Fusion protein containing monkeypox virus E8L antigen and preparation method thereof

Abstract

The present invention discloses a fusion protein containing monkeypox virus E8L antigen and a preparation method thereof. The present invention relates to the field of biotechnology, and specifically relates to a fusion protein containing monkeypox virus E8L antigen and a preparation method thereof. The fusion protein provided by the present invention is a protein obtained by fusing a polypeptide with the amino acid sequence of SEQ ID No.1 to the N-terminus of the monkeypox virus E8L antigen, and the amino acid sequence of the fusion protein is SEQ ID No.5, the 1-291st position of SEQ ID No.5 or the 1-296th position of SEQ ID No.5. The present invention uses a signal peptide to guide the eukaryotic cell secretion expression of monkeypox virus E8L. Among them, the albumin signal peptide Al guides the secretion expression level of monkeypox virus E8L antigen significantly better than the luciferase signal peptide Ga 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 particularly relates to a method for preparing the E8L antigen of monkeypox virus. Background Art

[0002] The basic infectious form of monkeypox virus is the mature virion (MV). In addition, there is another form: there is a lipid membrane derived from the endoplasmic reticulum membrane (extracellular enveloped, EV) surrounding the mature virion. Among them, the E8L antigen is the membrane protein of the mature virion of monkeypox virus and is also one of the important immune targets. Therefore, preparing the E8L protein is a key step in the research and development of key products for monkeypox virus detection reagents or vaccines. Summary of the Invention

[0003] The main problem to be solved by the present invention is how to prepare the E8L antigen of monkeypox virus.

[0004] To solve the above problems, the present invention provides an albumin signal peptide (signal peptide Al, SEQ ID No.1). Signal peptide Al can direct the secretion of the E8L antigen of monkeypox virus into the culture supernatant. After nickel column affinity chromatography, a high-purity antigen can be obtained, and its secretion and expression yield is significantly higher than that of the luciferase signal peptide (signal peptide Ga) and the antibody light chain signal peptide (signal peptide Lc).

[0005] The present invention first provides a fusion protein.

[0006] The fusion protein provided by the present invention is a protein obtained by fusing a polypeptide with the amino acid sequence of SEQ ID No.1 to the N-terminus of the E8L antigen of monkeypox virus.

[0007] Furthermore, the amino acid sequence of the fusion protein is SEQ ID No.5, the 1st to 291st positions of SEQ ID No.5, or the 1st to 296th positions of SEQ ID No.5.

[0008] The 1st to 16th positions of SEQ ID No.5 are signal peptide Al (i.e., SEQ ID No.1), the 18th to 291st positions are the E8L antigen of monkeypox virus, the 292nd to 296th positions are the Linker linker, and the 297th to 302nd positions are the histidine tag.

[0009] The present invention also provides a nucleic acid molecule encoding the above fusion protein.

[0010] Furthermore, the nucleic acid molecule is composed of the encoding gene of the above polypeptide and the encoding gene of the E8L antigen of monkeypox virus in sequence from the 5'-end to the 3'-end.

[0011] Furthermore, the coding gene of the polypeptide can be any of the following:

[0012] (a1) A DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 2;

[0013] (a2) A DNA molecule that hybridizes with the DNA molecule defined in (a1) under stringent conditions and encodes the polypeptide;

[0014] (a3) A DNA molecule that has a homology of more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% with the DNA sequence defined in (a1) or (a2) and encodes the polypeptide.

[0015] Furthermore, the coding gene of the monkeypox virus E8L antigen can be any of the following:

[0016] (b1) A DNA molecule whose nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 4;

[0017] (b2) A DNA molecule that hybridizes with the DNA molecule defined in (b1) under stringent conditions and encodes the same protein;

[0018] (b3) A DNA molecule that has a homology of more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% with the DNA sequence defined in (b1) or (b2) and encodes the same protein.

[0019] More specifically, the nucleic acid molecule can be a nucleotide sequence of the coding strand that is SEQ ID No. 8, positions 1-888 of SEQ ID No. 8, or positions 1-903 of SEQ ID No. 8.

