Method for preparing monkeypox virus M1R antigen secretory protein and nucleic acid molecule used therein

Abstract

The present invention discloses a method for preparing a secretory protein of a monkeypox virus M1R antigen and a nucleic acid molecule used therein. The present invention relates to the field of biotechnology, and in particular to a method for preparing a secretory protein of a monkeypox virus M1R antigen and a nucleic acid molecule used therein. The method provided by the present invention comprises the following steps: (A1) introducing a nucleic acid molecule into a host cell to obtain a recombinant cell; the nucleic acid molecule encodes a fusion protein, and the fusion protein is a protein obtained by fusing a polypeptide (amino acid sequence is SEQ ID No. 1) to the N-terminus of the monkeypox virus M1R antigen; (A2) culturing the recombinant cell to obtain the secretory protein of the monkeypox virus M1R antigen from the culture supernatant. The use of luciferase signal peptide Ga to guide the secretory expression level of the monkeypox virus M1R eukaryotic cell is significantly better than that of the albumin signal peptide Al and the antibody light chain signal peptide Lc, and is more suitable for large-scale industrial production, thereby reducing production costs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing a monkeypox virus M1R antigen secretory protein and a nucleic acid molecule used therein. Background Art

[0002] Monkeypox virus is a virus transmitted from animals to humans. It belongs to the same virus genus as smallpox, but is less toxic than smallpox, and the symptoms after infection are milder than smallpox. Since humans develop a rash after being infected with the monkeypox virus, the zoonotic infectious disease caused by the monkeypox virus is called monkeypox. It is a self-limiting disease. Most people recover 2 to 3 weeks after the onset of symptoms. Some people may develop complications, such as meningitis and pneumonia, which may cause serious consequences. Monkeypox is mainly prevalent in Central Africa, West Africa and other regions. In May 2022, a new round of "monkeypox" epidemic began to spread in non-epidemic areas, so it has attracted the attention of health regulatory authorities in many countries.

[0003] The basic infection form of monkeypox virus is mature virion (MV). In addition, there is another form: the mature virion is surrounded by a lipid membrane (extracellular enveloped, EV) derived from the endoplasmic reticulum membrane. Among them, M1R antigen is a membrane protein of mature virion (MV), which is necessary for the fusion of the virus core and penetration into the host cell. It participates in the assembly of virions and plays an important role in the entry and maturation of the virus. Therefore, the preparation of M1R 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 M1R antigen.

[0005] In order to solve the above problems, the present invention provides a method for preparing a secretory protein of monkeypox virus M1R antigen.

[0006] The method for preparing the monkeypox virus M1R antigen secretory protein provided by the present invention may include the following steps:

[0007] (A1) introducing a nucleic acid molecule into a host cell to obtain a recombinant cell; the nucleic acid molecule encodes a fusion protein, the fusion protein is a protein obtained by fusing a polypeptide to the N-terminus of the monkeypox virus M1R antigen, and the amino acid sequence of the polypeptide is SEQ ID No. 1;

[0008] (A2) culturing the recombinant cells and obtaining the monkeypox virus M1R antigen secretory protein from the culture supernatant.

[0009] In step (A1), the host cell is a eukaryotic host cell, such as HEK293 cells, CHO cells, yeast cells and insect cells.

[0010] In a specific embodiment of the present invention, the host cell is specifically an Expi293F cell.

[0011] In step (A2), the culturing is terminated when the cell viability drops to 65%-75%.

[0012] In step (A2), the secretory protein of monkeypox virus M1R antigen 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 and performing ultrafiltration concentration and nickel column purification.

[0013] In the above method, the amino acid sequence of the fusion protein is SEQ ID No.5, positions 1 to 202 of SEQ ID No.5, or positions 1 to 207 of SEQ ID No.5.

