A monkeypox virus A35R antigen and preparation method thereof

Abstract

The present invention discloses a monkeypox virus A35R antigen and a preparation method thereof. The present invention relates to the field of biotechnology, and in particular to a monkeypox virus A35R antigen and a preparation method thereof. The fusion protein provided by the present invention is a protein obtained by fusing a polypeptide having an amino acid sequence of SEQ ID No.1 to the N-terminus of the monkeypox virus A35R antigen, and the amino acid sequence of the fusion protein is SEQ ID No.5, the 1-141st position of SEQ ID No.5, or the 1-146th position of SEQ ID No.5. The present invention adopts a signal peptide to guide the secretory expression of the monkeypox virus A35R eukaryotic cell, wherein the luciferase signal peptide Ga guides the secretory expression level of the monkeypox virus A35R antigen to be significantly better than the albumin signal peptide Al 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 A35R antigen and a preparation method thereof. Background Art

[0002] In May 2022, a new round of "monkeypox" epidemic was first discovered in the UK and has spread to many countries including the UK, Portugal, Spain, Australia, Germany, France, etc. At present, the "monkeypox" virus epidemic is still spreading, so it has attracted the attention of health regulatory authorities in many countries.

[0003] The incubation period of monkeypox virus is about 6-16 days, and the symptoms are fever, severe headache, lymphadenopathy, back pain, myalgia, and severe fatigue. The transmission mode is animal-to-human and human-to-human transmission. Among them, human-to-human transmission is caused by close contact with infected respiratory secretions, skin lesions of infected people, or objects recently contaminated by patient body fluids or diseased materials. Usually, long-term face-to-face contact is required for respiratory droplet transmission to occur.

[0004] 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 (extracellularenveloped, EV) derived from the endoplasmic reticulum membrane. Among them, A35R is highly similar to A33R of vaccinia virus. It is a type II membrane glycoprotein specific to extracellular enveloped virus (EEV). It plays a key role in the formation of effective EEV and contributes to the effective cell-to-cell transmission of virus particles. It is a potential target for the development of therapeutic antibodies against monkeypox virus. Therefore, the preparation of A35R protein is a key step in the development of key products for monkeypox virus detection reagents or vaccines. Summary of the invention

[0005] The main problem to be solved by the present invention is how to prepare monkeypox virus A35R antigen.

[0006] In order to solve the above problems, the present invention provides a luciferase signal peptide (signal peptide Ga, SEQ ID No. 1), which can guide the secretion of monkeypox virus A35R 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 albumin signal peptide (signal peptide Al) and antibody light chain signal peptide (signal peptide Lc).

[0007] The present invention first provides a fusion protein.

[0008] The fusion protein provided by the present invention is a protein obtained by fusing a polypeptide having an amino acid sequence of SEQ ID No. 1 to the N-terminus of monkeypox virus A35R antigen.

[0009] Furthermore, the amino acid sequence of the fusion protein is SEQ ID No.5, positions 1-141 of SEQ ID No.5, or positions 1-146 of SEQ ID No.5.

[0010] Positions 1-17 of SEQ ID No.5 are signal peptide Ga (i.e., SEQ ID No.1), positions 18-141 are the monkeypox virus A35R antigen, positions 142-146 are linkers, and positions 147-152 are histidine tags.

[0011] The present invention also provides a nucleic acid molecule encoding the fusion protein.

[0012] Furthermore, the nucleic acid molecule is composed of the coding gene of the above polypeptide and the coding gene of monkeypox virus A35R antigen in sequence from the 5' end to the 3' end.

[0013] Furthermore, the polypeptide encoding gene may be any of the following:

[0014] (a1) a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 2;

[0015] (a2) a DNA molecule that hybridizes with the DNA molecule defined in (a1) under stringent conditions and encodes the polypeptide;

[0016] (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.

[0017] Furthermore, the gene encoding the monkeypox virus A35R antigen may be any of the following:

[0018] (b1) the nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 4;

[0019] (b2) a DNA molecule that hybridizes with the DNA molecule defined in (b1) under stringent conditions and encodes the same protein;

[0020] (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.