[0020] Positions 1-6 of SEQ ID No. 8 are the HindIII recognition site, positions 7-15 are the Kozak sequence, positions 16-63 are the albumin signal peptide Al coding gene, positions 64-888 are the E8L antigen coding gene, positions 889-903 are the linker gene, positions 904-921 are the histidine tag gene, positions 922-924 are the stop codon, and positions 925-932 are the PacI recognition site. The recombinant expression plasmid pCGS3-Al-E8L contains the coding gene of the recombinant protein Ga-E8L. The coding sequence of the coding gene of the recombinant protein Al-E8L is positions 16-924 of SEQ ID No. 7, and the coding gene of the recombinant protein Al-E8L encodes an amino acid sequence that is the protein of Sequence 5.

[0021] For the above-mentioned nucleic acid molecules or encoding 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 web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, the identity of a pair of nucleotide sequences can be retrieved, calculated, and then the identity value (%) can be obtained.

[0022] For the above-mentioned nucleic acid molecules or encoding genes, the stringent conditions may be as follows: Hybridize at 50 °C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4, and 1 mM EDTA, and wash in 2×SSC, 0.1% SDS at 50 °C; or: Hybridize at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, and wash in 1×SSC, 0.1% SDS at 50 °C; or: Hybridize at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, and wash in 0.5×SSC, 0.1% SDS at 50 °C; or: Hybridize at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, and wash in 0.1×SSC, 0.1% SDS at 50 °C; or: Hybridize at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, and wash in 0.1×SSC, 0.1% SDS at 65 °C; or: Hybridize at 65 °C in a solution of 6×SSC, 0.5% SDS, and then wash the membrane once each with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

[0023] The present invention also provides an expression cassette, recombinant vector, recombinant microorganism, or transgenic cell line of the above-mentioned nucleic acid molecule.

[0024] Among them, the expression cassette refers to DNA that can express the above-mentioned fusion protein in a host cell. This DNA not only includes a promoter that initiates the transcription of the target gene but may also include a terminator that terminates the transcription of the target gene. Further, the expression cassette may also include an enhancer sequence.

[0025] 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.8 (the coding gene of signal peptide Al is located at positions 16 - 63 of SEQ ID No.8, i.e., SEQ ID No.2) into the multiple cloning sites (such as HindIII and PacI) of the pCGS3 vector. Correspondingly, the transgenic cell line is obtained by introducing the recombinant plasmid into Expi293F cells.

[0026] The present invention also provides the use of a polypeptide with the amino acid sequence of SEQ ID No.1 or its related biological material in any of the following:

[0027] P1. Use in improving the secretion expression yield of monkeypox virus E8L antigen in host cells;

[0028] P2. Use in improving the secretion expression efficiency of monkeypox virus E8L antigen in host cells;

[0029] P3. Use in preparing a secreted protein product of monkeypox virus E8L antigen;

[0030] The related biological material is the coding gene of the polypeptide shown in SEQ ID No.1, or an expression cassette, recombinant vector, recombinant bacterium, or transgenic cell line containing the coding gene.

[0031] 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.8 (the coding gene of signal peptide Al is located at positions 16 - 63 of SEQ ID No.8, i.e., SEQ ID No.2) into the multiple cloning sites (such as HindIII and PacI) of the pCGS3 vector.

[0032] Furthermore, the host cell can be a eukaryotic host cell, such as: HEK293 cells, CHO cells, yeast cells, and insect cells, etc.

[0033] In a specific embodiment of the present invention, the host cell is specifically Expi293F cells.

[0034] Furthermore, the coding gene can be any of the following:

[0035] (a1) A DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No.2;

[0036] (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;

[0037] A DNA molecule having a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the DNA sequence defined by (a3), (a1) or (a2) and encoding the polypeptide.

[0038] For the above-mentioned proteins, homology refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search, the identity value (%) of a pair of amino acid sequences can be calculated.

[0039] For the above-mentioned proteins, the homology of more than 95% can be an identity of at least 96%, 97% or 98%. The homology of more than 90% can be an identity of at least 91%, 92%, 93% or 94%. The homology of more than 85% can be an identity of at least 86%, 87%, 88% or 89%. The homology of more than 80% can be an identity of at least 81%, 82%, 83% or 84%.