[0014] In the above method, the nucleic acid molecule consists of a gene encoding the polypeptide and a gene encoding the monkeypox virus M1R antigen;

[0015] The polypeptide encoding gene is any of the following:

[0016] (a1) a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 2;

[0017] (a2) a DNA molecule that hybridizes with the DNA molecule defined in (a1) under stringent conditions and encodes the polypeptide;

[0018] (a3) a DNA molecule encoding the polypeptide having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more to the DNA sequence defined in (a1) or (a2);

[0019] The gene encoding the monkeypox virus M1R antigen is any one of the following:

[0020] (b1) the nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 4;

[0021] (b2) a DNA molecule that hybridizes with the DNA molecule defined in (b1) under stringent conditions and encodes the same protein;

[0022] (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;

[0023] (b4) The nucleic acid molecule is a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No.7, positions 1 to 621, or positions 1 to 636 of SEQ ID No.7, or the like.

[0024] In the above proteins, homology refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0025] In the above proteins, the homology of more than 95% may be at least 96%, 97% or 98% identity. The homology of more than 90% may be at least 91%, 92%, 93% or 94% identity. The homology of more than 85% may be at least 86%, 87%, 88% or 89% identity. The homology of more than 80% may be at least 81%, 82%, 83% or 84% identity.

[0026] The present invention also provides the above fusion protein.

[0027] The fusion protein provided by the present invention is a protein obtained by fusing the polypeptide described in SEQ ID No. 1 to the N-terminus of the monkeypox virus M1R antigen.

[0028] Furthermore, the amino acid sequence of the fusion protein is SEQ ID No.5, positions 1-202 of SEQ ID No.5, or positions 1-207 of SEQ ID No.5.

[0029] Positions 1-17 of SEQ ID No.5 are signal peptide Ga (ie, SEQ ID No.1), positions 18-202 are the monkeypox virus M1R antigen, positions 203-207 are linkers, and positions 208-213 are histidine tags.

[0030] The present invention also provides a nucleic acid molecule encoding the above fusion protein.

[0031] In the above method, the nucleic acid molecule consists of a gene encoding the polypeptide and a gene encoding the monkeypox virus M1R antigen;

[0032] The polypeptide encoding gene is any of the following:

[0033] (a1) a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 2;

[0034] (a2) a DNA molecule that hybridizes with the DNA molecule defined in (a1) under stringent conditions and encodes the polypeptide;

[0035] (a3) a DNA molecule encoding the polypeptide having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more to the DNA sequence defined in (a1) or (a2);

[0036] The gene encoding the monkeypox virus M1R antigen is any one of the following:

[0037] (b1) the nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 4;

[0038] (b2) a DNA molecule that hybridizes with the DNA molecule defined in (b1) under stringent conditions and encodes the same protein;

[0039] (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;

[0040] (b4) The nucleic acid molecule is a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No.7, positions 1 to 621, or positions 1 to 636 of SEQ ID No.7, or the like.

[0041] The 1st to 6th positions of SEQ ID No.7 are HindIII recognition sites, the 7th to 15th positions are Kozak sequences, the 16th to 66th positions are luciferase signal peptide Ga coding genes, the 67th to 621st positions are M1R antigen coding genes, the 622th to 636th positions are linker genes, the 637th to 654th positions are histidine tag genes, the 655th to 657th positions are stop codons, and the 658th to 665th positions are PacI recognition sites. The recombinant expression plasmid pCGS3-Ga-M1R contains the coding gene of the recombinant protein Ga-M1R, the coding sequence of the coding gene of the recombinant protein Ga-M1R is the 16th to 657th positions of SEQ ID No.7, and the amino acid sequence of the coding gene of the recombinant protein Ga-M1R is the protein of sequence 5.

[0042] In the above nucleic acid molecules or coding genes, identity refers to the identity of nucleotide sequences. The identity of nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, searching for the identity of a pair of nucleotide sequences, performing calculations, and then obtaining the value (%) of identity.

[0043] In the above-mentioned nucleic acid molecules or encoding genes, the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4 and 1mM EDTA, and rinsing at 50°C, 2×SSC, 0.1% SDS; it may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and rinsing at 50°C, 1×SSC, 0.1% SDS; it may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and rinsing at 50°C, 0.5×SSC, 0.1% SDS; it may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and rinsing at 50°C, 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.