[0021] More specifically, the nucleotide sequence of the coding strand of the nucleic acid molecule may be SEQ ID No.7, positions 1 to 438 of SEQ ID No.7, or positions 1 to 453 of SEQ ID No.7.

[0022] 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 438th positions are A35R antigen coding genes, the 439th to 453th positions are linker genes, the 454th to 471st positions are histidine tag genes, the 472th to 474th positions are stop codons, and the 475th to 482nd positions are PacI recognition sites. The recombinant expression plasmid pCGS3-Ga-A35R contains the coding gene of the recombinant protein Ga-A35R, the coding sequence of the coding gene of the recombinant protein Ga-A35R is the 16th to 474th positions of SEQ ID No.7, and the amino acid sequence of the coding gene of the recombinant protein Ga-A35R is the protein of sequence 5.

[0023] 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.

[0024] In the above nucleic acid molecules or coding genes, the stringent conditions may be as follows: 50°C, in 7% sodium dodecyl sulfate (SDS), 0.5M Na 3 PO 4 Hybridize in a mixed solution of 1mM EDTA and rinse in 2×SSC, 0.1% SDS at 50°C; or in 7% SDS, 0.5M Na 3 PO 4 Hybridize in a mixed solution of 1 mM EDTA and rinse in 1×SSC, 0.1% SDS at 50°C; or in 7% SDS, 0.5 M Na 3 PO 4 Hybridize in a mixed solution of 1mM EDTA and rinse in 0.5×SSC, 0.1% SDS at 50°C; or wash in 7% SDS, 0.5M Na 3 PO 4Hybridize in a mixed solution of 1mM EDTA and rinse in 0.1×SSC, 0.1% SDS at 50°C; or in 7% SDS, 0.5M Na 3 PO 4 Hybridize in a mixed solution of 1mM EDTA and rinse in 0.1×SSC, 0.1% SDS at 65°C; or hybridize in a solution of 6×SSC, 0.5% SDS at 65°C, and then wash the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0025] The present invention also provides an expression box, a recombinant vector, a recombinant microorganism or a transgenic cell line of the nucleic acid molecule.

[0026] 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.

[0027] 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.

[0028] 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:

[0029] P1. Application of monkeypox virus A35R antigen in improving secretory expression yield in host cells;

[0030] P2. Application of improving the secretion expression efficiency of monkeypox virus A35R antigen in host cells;

[0031] P3, application in the preparation of monkeypox virus A35R antigen secretory protein products;

[0032] 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.

[0033] 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 signal peptide Ga, i.e., SEQ ID No.2) into the multiple cloning site (such as HindIII and Pac I) of the pCGS3 vector.

[0034] Furthermore, the host cell may be a eukaryotic host cell, such as HEK293 cells, CHO cells, yeast cells and insect cells.

[0035] In a specific embodiment of the present invention, the host cell is specifically an Expi293F cell.

[0036] Furthermore, the coding gene may be any of the following:

[0037] (a1) a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 2;

[0038] (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;

[0039] (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.

[0040] 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.

[0041] 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.

[0042] 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:

[0043] P1. Application of increasing the secretory expression yield of monkeypox virus A35R antigen in host cells;

[0044] P2. Application of improving the secretion expression efficiency of monkeypox virus A35R antigen in host cells;

[0045] P3. Application in the preparation of monkeypox virus A35R antigen secretory protein products.

[0046] The amino acid sequence of the monkeypox virus A35R antigen secretory protein is SEQ ID No.5.

[0047] Wherein, the host cell is a eukaryotic host cell.

[0048] Furthermore, the eukaryotic host cell can be HEK293 cell, CHO cell, yeast cell, insect cell and the like.

[0049] In a specific embodiment of the present invention, the host cell is specifically an Expi293F cell.

[0050] The method for preparing the monkeypox virus A35R antigen secretory protein also falls within the scope of protection claimed by the present invention.

[0051] The method for preparing the monkeypox virus A35R antigen secretory protein claimed in the present invention may include the following steps:

[0052] (A1) introducing the nucleic acid molecule described above into a host cell to obtain a recombinant cell;

[0053] (A2) culturing the recombinant cells and obtaining the monkeypox virus A35R antigen secretory protein from the culture supernatant.

