ga-B6R is described as follows: The recombinant expression vector is obtained by replacing the region between the HindIII and PacI recognition sites of the pCGS3 vector with the DNA molecule having the nucleotide sequence of SEQ ID No. 7, while keeping the other nucleotides of the pCGS3 vector unchanged. Positions 1-6 of SEQ ID No. 7 are HindIII recognition sites, positions 7-15 are Kozak sequences, positions 16-66 are the gene encoding the luciferase signal peptide Ga, positions 67-840 are the gene encoding the B6R antigen, positions 841-855 are the linker gene, positions 856-873 are the histidine tag gene, positions 874-876 are stop codons, and positions 877-884 are the PacI recognition site. The recombinant expression plasmid pCGS3-Ga-B6R contains the coding gene of the recombinant protein Ga-B6R. The coding sequence of the coding gene of the recombinant protein Ga-B6R is positions 16 to 876 of SEQ ID No. 7. The amino acid sequence of the coding gene of the recombinant protein Ga-B6R is a protein of sequence 5.

[0104] The structure of the recombinant expression plasmid pCGS3-Lc-B6R is described as follows: The recombinant expression vector is obtained by replacing the region between the restriction endonuclease HindIII and PacI recognition sites of the pCGS3 vector with the DNA molecule having the nucleotide sequence of SEQ ID No. 8, while keeping the other nucleotides of the pCGS3 vector unchanged. Positions 1-6 of SEQ ID No. 8 are the HindIII recognition site, positions 7-15 are the Kozak sequence, positions 16-75 are the gene encoding the signal peptide Lc, positions 76-849 are the gene encoding the B6R antigen, positions 850-864 are the linker gene, positions 865-882 are the histidine tag gene, positions 883-885 are the stop codon, and positions 886-893 are the PacI recognition site. The recombinant expression plasmid pCGS3-Lc-B6R contains the gene encoding the recombinant protein Lc-B6R. The coding sequence of the gene encoding the recombinant protein Lc-B6R is positions 16-885 of SEQ ID No. 8. The encoded recombinant protein Lc-B6R is a protein fused with the Lc signal peptide and positions 18-286 of sequence 5, and consists of 289 amino acids.

[0105] Example 2: Protein transient expression study

[0106] A. Expi293F cell transfection experiment

[0107] 1. Host Cell Culture

[0108] Host cells Expi293F (Expi293F) were taken from the liquid nitrogen tank. TM Rapidly thaw cells from a seed bank of cells in a 37°C water bath. Aseptically transfer the thawed cell suspension to a 125mL vial containing 30mL of prewarmed complete growth medium. Incubate on a shaker at 37°C, 8% CO2, 120 rpm, 25mm amplitude, and ≥80% humidity. After 15-30 minutes, collect the cell suspension and measure cell density and viability.

[0109] When the cell viability recovers to more than 90% and the cell density reaches 3-5×10 6 cells / mL, 0.3-0.5×10 6 cells / mL for inoculation and expansion.

[0110] 2. Cell Transfection

[0111] The recombinant vectors pCGS3-Ns-B6R, pCGS3-Ga-B6R, pCGS3-Lc-B6R and pCGS3 constructed in Example 1 were transfected into host cells separately.

[0112] 1. One day before transfection

[0113] 24 h before transfection, cells were plated at 2.5-3×10 6 cells / mL and culture for 24 h.

[0114] 2. On the day of transfection

[0115] (1) The cell density should reach 4.5-5.5×10 6 cells / mL, the viability should be ≥95%. Dilute the cells to 3×10 6 cells / mL.

[0116] (2) Prepare transfection reagent and DNA complex

[0117] 1) DNA dilution

[0118] The plasmids (pCGS3-Ns-B6R, pCGS3-Ga-B6R, and pCGS3-Lc-B6R constructed in Example 1) were diluted to 1 μg / μL with sterile water. According to the amount of plasmid to transfect 1 μg of 1 mL of cells, the amount of plasmid required for transfection of 50 mL of cells, i.e., 50 μL of plasmid was added to 3 mL of Opti-MEM™ I Reduced Serum medium for later use.

[0119] 2) Dilution of transfection reagent

[0120] Before use, add the transfection reagent ExpiFectamine293 TM Gently invert the Reagent upside down to mix thoroughly, and take the amount of transfection reagent required to transfect 50mL cells, i.e. 160μL ExpiFectamine293 TM The reagent was added to 2.8 mL of Opti-MEM™ I Reduced Serum medium by gently inverting and mixing, and then allowed to stand at room temperature for 5 minutes.