[0044] The present invention 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 M1R antigen in host cells;

[0046] P2. Application of improving the secretion expression efficiency of monkeypox virus M1R antigen in host cells;

[0047] P3, application in the preparation of secretory protein products of monkeypox virus M1R antigen;

[0048] The related biological material is the coding gene of the polypeptide described in SEQ ID No. 1 or an expression box or a recombinant vector or a recombinant bacterium or a 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.7 are the coding gene of the signal peptide Ga, i.e., SEQ ID No.2) into the multiple cloning site (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] The present invention also provides an expression box, a recombinant vector, a recombinant microorganism or a transgenic cell line of the nucleic acid molecule.

[0053] The expression cassette refers to a DNA capable of expressing the above fusion protein in a host cell, and the DNA 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.

[0054] 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 coding gene of the signal peptide Ga, i.e., SEQ ID No.2) into the multiple cloning site (such as HindIII and PacI) of the pCGS3 vector. Accordingly, the transgenic cell line is obtained by introducing the recombinant plasmid into Expi293F cells.

[0055] The present invention also provides the use of the above fusion protein or the nucleic acid molecule or the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line in any of the following:

[0056] P1. Application of increasing the secretory expression yield of monkeypox virus M1R antigen in host cells;

[0057] P2. Application of improving the secretion expression efficiency of monkeypox virus M1R antigen in host cells;

[0058] P3. Application in the preparation of monkeypox virus M1R antigen secretory protein products.

[0059] The amino acid sequence of the monkeypox virus M1R antigen secretory protein is SEQ ID No.5.

[0060] Wherein, the host cell is a eukaryotic host cell.

[0061] Furthermore, the eukaryotic host cell can be HEK293 cell, CHO cell, yeast cell, insect cell and the like.

[0062] In a specific embodiment of the present invention, the host cell is specifically an Expi293F cell.

[0063] The present invention uses albumin signal peptide Al, luciferase signal peptide Ga (SEQ ID No.1) and antibody signal peptide Lc to guide the eukaryotic cell secretory expression of monkeypox virus M1R antigen. Studies have shown that the secretory expression of monkeypox virus M1R antigen in the luciferase signal peptide Ga experimental group is significantly better than that in the albumin signal peptide Al 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 M1R 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 produces a protein sequence that meets expectations. The present invention is suitable for applications such as antigen preparation in vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The enzyme digestion identification diagram for the recombinant expression plasmid construction. Among them, 1 is pCGS3-Al-M1R (Al is the albumin signal peptide), 2 is pCGS3-Ga-M1R (Ga is the luciferase signal peptide), and 3 is pCGS3-Lc-M1R (Lc is the antibody light chain signal peptide).

[0065] Figure 2 This is the SDS-PAGE identification diagram of the supernatant secreted by the monkeypox virus M1R antigen cell. Among them, 1 is the albumin signal peptide Al secretion supernatant (pCGS3-Al-M1R transfected cell culture supernatant), 2 is the luciferase signal peptide Ga secretion supernatant (pCGS3-Ga-M1R transfected cell culture supernatant), 3 is the antibody light chain signal peptide Lc secretion supernatant (pCGS3-Lc-M1R transfected cell culture supernatant), and 4 is the negative group without transfected expression plasmid (pCGS3 vector transfected cell culture supernatant).

[0066] Figure 3The grayscale analysis diagram of the supernatant secreted by the monkeypox virus M1R antigen cell. Among them, 1 is the supernatant secreted by the albumin signal peptide Al (the supernatant of the cell culture transfected with pCGS3-Al-M1R), 2 is the supernatant secreted by the luciferase signal peptide Ga (the supernatant of the cell culture transfected with pCGS3-Ga-M1R), 3 is the supernatant secreted by the antibody light chain signal peptide Lc (the supernatant of the cell culture transfected with pCGS3-Lc-M1R), and 4 is the negative group without transfected expression plasmid (the supernatant of the cell culture transfected with pCGS3 vector).