[0054] Wherein, the nucleic acid molecule can be introduced into the host cell via the recombinant vector mentioned above.

[0055] In step (A1), the host cell is a eukaryotic host cell, such as HEK293 cells, CHO cells, yeast cells and insect cells.

[0056] In a specific embodiment of the present invention, the host cell is specifically an Expi293F cell.

[0057] In step (A2), the culturing is terminated when the cell viability drops to 65%-75%.

[0058] In step (A2), the secretory protein of monkeypox virus A35R 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.

[0059] 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 A35R antigen. Studies have shown that the secretory expression of monkeypox virus A35R 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 A35R 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

[0060] Figure 1 The enzyme digestion identification diagram for the recombinant expression plasmid construction. Among them, 1 is pCGS3-Al-A35R (Al is the albumin signal peptide), 2 is pCGS3-Ga-A35R (Ga is the luciferase signal peptide), and 3 is pCGS3-Lc-A35R (Lc is the antibody light chain signal peptide).

[0061] Figure 2 The SDS-PAGE identification diagram of the supernatant secreted by the monkeypox virus A35R antigen cell. Among them, 1 is the albumin signal peptide Al secretion supernatant (pCGS3-Al-A35R transfected cell culture supernatant), 2 is the luciferase signal peptide Ga secretion supernatant (pCGS3-Ga-A35R transfected cell culture supernatant), 3 is the antibody light chain signal peptide Lc secretion supernatant (pCGS3-Lc-A35R transfected cell culture supernatant), and 4 is the negative group without transfected expression plasmid (pCGS3 vector transfected cell culture supernatant).

[0062] Figure 3 The grayscale analysis of the supernatant secreted by the monkeypox virus A35R antigen cell. Among them, 1 is the albumin signal peptide Al secretion supernatant (pCGS3-Al-A35R transfected cell culture supernatant), 2 is the luciferase signal peptide Ga secretion supernatant (pCGS3-Ga-A35R transfected cell culture supernatant), 3 is the antibody light chain signal peptide Lc secretion supernatant (pCGS3-Lc-A35R transfected cell culture supernatant), and 4 is the negative group without transfected expression plasmid (pCGS3 vector transfected cell culture supernatant).

[0063] Figure 4The purified SDS-PAGE purification and identification of monkeypox virus A35R 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

[0064] 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.

[0065] 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.

[0066] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0067] The main reagents and their manufacturers in the following examples are as follows:

[0068] pCGS3 vector: Merck;

[0069] HindIII endonuclease: NEB;

[0070] PacI endonuclease: NEB;

[0071] Prime GXL Premix: TAKARA Corporation;

[0072] DNA Ligation Kit Ver.2.1: TAKARA;

[0073] Expi293F TM Cells: Thermo Fisher Scientific.

[0074] Expi293 TM Expression Medium: Thermo Fisher Scientific;

[0075] ExpiFectamine TM 293 Transfection Kit: Thermo Fisher;

[0076] Opti-MEM TMI Reduced Serum Medium: Thermo Fisher Company;

[0077] Ni-NTA protein purification kit: Sangon Biotech (Shanghai) Co., Ltd.;

[0078] Amicon Ultra-15 centrifugal filter device: Millipore;

[0079] Amicon Ultra-0.5 centrifugal filter device: Millipore;

[0080] PBS pH 7.4 (1×): Gibco;

[0081] Gel imaging system: Protein Simple;

[0082] Cell counter: Roche;

[0083] Clean bench: Suzhou Antai Air Technology Co., Ltd.;

[0084] Electric constant temperature water bath: Fisher Scientific;

[0085] CO2 constant temperature shaker: CRYSTAL;

[0086] HYG-A fully constant temperature bottle shaking cabinet: Taicang Experimental Equipment Factory;

[0087] DYY-6C electrophoresis apparatus: Beijing Liuyi Instrument Factory;

[0088] DYCP-31DN horizontal electrophoresis tank: Beijing Liuyi Instrument Factory;

[0089] Micropipette: Eppendorf.

