Application of luciferase signal peptide, fusion protein containing luciferase signal peptide and preparation method thereof

Abstract

The present invention discloses the application of luciferase signal peptide and fusion protein containing luciferase signal peptide and preparation method thereof. The present invention provides the application of polypeptide whose amino acid sequence is SEQ ID No.1 or its related biological material in any of the following: P1, improving the secretion expression yield of monkeypox virus A29L antigen in host cells; P2, improving the secretion expression efficiency of monkeypox virus A29L antigen in host cells; P3, preparing monkeypox virus A29L antigen secretory protein products. The present invention adopts signal peptide to guide the eukaryotic cell secretion expression of monkeypox virus A29L, wherein the secretion expression level of monkeypox virus A29L antigen guided by luciferase signal peptide Ga is significantly better than that of albumin signal peptide Al and 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 the application of a luciferase signal peptide, a fusion protein containing the luciferase signal peptide, and a preparation method thereof. Background Art

[0002] In 1958, a poxvirus was discovered that caused a disease similar to human smallpox in experimental macaques, so it was called monkeypox virus. In 1970, the monkeypox virus was first found in a 9-year-old boy in Congo. The symptoms were blisters or pustules, and it was less contagious than smallpox. Since then, cases of monkeypox have been continuously found in Congo, Central Africa, and West Africa. 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, and France. At present, the "monkeypox" virus epidemic is still tending to spread, 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, A29L antigen is a membrane protein of mature virions of monkeypox virus and is also one of the important immune targets. Therefore, how to prepare A29L protein has become an urgent problem to be solved in the research and 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 A29L antigen.

[0005] 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 A29L 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).

[0006] The present invention first 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:

[0007] P1. Application of monkeypox virus A29L antigen in improving secretory expression yield in host cells;

[0008] P2. Application of improving the secretion expression efficiency of monkeypox virus A29L antigen in host cells;

[0009] P3, application in the preparation of monkeypox virus A29L antigen secretory protein products;

[0010] 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.

[0011] 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 PacI) of the pCGS3 vector.

[0012] Furthermore, the host cell may be a eukaryotic host cell, such as HEK293 cells, CHO cells, yeast cells and insect cells.

[0013] In a specific embodiment of the present invention, the host cell is specifically an Expi293F cell.

[0014] Furthermore, the coding gene may be any of the following:

[0015] (a1) a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 2;

[0016] (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;

[0017] (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.

[0018] 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.

[0019] 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.

[0020] The invention also provides a fusion protein.

[0021] 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 A29L antigen described in SEQ ID No.3.

[0022] Furthermore, the amino acid sequence of the fusion protein is SEQ ID No.5, positions 1 to 127 of SEQ ID No.5, or positions 1 to 132 of SEQ ID No.5.

[0023] Positions 1-17 of SEQ ID No.5 are signal peptide Ga (i.e., SEQ ID No.1), positions 18-127 are the monkeypox virus A29L antigen, positions 128-132 are linkers, and positions 133-138 are histidine tags.

[0024] The present invention also provides a nucleic acid molecule encoding the fusion protein.

[0025] Furthermore, the nucleic acid molecule is composed of the coding gene of the above polypeptide and the coding gene of monkeypox virus A29L antigen in sequence from the 5' end to the 3' end.

[0026] Furthermore, the polypeptide encoding gene may be any of the following:

[0027] (a1) a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 2;

[0028] (a2) a DNA molecule that hybridizes with the DNA molecule defined in (a1) under stringent conditions and encodes the polypeptide;

[0029] (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.

[0030] Furthermore, the gene encoding the monkeypox virus A29L antigen may be any of the following:

[0031] (b1) the nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 4;

[0032] (b2) a DNA molecule that hybridizes with the DNA molecule defined in (b1) under stringent conditions and encodes the same protein;

[0033] (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.

[0034] More specifically, the nucleotide sequence of the coding strand of the nucleic acid molecule may be SEQ ID No.7, positions 1 to 396 of SEQ ID No.7, or positions 1 to 411 of SEQ ID No.7.