[0067] Figure 4 The purified SDS-PAGE purification and identification of monkeypox virus M1R antigen protein. Among them, 1 is the purified sample of albumin signal peptide Al secretion expression, 2 is the purified sample of luciferase signal peptide Ga secretion expression, and 3 is the purified sample of antibody light chain signal peptide Lc secretion expression. DETAILED DESCRIPTION

[0068] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0069] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0070] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0071] The main reagents and their manufacturers in the following examples are as follows:

[0072] pCGS3 vector: Merck;

[0073] HindIII endonuclease: NEB;

[0074] PacI endonuclease: NEB;

[0075] GXL Premix: TAKARA Corporation;

[0076] DNA Ligation Kit Ver.2.1: TAKARA;

[0077] Expi293F TM Cells: Thermo Fisher Scientific.

[0078] Expi293 TM Expression Medium: Thermo Fisher Scientific;

[0079] ExpiFectamine TM 293 Transfection Kit: Thermo Fisher;

[0080] Opti-MEM TM I Reduced Serum Medium: Thermo Fisher Company;

[0081] Ni-NTA protein purification kit: Sangon Biotech (Shanghai) Co., Ltd.;

[0082] Amicon Ultra-15 centrifugal filter device: Millipore;

[0083] Amicon Ultra-0.5 centrifugal filter device: Millipore;

[0084] PBS pH 7.4 (1×): Gibco;

[0085] Gel imaging system: Protein Simple;

[0086] Cell counter: Roche.

[0087] Example 1: Construction of recombinant expression plasmid

[0088] In this example, recombinant expression plasmids containing three different signal peptides were constructed, namely pCGS3-Al-M1R, pCGS3-Ga-M1R and pCGS3-Lc-M1R.

[0089] The recombinant expression plasmid pCGS3-A1-M1R is a recombinant expression vector obtained by replacing the fragment between the restriction endonuclease HindIII recognition site and the restriction endonuclease PacI recognition site of the pCGS3 vector with a DNA molecule having a nucleotide sequence of SEQ ID No.6, while keeping other nucleotides of the pCGS3 vector unchanged. Positions 1-6 of SEQ ID No.6 are HindIII recognition sites, positions 7-15 are Kozak sequences, positions 16-63 are albumin signal peptide A1 encoding genes, positions 64-618 are M1R antigen encoding genes, positions 619-633 are linker genes, positions 634-651 are histidine tag genes, positions 652-654 are stop codons, and positions 655-662 are PacI recognition sites. The recombinant expression plasmid pCGS3-Al-M1R contains the coding gene of the recombinant protein Al-M1R. The coding sequence of the coding gene of the recombinant protein Al-M1R is the 16th to 654th positions of SEQ ID No. 6. The encoded recombinant protein Al-M1R is a protein fused with the Al signal peptide and the 18th to 213th positions of sequence 5, and consists of 212 amino acids.

[0090] The recombinant expression plasmid pCGS3-Ga-M1R is a recombinant expression vector obtained by replacing the fragment between the restriction endonuclease HindIII recognition site and the restriction endonuclease PacI recognition site of the pCGS3 vector with a DNA molecule having a nucleotide sequence of SEQ ID No. 7, while keeping 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 luciferase signal peptide Ga encoding genes, positions 67-621 are M1R antigen encoding genes, positions 622-636 are linker genes, positions 637-654 are histidine tag genes, positions 655-657 are stop codons, and positions 658-665 are PacI recognition sites. The recombinant expression plasmid pCGS3-Ga-M1R contains the coding gene of the recombinant protein Ga-M1R. The coding sequence of the coding gene of the recombinant protein Ga-M1R is the 16th to 657th positions of SEQ ID No. 7. The amino acid sequence of the coding gene of the recombinant protein Ga-M1R is the protein of sequence 5.