[0090] Example 1: Construction of recombinant expression plasmid

[0091] The monkeypox virus is selected from the latest monkeypox virus in 2022 (NCBI genome accession number is ON563414.3), among which the NCBI accession number of the A35R antigen is URK20584.1. The present invention selects the extracellular region of the A35R antigen sequence, that is, the amino acid sequence of positions 58-181. The coding genes of the albumin signal peptide Al, the luciferase signal peptide Ga, and the antibody light chain signal peptide Lc are fused to the 5' end of the coding gene of the monkeypox virus A35R antigen protein, and the corresponding fragment names are Al-A35R, Ga-A35R and Lc-A35R, respectively, cloned into the pCGS3 vector, and a eukaryotic recombinant expression plasmid is constructed. The specific operation process is described as follows.

[0092] Al-A35R gene synthesis: The carbon terminus of the albumin signal peptide Al was fused with monkeypox antigen A35R (the amino acid sequence of monkeypox antigen A35R is SEQ ID No. 3, and the coding sequence of the encoding gene is SEQ ID No. 4). Sangon Biotechnology was commissioned to perform codon optimization and synthesize the Al-A35R gene (the nucleotide sequence is SEQ ID No. 6). The synthetic plasmid delivered by Sangon Biotechnology was pUC57-Al-A35R (containing the Al-A35R gene).

[0093] pUC57-Al-A35R was used as template and polymerase Prime was used GXL Premix (TAKARA) was used to amplify Ga-A35R and Lc-A35R target fragments. The details are as follows:

[0094] 1) Ga-A35R gene fragment amplification: Primer 1 and primer 5 are used to amplify fragment A, fragment A is used as the second round template, and primer 2 and primer 5 are used for the second round of amplification to obtain fragment B, which is the Ga-A35R target fragment;

[0095] 2) Amplification of Lc-A35R gene fragment: 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-A35R.

[0096] The primer sequences used in the above PCR amplification are as follows:

[0097] Primer 1: 5'-TGTTTGCTCTGATTTGTATTGCCGTGGCTGAGGCCAGACTGAACCAGTGTATGTC-3';

[0098] Primer 2: 5'-CCCAAGCTTGCCGCCACCATGGGGGTGAAGGTGTTGTTGCTCTGATTTGTATTG-3';

[0099] Primer 3: 5'-CTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAGATGCAGACTGAACCAGTGTATGTC-3';

[0100] Primer 4: 5'-CCCAAGCTTGCCGCCACCATGAGCGTGCCAACCCAGGTGCTGGGCCTGCTGCTGCTGTG-3';

[0101] Primer 5: 5'-CCTTAATTAATCAGTGGTGGTGGTGATGGTGAGAG-3'.

[0102] The pCGS3 vector was double-digested with HindIII (NEB) and PacI (NEB) to obtain the vector fragment, the fully synthesized gene pUC57-Al-A35R, and the amplified fragments Ga-A35R and Lc-A35R 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-A35R, pCGS3-Ga-A35R, and pCGS3-Ga-A35R were obtained.

[0103] 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-A35R expression plasmid, lane 2 is the double enzyme digestion identification of pCGS3-Ga-A35R expression plasmid, and lane 3 is the double enzyme digestion identification of pCGS3-Lc-A35R expression plasmid. After digestion, the vector fragment size is about 7100bp, and the target gene size is about 480bp; the size of the digestion band is in line with expectations.

[0104] The structural description of the recombinant expression plasmid pCGS3-A1-A35R is as follows: the recombinant expression vector is 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 the other nucleotides of the pCGS3 vector unchanged. The 1st to 6th positions of SEQ ID No.6 are HindIII recognition sites, the 7th to 15th positions are Kozak sequences, the 16th to 63rd positions are albumin signal peptide A1 encoding genes, the 64th to 435th positions are A35R antigen encoding genes, the 436th to 450th positions are linker genes, the 451st to 468th positions are histidine tag genes, the 469th to 471st positions are stop codons, and the 472th to 479th positions are PacI recognition sites. The recombinant expression plasmid pCGS3-Al-A35R contains a gene encoding a recombinant protein Al-A35R, the coding sequence of the gene encoding the recombinant protein Al-A35R is the 16th to 471st positions of SEQ ID No.6, and the encoded recombinant protein Al-A35R is a protein obtained by fusion of the Al signal peptide and the 18th to 152th positions of sequence 5, and consists of 151 amino acids.