[0035] 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 396th positions are A29L antigen coding genes, the 397th to 411th positions are linker genes, the 412th to 429th positions are histidine tag genes, the 430th to 432th positions are stop codons, and the 433th to 440th positions are PacI recognition sites. The recombinant expression plasmid pCGS3-Ga-A29L contains the coding gene of the recombinant protein Ga-A29L, the coding sequence of the coding gene of the recombinant protein Ga-A29L is the 16th to 432th positions of SEQ ID No.7, and the amino acid sequence of the coding gene of the recombinant protein Ga-A29L is the protein of sequence 5.

[0036] 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.

[0037] 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.

[0038] The present invention also provides an expression box, a recombinant vector, a recombinant microorganism or a transgenic cell line of the nucleic acid molecule.

[0039] 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.

[0040] 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.

[0041] 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:

[0042] P1. Application of monkeypox virus A29L antigen in improving secretory expression yield in host cells;

[0043] P2. Application of improving the secretion expression efficiency of monkeypox virus A29L antigen in host cells;

[0044] P3. Application in the preparation of monkeypox virus A29L antigen secretory protein products.

[0045] The amino acid sequence of the monkeypox virus A29L antigen secretory protein is SEQ ID No.5.

[0046] Wherein, the host cell is a eukaryotic host cell.

[0047] Furthermore, the eukaryotic host cell can be HEK293 cell, CHO cell, yeast cell, insect cell and the like.

[0048] In a specific embodiment of the present invention, the host cell is specifically an Expi293F cell.

[0049] The method for preparing the secretory protein of monkeypox virus A29L antigen also falls within the scope of protection claimed by the present invention.

[0050] The method for preparing the monkeypox virus A29L antigen secretory protein claimed in the present invention may include the following steps:

[0051] (A1) introducing the nucleic acid molecule described above into a host cell to obtain a recombinant cell;

[0052] (A2) culturing the recombinant cells and obtaining the monkeypox virus A29L antigen secretory protein from the culture supernatant.

[0053] Wherein, the nucleic acid molecule can be introduced into the host cell via the recombinant vector mentioned above.

[0054] In step (A1), the host cell is a eukaryotic host cell, such as HEK293 cells, CHO cells, yeast cells and insect cells.

[0055] In a specific embodiment of the present invention, the host cell is specifically an Expi293F cell.

[0056] In step (A2), the culturing is terminated when the cell viability drops to 65%-75%.

[0057] In step (A2), the secretory protein of monkeypox virus A29L 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 for ultrafiltration concentration and nickel column purification.

[0058] 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 A29L antigen. Studies have shown that the secretory expression of monkeypox virus A29L 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 A29L 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 expected protein sequence is produced. 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. Among them, 1 is pCGS3-Al-A29L (Al is the albumin signal peptide), 2 is pCGS3-Ga-A29L (Ga is the luciferase signal peptide), and 3 is pCGS3-Lc-A29L (Lc is the antibody light chain signal peptide).

[0060] Figure 2 The SDS-PAGE identification diagram of the supernatant secreted by the monkeypox virus A29L antigen cell. Among them, 1 is the supernatant secreted by the albumin signal peptide Al (pCGS3-Al-A29L transfected cell culture supernatant), 2 is the supernatant secreted by the luciferase signal peptide Ga (pCGS3-Ga-A29L transfected cell culture supernatant), 3 is the supernatant secreted by the antibody light chain signal peptide Lc (pCGS3-Lc-A29L transfected cell culture supernatant), and 4 is the negative group without transfected expression plasmid (pCGS3 vector transfected cell culture supernatant).

[0061] Figure 3 The grayscale analysis diagram of the supernatant secreted by the monkeypox virus A29L antigen cell. Among them, 1 is the albumin signal peptide Al secretion supernatant (pCGS3-Al-A29L transfected cell culture supernatant), 2 is the luciferase signal peptide Ga secretion supernatant (pCGS3-Ga-A29L transfected cell culture supernatant), 3 is the antibody light chain signal peptide Lc secretion supernatant (pCGS3-Lc-A29L transfected cell culture supernatant), and 4 is the negative group without transfected expression plasmid (pCGS3 vector transfected cell culture supernatant).