[0091] The recombinant expression plasmid pCGS3-Lc-M1R is a recombinant expression vector obtained by replacing the fragment between the restriction endonuclease HindIII recognition site and the restriction endonuclease PacI recognition site of the pCGS3 vector with a DNA molecule having a nucleotide sequence of SEQ ID No. 8, while keeping other nucleotides of the pCGS3 vector unchanged. Positions 1-6 of SEQ ID No. 8 are HindIII recognition sites, positions 7-15 are Kozak sequences, positions 16-75 are signal peptide Lc encoding genes, positions 76-630 are M1R antigen encoding genes, positions 631-645 are linker genes, positions 646-663 are histidine tag genes, positions 664-666 are stop codons, and positions 667-674 are PacI recognition sites. The recombinant expression plasmid pCGS3-Lc-M1R contains the coding gene of the recombinant protein Lc-M1R. The coding sequence of the coding gene of the recombinant protein Lc-M1R is the 16th to 666th positions of SEQ ID No.8. The encoded recombinant protein Lc-M1R is a protein fused with the Lc signal peptide and the 18th to 213th positions of sequence 5, and consists of 216 amino acids.

[0092] The extracellular region of the M1R antigen (NCBI accession number URK20517.1) sequence of monkeypox virus (NCBI genome accession number ON563414.3) was selected (the amino acid sequence of the M1R antigen protein is SEQ ID No. 3, and the coding sequence of the encoding gene is SEQ ID No. 4), that is, the fusion proteins obtained by fusion of the 1-185 amino acid sequence with different signal peptides are named Al-M1R, Ga-M1R and Lc-M1R.

[0093] The above three recombinant expression plasmids are respectively obtained by fusing the coding genes of albumin signal peptide Al, luciferase signal peptide Ga and antibody light chain signal peptide Lc to the 5' end of the coding gene of monkeypox virus M1R antigen protein, and the corresponding fragment names are Al-M1R, Ga-M1R and Lc-M1R, which are cloned into pCGS3 vector to construct the eukaryotic recombinant expression plasmids. The specific method is as follows:

[0094] Al-M1R gene synthesis: The carbon terminus of the albumin signal peptide Al was fused with the monkeypox antigen M1R. Sangon Biotechnology was commissioned to perform codon optimization and synthesize the Al-M1R gene (the nucleotide sequence is SEQ ID No. 6). The synthetic plasmid delivered by Sangon Biotechnology was pUC57-Al-M1R (containing the Al-M1R gene).

[0095] pUC57-Al-M1R was used as template and polymerase GXL Premix (TAKARA) was used to amplify Ga-M1R and Lc-M1R target fragments. The details are as follows:

[0096] 1) Ga-M1R gene fragment amplification: Primer 1 and primer 5 are used to amplify fragment A, and fragment A is used as the second round template. Primer 2 and primer 5 are used for the second round of amplification to obtain fragment B, which is the Ga-M1R target fragment;

[0097] 2) Lc-M1R gene fragment amplification: Primers 3 and 5 were used to amplify fragment C, which was used as the second round template. Primers 4 and 5 were used to perform the second round of amplification to obtain fragment D, which was the target fragment of Lc-M1R.

[0098] The primer sequences used in the above PCR amplification are as follows:

[0099] Primer 1: 5'-TGTTTGCCTGATTTGTATTGCCGTGGCTGAGGCCATGGGCGCCGCTGCTTCCAT-3';

[0100] Primer 2: 5'-CCCAAGCTTGCCGCCACCATGGGGGTGAAGGTGTTGTTGCTCTGATTTGTATTG-3';

[0101] Primer 3: 5'-CTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAGATGCATGGGCGCCGCTGCTTCCAT-3';

[0102] Primer 4: 5'-CCCAAGCTTGCCGCCACCATGAGCGTGCCAACCCAGGTGCTGGGCCTGCTGCTGCTGTG-3';

[0103] Primer 5: 5'-CCTTAATTAATCAGTGGTGGTGATGGTGATGAGAG-3'.

[0104] The pCGS3 vector was double-digested with HindIII (NEB) and PacI (NEB) to obtain the vector fragment, the fully synthesized gene pUC57-Al-M1R, and the amplified fragments Ga-M1R and Lc-M1R were double-digested with HindIII and PacI to obtain the target fragment. DNA Ligation Kit Ver.2.1 (TAKARA) was used for ligation, transformation, plasmid extraction, and identification. Three recombinant expression plasmids pCGS3-Al-M1R, pCGS3-Ga-M1R, and pCGS3-Ga-M1R were obtained.