[0105] The structural description of the recombinant expression plasmid pCGS3-Ga-A35R is as follows: the recombinant expression vector is 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 the other nucleotides of the pCGS3 vector unchanged. 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 encoding genes, the 67th to 438th positions are A35R antigen encoding genes, the 439th to 453th positions are linker genes, the 454th to 471th positions are histidine tag genes, the 472th to 474th positions are stop codons, and the 475th to 482nd positions are PacI recognition sites. The recombinant expression plasmid pCGS3-Ga-A35R contains the coding gene of the recombinant protein Ga-A35R, the coding sequence of the coding gene of the recombinant protein Ga-A35R is the 16th to 474th positions of SEQ ID No.7, and the amino acid sequence encoded by the coding gene of the recombinant protein Ga-A35R is a protein of sequence 5.

[0106] The structural description of the recombinant expression plasmid pCGS3-Lc-A35R is as follows: the recombinant expression vector is 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 the other nucleotides of the pCGS3 vector unchanged. The 1st to 6th positions of SEQ ID No.8 are HindIII recognition sites, the 7th to 15th positions are Kozak sequences, the 16th to 75th positions are signal peptide Lc encoding genes, the 76th to 447th positions are A35R antigen encoding genes, the 448th to 462th positions are linker genes, the 463th to 480th positions are histidine tag genes, the 481st to 483rd positions are stop codons, and the 484th to 491st positions are PacI recognition sites. The recombinant expression plasmid pCGS3-Lc-A35R contains the coding gene of the recombinant protein Lc-A35R. The coding sequence of the coding gene of the recombinant protein Lc-A35R is the 16th to 483rd positions of SEQID No.8. The encoded recombinant protein Lc-A35R is a protein obtained by fusion of the Lc signal peptide and the 18th to 152th positions of sequence 5, and consists of 155 amino acids.

[0107] Example 2: Study on transient protein expression

[0108] A. Cell Expi293F transfection experiment

[0109] 1. Host Cell Culture

[0110] 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 125 mL vial containing 30 mL of pre-warmed complete growth medium. The shaking incubator was cultured at 37°C and 8% CO 2 , 120rpm, amplitude 25mm, humidity ≥80%. After 15-30min, take the cell suspension to test the cell density and viability.

[0111] 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.

[0112] 2. Cell Transfection

[0113] The recombinant vectors pCGS3-Al-A35R, pCGS3-Ga-A35R, pCGS3-Lc-A35R and pCGS3 constructed in Example 1 were transfected into host cells separately.

[0114] 1. One day before transfection

[0115] 24 h before transfection, cells were plated at 2.5-3×10 6 cells / mL and cultured for 24 h.

[0116] 2. On the day of transfection

[0117] (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.

[0118] (2) Prepare transfection reagent and DNA complex

[0119] 1) DNA dilution

[0120] The plasmid (pCGS3-Al-A35R, pCGS3-Ga-A35R and pCGS3-Lc-A35R 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.

[0121] 2) Dilution of transfection reagent

[0122] 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.

[0123] 3) Add the diluted transfection reagent to the plasmid and gently mix by inverting up and down. Incubate at room temperature for 10 to 20 minutes. Slowly add the mixed transfection reagent and DNA complex to the cell culture. 37°C, 8% CO 2 , 120 rpm, amplitude 25 mm, humidity ≥ 80%.

[0124] 3. First day after transfection

[0125] 18-22h after transfection, add enhancer according to the amount of transfection 50mL cells. That is, take 300μL ExpiFectamineTM 293Transfection Enhancer 1 and 3mL ExpiFectamineTM293Transfection Enhancer 2, mix well and slowly add to the cell culture to obtain pCGS3-Al-A35R transfected cells, pCGS3-Ga-A35R transfected cells, pCGS3-Lc-A35R transfected cells and pCGS3 vector transfected cells respectively.