[0062] Figure 4 The purified SDS-PAGE purification and identification of monkeypox virus A29L 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

[0063] 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.

[0064] 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.

[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 Corporation;

[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 bottle shaking 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 NCBI accession number of the A29L antigen is URK20577.1. The coding genes of the albumin signal peptide Al, the luciferase signal peptide Ga and the antibody light chain signal peptide Lc were fused to the 5' end of the coding gene of the monkeypox virus A29L antigen protein, and the corresponding fragment names were Al-A29L, Ga-A29L and Lc-A29L, respectively, and cloned into the pCGS3 vector to construct a eukaryotic recombinant expression plasmid. The specific operation process is described as follows.

[0091] Al-A29L gene synthesis: The carbon terminus of the albumin signal peptide Al was fused with monkeypox antigen A29L (the amino acid sequence of monkeypox antigen A29L 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-A29L gene (the nucleotide sequence is SEQ ID No.6). The synthetic plasmid delivered by Sangon Biotechnology was pUC57-Al-A29L (containing the Al-A29L gene).

[0092] pUC57-Al-A29L was used as template and polymerase GXL Premix (TAKARA) was used to amplify Ga-A29L and Lc-A29L target fragments. The details are as follows:

[0093] 1) Ga-A29L 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-A29L target fragment;

[0094] 2) Lc-A29L 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-A29L.

[0095] The primer sequences used in the above PCR amplification are as follows:

[0096] Primer 1: 5'-TGTTTGCCTGATTTGTATTGCCGTGGCTGAGGCCATGGATGGCACCCTGTTTCC-3';

[0097] Primer 2: 5'-CCCAAGCTTGCCGCCACCATGGGGGTGAAGGTGTTGTTGCTCTGATTTGTATTG-3';

[0098] Primer 3: 5'-CTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAGATGCATGGATGGCACCCTGTTTCC-3';

[0099] Primer 4: 5'-CCCAAGCTTGCCGCCACCATGAGCGTGCCAACCCAGGTGCTGGGCCTGCTGCTGCTGTG-3';

[0100] Primer 5: 5'-CCTTAATTAATCAGTGGTGATGGTGGTGGTGAGAG-3'.

[0101] The pCGS3 vector was double-digested with HindIII (NEB) and PacI (NEB) to obtain the vector fragment, the fully synthesized gene pUC57-Al-A29L, and the amplified fragments Ga-A29L and Lc-A29L 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-A29L, pCGS3-Ga-A29L, and pCGS3-Ga-A29L were obtained.

[0102] 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-A29L expression plasmid, lane 2 is the double enzyme digestion identification of pCGS3-Ga-A29L expression plasmid, and lane 3 is the double enzyme digestion identification of pCGS3-Lc-A29L 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.

[0103] The structural description of the recombinant expression plasmid pCGS3-A1-A29L 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 393rd positions are A29L antigen encoding genes, the 394th to 408th positions are linker genes, the 409th to 426th positions are histidine tag genes, the 427th to 429th positions are stop codons, and the 429th to 437th positions are PacI recognition sites. The recombinant expression plasmid pCGS3-Al-A29L contains a gene encoding a recombinant protein Al-A29L. The coding sequence of the gene encoding the recombinant protein Al-A29L is the 16th to 429th positions of SEQ ID No.6. The encoded recombinant protein Al-A29L is a protein fused with the Al signal peptide and the 18th to 138th positions of sequence 5, and consists of 137 amino acids.

[0104] The structural description of the recombinant expression plasmid pCGS3-Ga-A29L 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 396th positions are A29L antigen encoding genes, the 397th to 411th positions are linker genes, the 412th to 429th positions are histidine tag genes, the 430th to 432th positions are stop codons, and the 433th to 440th positions are PacI recognition sites. The recombinant expression plasmid pCGS3-Ga-A29L contains the coding gene of the recombinant protein Ga-A29L. The coding sequence of the coding gene of the recombinant protein Ga-A29L is the 16th to 432nd positions of SEQ ID No.7. The amino acid sequence of the coding gene of the recombinant protein Ga-A29L is the protein of sequence 5.