[0040] The present invention also provides the application of the above-mentioned fusion protein, nucleic acid molecule, expression cassette, recombinant vector, recombinant microorganism or transgenic cell line in any of the following:

[0041] P1. Application in improving the secretion expression yield of monkeypox virus E8L antigen in host cells;

[0042] P2. Application in improving the secretion expression efficiency of monkeypox virus E8L antigen in host cells;

[0043] P3. Application in preparing a secreted protein product of monkeypox virus E8L antigen.

[0044] The amino acid sequence of the secreted protein of monkeypox virus E8L antigen is SEQ ID No. 5.

[0045] Among them, the host cell is a eukaryotic host cell.

[0046] Furthermore, the eukaryotic host cell can be HEK293 cells, CHO cells, yeast cells, insect cells, etc.

[0047] In a specific embodiment of the present invention, the host cell is specifically Expi293F cell.

[0048] The method for preparing the secreted protein of monkeypox virus E8L antigen also belongs to the scope of protection of the present invention.

[0049] The method for preparing the secreted protein of monkeypox virus E8L antigen as claimed in the present invention may include the following steps:

[0050] (A1) Introduce the nucleic acid molecule described above into a host cell to obtain a recombinant cell;

[0051] (A2) Culture the recombinant cell and obtain the secreted protein of monkeypox virus E8L antigen from the culture supernatant.

[0052] Among them, the nucleic acid molecule can be introduced into the host cell through the recombinant vector described above.

[0053] In step (A1), the host cell is a eukaryotic host cell, such as HEK293 cell, CHO cell, yeast cell, insect cell, etc.

[0054] In a specific embodiment of the present invention, the host cell is specifically Expi293F cell.

[0055] In step (A2), the culture is terminated when the cell viability drops to 65%-75%.

[0056] In step (A2), the method for obtaining the secreted protein of monkeypox virus E8L antigen from the culture supernatant includes the following steps: collect the culture and centrifuge it at 3500g for 30 min, collect the supernatant and perform ultrafiltration concentration and nickel column purification.

[0057] The present invention uses albumin signal peptide Al (SEQ ID No.1), luciferase signal peptide Ga and antibody signal peptide Lc to guide the eukaryotic cell secretion and expression of monkeypox virus E8L antigen. Research has shown that: the secretion and expression level of monkeypox virus E8L antigen in the experimental group with albumin signal peptide Al is significantly better than that in the experimental groups with luciferase signal peptide Ga and antibody light chain signal peptide Lc, and it is more suitable for large-scale industrial production, reducing production costs. The present invention uses the eukaryotic cell secretion expression method to prepare E8L protein, which has the following advantages: 1) The background of the secreted supernatant protein is low and it is easy to purify; 2) Soluble expression, avoiding the formation of inclusion bodies; 3) Precise cleavage by signal peptidase, no redundant Met residues at the N-terminus, and a protein sequence that meets expectations is produced. The present invention is applicable to applications such as antigen preparation in vaccine development. Brief Description of the Drawings

[0058] Figure 1It is a restriction enzyme digestion identification diagram for the construction of recombinant expression plasmids. Among them, 1 is pCGS3-Lc-E8L (Lc is the antibody light chain signal peptide), 2 is pCGS3-Ga-E8L (Ga is the luciferase signal peptide), and 3 is pCGS3-Al-E8L (Al is the albumin signal peptide).

[0059] Figure 2 It is an SDS-PAGE identification diagram of the cell secreted supernatant of monkeypox virus E8L antigen. Among them, 1 is the secreted supernatant of the antibody light chain signal peptide Lc (the cell culture supernatant transfected with pCGS3-Lc-E8L), 2 is the secreted supernatant of the luciferase signal peptide Ga (the cell culture supernatant transfected with pCGS3-Ga-E8L), 3 is the secreted supernatant of the albumin signal peptide Al (the cell culture supernatant transfected with pCGS3-Al-E8L), and 4 is the negative group without transfected expression plasmid (the cell culture supernatant transfected with the pCGS3 vector).

[0060] Figure 3 It is a gray scale analysis diagram of the cell secreted supernatant of monkeypox virus E8L antigen. Among them, 1 is the secreted supernatant of the antibody light chain signal peptide Lc (the cell culture supernatant transfected with pCGS3-Lc-E8L), 2 is the secreted supernatant of the luciferase signal peptide Ga (the cell culture supernatant transfected with pCGS3-Ga-E8L), 3 is the secreted supernatant of the albumin signal peptide Al (the cell culture supernatant transfected with pCGS3-Al-E8L), and 4 is the negative group without transfected expression plasmid (the cell culture supernatant transfected with the pCGS3 vector).