[0105] The results of enzyme digestion identification of the above three recombinant expression plasmids are as follows Figure 1 As shown, lane 1 is the double enzyme digestion identification of pCGS3-Al-M1R expression plasmid, lane 2 is the double enzyme digestion identification of pCGS3-Ga-M1R expression plasmid, and lane 3 is the double enzyme digestion identification of pCGS3-Lc-M1R expression plasmid. After digestion, the vector fragment size is about 7100bp, and the target gene size is about 440bp; the size of the digestion band is in line with expectations.

[0106] Example 2: Protein transient expression study

[0107] A. Cell Expi293F transfection experiment

[0108] 1. Host Cell Culture

[0109] Take host cells Expi293F (Expi293F TM Cells were rapidly thawed in a 37°C water bath, and the thawed cell suspension was aseptically transferred to a 125mL vial containing 30mL preheated complete growth medium. The shaking incubator was cultured at 37°C, 8% CO2, 120rpm, amplitude 25mm, and humidity ≥80%. After 15-30 minutes, the cell suspension was taken to test the cell density and viability.

[0110] 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 amplification.

[0111] 2. Cell Transfection

[0112] The recombinant vectors pCGS3-Al-M1R, pCGS3-Ga-M1R, pCGS3-Lc-M1R and pCGS3 constructed in Example 1 were transfected into host cells separately.

[0113] 1. One day before transfection

[0114] 24 h before transfection, cells were plated at 2.5-3×10 6 cells / mL and cultured for 24 h.

[0115] 2. On the day of transfection

[0116] (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 cells / mL with fresh pre-warmed complete growth medium. 6 cells / mL.

[0117] (2) Prepare transfection reagent and DNA complex

[0118] 1) DNA dilution

[0119] The plasmid (pCGS3-Al-M1R, pCGS3-Ga-M1R and pCGS3-Lc-M1R constructed in Example 1) was diluted to 1 μg / μL with sterile water. According to the amount of 1 μg of plasmid for transfection 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-MEMTM I Reduced Serum medium for later use.

[0120] 2) Dilution of transfection reagent

[0121] Before use, add the transfection reagent ExpiFectamine293 TM Gently invert the Reagent upside down to mix well, and take the amount of transfection reagent required to transfect 50mL of cells, i.e. 160μL ExpiFectamine293 TM The reagent was added to 2.8 mL Opti-MEMTM I Reduced Serum medium by gently inverting to mix well and allowed to stand at room temperature for 5 min.

[0122] 3) Add the diluted transfection reagent to the plasmid and gently mix by inverting up and down. Incubate at room temperature for 10-20 minutes. Slowly add the mixed transfection reagent and DNA complex to the cell culture. Incubate at 37°C, 8% CO2, 120 rpm, amplitude 25 mm, humidity ≥ 80%.

[0123] 3. First day after transfection

[0124] 18-22h after transfection, add enhancer according to the amount of transfection 50mL cells. That is, take 300μL ExpressiFectamineTM 293 Transfection Enhancer 1 and 3mL ExpiFectamineTM 293 Transfection Enhancer 2, mix them well and slowly add them to the cell culture to obtain pCGS3-Al-M1R transfected cells, pCGS3-Ga-M1R transfected cells, pCGS3-Lc-M1R transfected cells and pCGS3 vector transfected cells respectively.

[0125] 4. Collection of culture supernatant

[0126] The cell viability was monitored every day after transfection. The culture was terminated when the viability dropped to 65%-75% on the 4th day. The culture was collected and centrifuged at 3500g for 30 min to collect the supernatant, and the culture supernatant of pCGS3-Al-M1R transfected cell culture supernatant, pCGS3-Ga-M1R transfected cell culture supernatant, pCGS3-Lc-M1R transfected cell culture supernatant and pCGS3 vector transfected cell culture supernatant were obtained respectively.