[0105] The structural description of the recombinant expression plasmid pCGS3-Lc-A29L 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 405th positions are A29L antigen encoding genes, the 406th to 420th positions are linker genes, the 421st to 438th positions are histidine tag genes, the 439th to 441th positions are stop codons, and the 442th to 449th positions are PacI recognition sites. The recombinant expression plasmid pCGS3-Lc-A29L contains the coding gene of the recombinant protein Lc-A29L. The coding sequence of the coding gene of the recombinant protein Lc-A29L is the 16th to 441th positions of SEQID No.8. The encoded recombinant protein Lc-A29L is a protein fused with the Lc signal peptide and the 18th to 138th positions of sequence 5, and consists of 141 amino acids.

[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-A29L, pCGS3-Ga-A29L, pCGS3-Lc-A29L 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-A29L, pCGS3-Ga-A29L and pCGS3-Lc-A29L 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 well and slowly add to the cell culture to obtain pCGS3-Al-A29L transfected cells, pCGS3-Ga-A29L transfected cells, pCGS3-Lc-A29L 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 pCGS3-Al-A29L transfected cell culture supernatant, pCGS3-Ga-A29L transfected cell culture supernatant, pCGS3-Lc-A29L 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-A29L transfected cell culture supernatant), the secretion supernatant of luciferase signal peptide Ga (pCGS3-Ga-A29L transfected cell culture supernatant), and the secretion supernatant of antibody light chain signal peptide Lc (pCGS3-Lc-A29L 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 Ga monkeypox virus A29L antigen guided by the luciferase signal peptide of the present invention has a higher secretory expression amount.

[0130] Example 3: Protein purification of A29L antigen

[0131] 1. Ultrafiltration concentration

[0132] The pCGS3-Al-A29L transfected cell culture supernatant, pCGS3-Ga-A29L transfected cell culture supernatant and pCGS3-Lc-A29L 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 Ga-A29L in the culture supernatant of pCGS3-Ga-A29L transfected cells was 161.78 mg / L, the yield of recombinant protein Lc-A29L in the culture supernatant of pCGS3-Lc-A29L transfected cells was 142.15 mg / L, and the yield of recombinant protein Al-A29L in the culture supernatant of pCGS3-Al-A29L transfected cells was 38.62 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 A29L 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. 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 monkeypox virus A29L antigen in improving secretory expression yield in host cells; P2. Application of improving the secretion expression efficiency of monkeypox virus A29L antigen in host cells; P3, application in the preparation of monkeypox virus A29L antigen secretory protein products; The host cell is Expi293F cell; 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.

2. The use according to claim 1, 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.

3. A fusion protein, which is a protein obtained by fusing the polypeptide in the application of claim 1 to the N-terminus of monkeypox virus A29L antigen.

4. The fusion protein according to claim 3, characterized in that: The amino acid sequence of the fusion protein is SEQ ID No. 5, positions 1 to 127 of SEQ ID No. 5, or positions 1 to 132 of SEQ ID No.

5.

5. A nucleic acid molecule encoding the fusion protein of claim 3 or 4.

6. The nucleic acid molecule according to claim 5, characterized in that: The nucleic acid molecule consists of a gene encoding the polypeptide and a gene encoding the monkeypox virus A29L 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 A29L 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 396, or positions 1 to 411 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 3 or 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 monkeypox virus A29L antigen in improving secretory expression yield in host cells; P2. Application of improving the secretion expression efficiency of monkeypox virus A29L antigen in host cells; P3, application in the preparation of monkeypox virus A29L antigen secretory protein products; The host cell is Expi293F cell.

9. A method for preparing a secretory protein of monkeypox virus A29L antigen, comprising the following steps: (A1) introducing the nucleic acid molecule according to claim 5 or 6 into a host cell to obtain a recombinant cell; (A2) culturing the recombinant cells and obtaining the monkeypox virus A29L antigen secretory protein from the culture supernatant.

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