[0121] 3) Add the diluted transfection reagent to the plasmid and gently invert to mix. Incubate at room temperature for 10-20 minutes. Slowly add the transfection reagent and DNA complex to the cell culture. Incubate at 37°C, 8% CO2, 120 rpm, 25 mm amplitude, and ≥80% humidity.

[0122] 3. First day after transfection

[0123] 18-22 hours after transfection, add the enhancer at a volume sufficient to transfect 50 mL of cells. Specifically, mix 300 μL of ExpressiFectamine™ 293 Transfection Enhancer 1 with 3 mL of ExpiFectamine™ 293 Transfection Enhancer 2 and slowly add to the cell culture to obtain pCGS3-Ns-B6R-transfected cells, pCGS3-Ga-B6R-transfected cells, pCGS3-Lc-B6R-transfected cells, and pCGS3 vector-transfected cells, respectively.

[0124] 4. Collection of culture supernatant

[0125] The cell viability was monitored every day after transfection. The culture was terminated on the 4th day when the viability dropped to 65%-75%. The culture was collected and centrifuged at 3500g for 30 minutes to collect the supernatant, and the culture supernatant of the pCGS3-Ns-B6R transfected cell culture supernatant, the pCGS3-Ga-B6R transfected cell culture supernatant, the pCGS3-Lc-B6R transfected cell culture supernatant and the pCGS3 vector transfected cell culture supernatant were obtained respectively.

[0126] B. SDS protein electrophoresis and grayscale analysis

[0127] The supernatant was analyzed by protein electrophoresis, and grayscale analysis of the SDS protein electrophoresis pattern was performed using Image J software. The following steps were used: Image → Type → Convert to 32-Bit Grayscale; Process → Subtract Background → OK to remove the background color; Select the lane using the Rectangle tool → Analyze → Gel → Select First Lane to determine the analysis lane, and repeat to select multiple lanes for simultaneous analysis; Analyze → Gel → Plot Lane to generate the peak area; Select the peak corresponding to the target band using the Linear tool, and calculate the corresponding peak area using the Wand tool to determine the percentage of the target protein in the total protein.

[0128] The results of SDS-PAGE identification and grayscale analysis showed that the target protein expression levels in the secretion supernatant of natural signal peptide Ns (culture supernatant of cells transfected with pCGS3-Ns-B6R), the secretion supernatant of luciferase signal peptide Ga (culture supernatant of cells transfected with pCGS3-Ga-B6R), and the secretion supernatant of antibody light chain signal peptide Lc (culture supernatant of cells transfected with pCGS3-Lc-B6R) accounted for 12.32%, 20.66% and 20.50% of the total protein, respectively. Figure 2 and Figure 3 In summary, compared with the natural signal peptide Ns and signal peptide Lc, the monkeypox virus B6R antigen guided by the luciferase signal peptide Ga has a higher secretory expression level.

[0129] Example 3: Protein purification of B6R antigen

[0130] 1. Ultrafiltration concentration

[0131] The culture supernatant of the pCGS3-Ns-B6R transfected cells, the culture supernatant of the pCGS3-Ga-B6R transfected cells, and the culture supernatant of the pCGS3-Lc-B6R transfected cells of Example 2 were respectively subjected to ultrafiltration and concentration by centrifugation at 6000 g for 20 min at 4°C using an Amicon Ultra-15 centrifugal filter device (Millipore), and the final cell supernatant was concentrated to 20-30 mL.

[0132] 2. Nickel column purification

[0133] Mix the ultrafiltration concentrated supernatant obtained in step 1 and Binding / Wash Buffer in a volume ratio of 1:1 and let it stand for 20 minutes to fully incubate. Equilibrate the column with two column volumes of Binding / Wash Buffer, and let the buffer flow through the pre-packed column by gravity. Add the ultrafiltration concentrated supernatant and Binding / Wash Buffer mixture to the column and let it flow through the pre-packed column by gravity; if there is any residual sample, re-load the sample, re-circulate once, and collect the flow-through into a centrifuge tube. Wash the column with two column volumes of Binding / wash buffer and collect the flow-through until the absorbance of the flow-through at 280nm is close to the baseline. Elute the histidine-tagged protein on the column with two column volumes of Elution Buffer. Repeat this step until the absorbance of the flow-through at 280nm is close to the baseline, and collect the eluate for purification.