[0127] B. SDS protein electrophoresis and grayscale analysis

[0128] The above supernatant was subjected to protein electrophoresis analysis, and the grayscale analysis of the SDS protein electrophoresis image was performed using Image J software. The operation steps were Image→Type→32-Bit to convert to grayscale; Process→Subtract Background→OK to remove the background color; the rectangular tool selected the lane→Analyze→Gel→Select First Lane to determine the analysis lane, and repeatedly selected multiple lanes for simultaneous analysis; Analyze→Gel→Plot Lane to generate the peak area; the linear tool selected the peak map corresponding to the target band, and the Wand tool calculated the corresponding peak map area to obtain the percentage of the target protein in the total protein.

[0129] The results of SDS-PAGE identification and grayscale analysis showed that the expression levels of the target proteins in the secretion supernatant of albumin signal peptide Al (pCGS3-Al-M1R transfected cell culture supernatant), the secretion supernatant of luciferase signal peptide Ga (pCGS3-Ga-M1R transfected cell culture supernatant), and the secretion supernatant of antibody light chain signal peptide Lc (pCGS3-Lc-M1R transfected cell culture supernatant) accounted for 2.16%, 12.09% and 11.42% of the total protein ( Figure 2 and Figure 3 ). In summary, compared with signal peptide Al and signal peptide Lc, the monkeypox virus M1R antigen guided by luciferase signal peptide Ga has a higher secretory expression level.

[0130] Example 3: Protein purification of M1R antigen

[0131] 1. Ultrafiltration concentration

[0132] The pCGS3-Al-M1R transfected cell culture supernatant, pCGS3-Ga-M1R transfected cell culture supernatant and pCGS3-Lc-M1R transfected cell culture supernatant of Example 2 were respectively subjected to ultrafiltration and concentration by centrifugation at 6000g 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.

[0133] 2. Nickel column purification

[0134] The ultrafiltration concentrated supernatant obtained in step 1 and Binding / Wash Buffer are mixed in a volume ratio of 1:1 and allowed to stand for 20 minutes for full incubation. Two column volumes of Binding / Wash Buffer are used to balance the column, and the buffer flows through the pre-loaded column by gravity. The ultrafiltration concentrated supernatant and Binding / Wash Buffer mixture are added to the column and flowed through the pre-loaded column by gravity; if there is any remaining sample, it can be loaded again, re-circulated once, and the flow-through liquid is collected into a centrifuge tube. Two column volumes of Binding / wash buffer are used to wash the column and the flow-through liquid is collected until the absorbance of the flow-through liquid at 280nm is close to the baseline. Two column volumes of Elution Buffer are used to elute the histidine-tagged protein on the column. Repeat this step until the absorbance of the flow-through liquid at 280nm is close to the baseline, and the eluate is collected for purification.

[0135] 3. Ultrafiltration replacement

[0136] The protein solution after nickel column purification was added to Amicon Ultra-15 centrifugal filter device (Millipore), and centrifuged at 10000g for 3min in batches until about 150μL of the solution remained. 300μL PBS (pH7.4) was gently added, and centrifuged at 10000g until 150μL remained, and repeated three times. PBS (pH7.4) was eluted into the ultrafiltration tube to collect the sample, and the final volume was about 1-2mL. 5μL of the sample was retained for protein concentration determination and SDS-PAGE protein electrophoresis detection.

[0137] After Ni column purification, the yield of recombinant protein Al-M1R in the culture supernatant of pCGS3-Al-M1R transfected cells was 12.19 mg / L, the yield of recombinant protein Ga-M1R in the culture supernatant of pCGS3-Ga-M1R transfected cells was 73.43 mg / L, and the yield of recombinant protein Lc-M1R in the culture supernatant of pCGS3-Lc-M1R transfected cells was 14.06 mg / L. Figure 4 ), among which, the signal peptide Ga experimental group had the highest yield after purification.

[0138] Aluminum salt; or CpG; or liposome; or oily adjuvant can be used to produce monkeypox virus immune composition for preventing monkeypox infection.

[0139] Based on the results of the above examples, the present invention uses three signal peptides, signal peptide Al, signal peptide Ga and signal peptide Lc, to guide the eukaryotic cell secretory expression of monkeypox virus M1R antigen. 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.