[0061] Figure 4 It is an SDS-PAGE purification identification of the monkeypox virus E8L antigen protein purification. Among them, 1 is the secreted expression and purification sample of the antibody light chain signal peptide Lc, 2 is the secreted expression and purification sample of the luciferase signal peptide Ga, and 3 is the secreted expression and purification sample of the albumin signal peptide Al. Specific Embodiments

[0062] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0063] The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0064] In the following quantitative experiments, unless otherwise specified, three repeated experiments are set up.

[0065] The main reagents and their manufacturer information in the following examples are as follows:

[0066] pCGS3 vector: Merck;

[0067] HindIII endonuclease: NEB;

[0068] PacI endonuclease: NEB;

[0069] GXL Premix: TAKARA;

[0070] DNA Ligation Kit Ver.2.1: TAKARA;

[0071] Expi293F TM Cells: Thermo Fisher;

[0072] Expi293 TM Expression Medium: Thermo Fisher;

[0073] ExpiFectamine TM 293Transfection Kit: Thermo Fisher;

[0074] Opti-MEM TM I Reduced Serum Medium: Thermo Fisher;

[0075] Ni-NTA protein purification kit: Sangon Biotech (Shanghai) Co., Ltd.;

[0076] Amicon Ultra-15 centrifugal filtration device: Millipore;

[0077] Amicon Ultra-0.5 centrifugal filtration device: Millipore;

[0078] PBS pH7.4 (1×): Gibco;

[0079] Gel imaging system: Protein Simple.

[0080] Example 1. Construction of recombinant expression plasmid

[0081] The extracellular region at positions 1-275 of the E8L antigen (NCBI accession number URK20542.1) of the novel monkeypox virus (NCBI genome accession number ON563414.3) was selected in the present invention (the amino acid sequence of the E8L antigen protein is SEQ ID No. 3, and the coding sequence of the coding gene is SEQ ID No. 4). The coding genes of the antibody light chain signal peptide Lc, the luciferase signal peptide Ga, and the albumin signal peptide Al were fused to the 5' end of the coding gene of the monkeypox virus E8L antigen protein, and the corresponding fragment names were Lc-E8L, Ga-E8L, and Al-E8L, respectively, which were cloned into the pCGS3 vector to construct eukaryotic recombinant expression plasmids pCGS3-Lc-E8L, pCGS3-Ga-E8L, and pCGS3-Al-E8L.

[0082] The recombinant expression plasmid pCGS3-Lc-E8L was obtained by replacing the fragment between the recognition sites of the restriction endonuclease HindIII and the restriction endonuclease PacI of the pCGS3 vector with a DNA molecule having the nucleotide sequence of SEQ ID No. 6, while keeping the other nucleotides of the pCGS3 vector unchanged. The 1-6th positions of SEQ ID No. 6 are the HindIII recognition site, the 7-15th positions are the Kozak sequence, the 16-75th positions are the coding gene of the signal peptide Lc, the 76-900th positions are the coding gene of the E8L antigen, the 901-915th positions are the linker gene, the 916-933rd positions are the histidine tag gene, the 934-936th positions are the stop codon, and the 937-944th positions are the PacI recognition site. The recombinant expression plasmid pCGS3-Lc-E8L contains the coding gene of the recombinant protein Lc-E8L. The coding sequence of the coding gene of the recombinant protein Lc-E8L is the 16th-936th positions of SEQ ID No. 6, and the encoded recombinant protein Lc-E8L is a protein fused by the Lc signal peptide and the 17-302nd positions of SEQ ID No. 5, consisting of 306 amino acids.