[0134] 3. Ultrafiltration replacement

[0135] After nickel column purification, the protein solution was added to an Amicon Ultra-15 centrifugal filter device (Millipore) and centrifuged in batches at 10,000 g for 3 min until approximately 150 μL of solution remained. Then, 300 μL of PBS (pH 7.4) was gently added and centrifuged at 10,000 g until 150 μL remained. This was repeated three times. The sample was eluted with PBS (pH 7.4) and collected in an ultrafiltration tube to a final volume of approximately 1-2 mL. A 5 μL sample was retained for protein concentration determination and SDS-PAGE analysis.

[0136] After Ni column purification and identification analysis, the yield of recombinant protein Ga-B6R in the culture supernatant of pCGS3-Ga-B6R transfected cells was 50.84 mg / L, the yield of recombinant protein Lc-B6R in the culture supernatant of pCGS3-Lc-B6R transfected cells was 15.87 mg / L, and the yield of recombinant protein Ns-B6R in the culture supernatant of pCGS3-Ns-B6R transfected cells was 42.93 mg / L ( Figure 4), among which, the signal peptide Ga experimental group had the highest yield after purification, and the expression level of the recombinant protein Lc-B6R in the signal peptide Lc experimental group was higher in the supernatant, but the loss during the purification process was large, and the protein yield after purification was low.

[0137] A monkeypox virus immune composition can be produced by using aluminum salt; or CpG; or liposome; or oily adjuvant to prevent monkeypox virus infection.

[0138] Based on the results of the above examples, the present invention uses three signal peptides, signal peptide Ns, signal peptide Ga and signal peptide Lc, to guide the secretory expression of monkeypox virus B6R antigen in eukaryotic cells. The secretory expression level of the luciferase signal peptide Ga of the present invention is significantly better than that of the other two signal peptides.

[0139] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes a fusion protein, which is a protein obtained by fusing a polypeptide with an amino acid sequence of SEQ ID No.1 to the N-terminus of the monkeypox virus B6R antigen.

2. The nucleic acid molecule according to claim 1, characterized in that: The amino acid sequence of the fusion protein is SEQ ID No.5, positions 1-275 of SEQ ID No.5, or positions 1-280 of SEQ ID No.

5.

3. The nucleic acid molecule according to claim 1 or 2, wherein: The coding gene of the polypeptide is a DNA molecule with a nucleotide sequence of SEQ ID No.2 for the coding strand; The coding gene of the monkeypox virus B6R antigen is a DNA molecule with a nucleotide sequence shown in SEQ ID No.4 for the coding strand.

4. The nucleic acid molecule according to claim 1 or 2, characterized in that: The nucleic acid molecule is a DNA molecule with a nucleotide sequence of SEQ ID No.7, positions 1-840 of SEQ ID No.7, or positions 1-855 of SEQ ID No.7 for the coding strand.

5. An expression cassette, recombinant vector, recombinant microorganism, or transgenic cell line containing any one of the nucleic acid molecules according to claims 1-4.

6. Use of any one of the nucleic acid molecules according to claims 1-4 or the expression cassette, recombinant vector, recombinant microorganism, or transgenic cell line according to claim 5 in any of the following: P1. Use in increasing the secretion expression yield of the monkeypox virus B6R antigen in host cells; P2. Use in increasing the secretion expression efficiency of the monkeypox virus B6R antigen in host cells; P3. Use in preparing a secretion protein product of the monkeypox virus B6R antigen; The host cell is an Expi293F eukaryotic cell.

7. A method for preparing a secretion protein of the monkeypox virus B6R antigen, comprising the following steps: (A1) Introducing any one of the nucleic acid molecules according to claims 1-4 into a host cell to obtain a recombinant cell; the host cell is an Expi293F eukaryotic cell; (A2) Culturing the recombinant cell and obtaining the monkeypox virus B6R antigen secretion protein from the culture supernatant.

8. Use of a polypeptide with an amino acid sequence of SEQ ID No.1 or its related biological material in any of the following: P1. Use in increasing the secretion expression yield of the monkeypox virus B6R antigen in host cells; P2. Use in increasing the secretion expression efficiency of the monkeypox virus B6R antigen in host cells; P3. Use in preparing a secretion protein product of the monkeypox virus B6R antigen; The related biological material is the coding gene of the polypeptide or an expression cassette or recombinant vector or recombinant bacterium or transgenic cell line containing the coding gene; The host cell is an Expi293F eukaryotic cell.

9. The application according to claim 8, characterized in that: The coding gene is a DNA molecule with a nucleotide sequence of SEQ ID No.2 for the coding strand.

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