[0126] 4. Collection of culture supernatant

[0127] 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-A35R transfected cells, pCGS3-Ga-A35R transfected cells, pCGS3-Lc-A35R transfected cells and pCGS3 vector transfected cells were obtained respectively.

[0128] B. SDS protein electrophoresis and grayscale analysis

[0129] 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.

[0130] 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-A35R transfected cell culture supernatant), the secretion supernatant of luciferase signal peptide Ga (pCGS3-Ga-A35R transfected cell culture supernatant), and the secretion supernatant of antibody light chain signal peptide Lc (pCGS3-Lc-A35R transfected cell culture supernatant) accounted for 2.62%, 10.63% and 2.33% of the total protein ( Figure 2 and Figure 3 ). In summary, compared with signal peptide Al and signal peptide Lc, the Ga monkeypox virus A35R antigen guided by the luciferase signal peptide of the present invention has a higher secretory expression amount.

[0131] Example 3: Protein purification of A35R antigen

[0132] 1. Ultrafiltration concentration

[0133] The culture supernatant of the pCGS3-Al-A35R transfected cells, the culture supernatant of the pCGS3-Ga-A35R transfected cells and the culture supernatant of the pCGS3-Lc-A35R transfected cells 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.

[0134] 2. Nickel column purification

[0135] 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.

[0136] 3. Ultrafiltration replacement

[0137] 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.

[0138] After Ni column purification, the yield of recombinant protein Ga-A35R in the culture supernatant of pCGS3-Ga-A35R transfected cells was 79.78 mg / L, the yield of recombinant protein Lc-A35R in the culture supernatant of pCGS3-Lc-A35R transfected cells was 18.13 mg / L, and the yield of recombinant protein Al-A35R in the culture supernatant of pCGS3-Al-A35R transfected cells was 22.18 mg / L ( Figure 4 ), among which, the signal peptide Ga experimental group had the highest yield after purification.

[0139] Aluminum salt; or CpG; or liposome; or oily adjuvant can be used to produce monkeypox virus immune composition for preventing monkeypox infection.

[0140] 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 A35R 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.

[0141] 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 nucleic acid molecule, characterized in that: The nucleic acid molecule consists of a polypeptide coding gene and a monkeypox virus A35R antigen coding gene; The polypeptide encoding gene is a DNA molecule whose nucleotide sequence is SEQ ID No. 2; The gene encoding the monkeypox virus A35R 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) The nucleic acid molecule is a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No.7, positions 1 to 438 of SEQ ID No.7, or positions 1 to 453 of SEQ ID No.

7.

2. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule according to claim 1.

3. Application of polypeptides or related biological materials in any of the following: P1. Application of monkeypox virus A35R antigen in improving secretory expression yield in host cells; P2. Application of improving the secretion expression efficiency of monkeypox virus A35R antigen in host cells; P3, application in the preparation of monkeypox virus A35R antigen secretory protein products; The polypeptide encoding gene is a DNA molecule whose nucleotide sequence is SEQ ID No. 2; The related biological material is the coding gene of the polypeptide or an expression cassette or a recombinant vector or a recombinant bacterium or a transgenic cell line containing the coding gene; The host cell is Expi293F cell.

4. Use of the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line according to claim 2 in any of the following: P1. Application of monkeypox virus A35R antigen in improving secretory expression yield in host cells; P2. Application of improving the secretion expression efficiency of monkeypox virus A35R antigen in host cells; P3, application in the preparation of monkeypox virus A35R antigen secretory protein products; The host cell is Expi293F cell.

5. A method for preparing a secretory protein of monkeypox virus A35R antigen, comprising the following steps: (A1) introducing the nucleic acid molecule of claim 1 into a host cell to obtain a recombinant cell; (A2) culturing the recombinant cells and obtaining the monkeypox virus A35R antigen secretory protein from the culture supernatant; The host cell is Expi293F cell.

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