[0140] 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 implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for preparing a secretory protein of monkeypox virus M1R antigen, comprising the following steps: (A1) introducing a nucleic acid molecule into a host cell to obtain a recombinant cell; the nucleic acid molecule encodes a fusion protein, the fusion protein is a protein obtained by fusing a polypeptide to the N-terminus of the monkeypox virus M1R antigen, and the amino acid sequence of the polypeptide is SEQ ID No. 1; (A2) culturing the recombinant cells and obtaining the monkeypox virus M1R antigen secretory protein from the culture supernatant; The amino acid sequence of the monkeypox virus M1R antigen is positions 18-202 of SEQ ID No.

5.

2. The method according to claim 1, characterized in that: The amino acid sequence of the fusion protein is SEQ ID No. 5, positions 1 to 202 of SEQ ID No. 5, or positions 1 to 207 of SEQ ID No.

5.

3. The method according to claim 1 or 2, characterized in that: The nucleic acid molecule is composed of a gene encoding the polypeptide and a gene encoding the monkeypox virus M1R antigen; The polypeptide encoding gene is any of the following: (a1) a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 2; (a2) a DNA molecule that hybridizes with the DNA molecule defined in (a1) under stringent conditions and encodes the polypeptide; (a3) a DNA molecule having more than 80% identity with the DNA sequence defined in (a1) or (a2) and encoding the polypeptide; The gene encoding the monkeypox virus M1R antigen is any one of the following: (b1) the nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 4; (b2) a DNA molecule that hybridizes with the DNA molecule defined in (b1) under stringent conditions and encodes the same protein; (b3) a DNA molecule having more than 80% identity with the DNA sequence defined in (b1) or (b2) and encoding the same protein; (b4) The nucleic acid molecule is a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No.7, positions 1 to 621, or positions 1 to 636 of SEQ ID No.7, or the like.

4. The fusion protein according to claim 1 or 2.

5. A nucleic acid molecule encoding the fusion protein of claim 4.

6. The nucleic acid molecule according to claim 5, characterized in that: The nucleic acid molecule is composed of a gene encoding the polypeptide and a gene encoding the monkeypox virus M1R antigen; The polypeptide encoding gene is any of the following: (a1) a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 2; (a2) a DNA molecule that hybridizes with the DNA molecule defined in (a1) under stringent conditions and encodes the polypeptide; (a3) a DNA molecule having more than 80% identity with the DNA sequence defined in (a1) or (a2) and encoding the polypeptide; The gene encoding the monkeypox virus M1R antigen is any one of the following: (b1) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 4; (b2) a DNA molecule that hybridizes with the DNA molecule defined in (b1) under stringent conditions and encodes the same protein; (b3) a DNA molecule having more than 80% identity with the DNA sequence defined in (b1) or (b2) and encoding the same protein; (b4) The nucleic acid molecule is a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No.7, positions 1 to 621, or positions 1 to 636 of SEQ ID No.7, or the like.

7. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule of claim 6.

8. Use of the fusion protein according to claim 4, the nucleic acid molecule according to claim 5 or 6, or the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line according to claim 7 in any of the following: P1. Application of increasing the secretory expression yield of monkeypox virus M1R antigen in host cells; P2. Application of improving the secretion expression efficiency of monkeypox virus M1R antigen in host cells; P3, application in the preparation of secretory protein products of monkeypox virus M1R antigen; The host cell is Expi293F cell.

9. 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: P1. Application of increasing the secretory expression yield of monkeypox virus M1R antigen in host cells; P2. Application of improving the secretion expression efficiency of monkeypox virus M1R antigen in host cells; P3, application in the preparation of secretory protein products of monkeypox virus M1R antigen; The related biological material is the coding gene of the polypeptide or an expression box or a recombinant vector or a recombinant bacterium or a transgenic cell line containing the coding gene; the host cell is an Expi293F cell.

10. The use according to claim 9, characterized in that: The coding gene is any of the following: (a1) a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 2; (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; (a3) A DNA molecule having 80% or more identity with the DNA sequence defined in (a1) or (a2) and encoding the polypeptide.

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