[0083] The recombinant expression plasmid pCGS3-Ga-E8L is a recombinant expression vector obtained by replacing the fragment between the recognition sites of restriction endonuclease HindIII and restriction endonuclease PacI of the pCGS3 vector with a DNA molecule having the 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 the recognition site of HindIII, the 7th to 15th positions are the Kozak sequence, the 16th to 66th positions are the coding gene of the luciferase signal peptide Ga, the 67th to 891st positions are the coding gene of the E8L antigen, the 892nd to 906th positions are the linker gene, the 907th to 924th positions are the histidine tag gene, the 925th to 927th positions are the stop codon, and the 928th to 935th positions are the recognition site of PacI. The recombinant expression plasmid pCGS3-Ga-E8L contains the coding gene of the recombinant protein Ga-E8L. The coding sequence of the coding gene of the recombinant protein Ga-E8L is the 16th to 927th positions of SEQ ID No.7, and the encoded recombinant protein Ga-E8L is a protein fused by the Ga signal peptide and the 17th to 302nd positions of Sequence 5, consisting of 303 amino acids.

[0084] The recombinant expression plasmid pCGS3-Al-E8L is a recombinant expression vector obtained by replacing the fragment between the recognition sites of restriction endonuclease HindIII and restriction endonuclease PacI of the pCGS3 vector with a DNA molecule having the 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 the recognition site of HindIII, the 7th to 15th positions are the Kozak sequence, the 16th to 63rd positions are the coding gene of the albumin signal peptide Al, the 64th to 888th positions are the coding gene of the E8L antigen, the 889th to 903rd positions are the linker gene, the 904th to 921st positions are the histidine tag gene, the 922nd to 924th positions are the stop codon, and the 925th to 932nd positions are the recognition site of PacI. The recombinant expression plasmid pCGS3-Al-E8L contains the coding gene of the recombinant protein Al-E8L. The coding sequence of the coding gene of the recombinant protein Al-E8L is the 16th to 924th positions of SEQ ID No.8, and the coding gene of the recombinant protein Al-E8L encodes the protein of Amino Acid Sequence 5.

[0085] The construction methods of pCGS3-Lc-E8L, pCGS3-Ga-E8L and pCGS3-Al-E8L are as follows.

[0086] Synthesis of the Lc-E8L gene: The carboxyl terminus of the antibody light chain signal peptide Lc was fused with the monkeypox antigen E8L. GeneScript was commissioned to perform codon optimization, and the synthesized sequence was the Lc-E8L nucleic acid sequence (SEQ ID No. 6). The synthesized plasmid delivered by GeneScript was pUC57-Lc-E8L.

[0087] Using pUC57-Lc-E8L as a template, polymerase GXL Premix (from TAKARA) was used to amplify the target fragments of Ga-E8L and Al-E8L:

[0088] 1) Amplification of the Ga-E8L gene fragment: Primer 1 and Primer 5 were used to amplify fragment A. Fragment A was used as the template for the second round, and Primer 2 and Primer 5 were used for the second-round amplification to obtain fragment B, which was the Ga-E8L target fragment;

[0089] 2) Amplification of the Al-E8L gene fragment: Primer 3 and Primer 5 were used to amplify fragment C. Fragment C was used as the template for the second round, and Primer 4 and Primer 5 were used for the second-round amplification to obtain fragment D, which was the Al-E8L target fragment.

[0090] The primer sequences used in the above PCR amplification are as follows:

[0091] Primer 1: 5’-TGTTTGCTCTGATTTGTATTGCCGTGGCTGAGGCCATGCCTCAGCAGCTGTCTCC-3’;

[0092] Primer 2: 5’-CCCAAGCTTGCCGCCACCATGGGGGTGAAGGTGTTGTTTGCTCTGATTTGTATTG-3’;

[0093] Primer 3: 5’-TTTATTTCCCTGCTGTTTAGCTCCGCCTATAGTATGCCTCAGCAGCTGTCTCC-3’;

[0094] Primer 4: 5’-CCCAAGCTTGCCGCCACCATGAAGTGGGTGACATTTATTTCCCTGCTGTTTAG-3’;

[0095] Primer 5: 5’-CCTTAATTAATCAGTGGTGGTGGTGATGGTGGGAG-3’.

[0096] The pCGS3 vector was digested with HindIII (NEB) and PacI (NEB) to obtain a vector fragment. The gene pUC57-Lc-E8L was fully synthesized. The amplified fragments Ga-E8L and Al-E8L were digested with HindIII and PacI to obtain the target fragments. Ligation was performed using DNA Ligation Kit Ver.2.1 (TAKARA), followed by transformation, plasmid extraction, and identification. Three recombinant expression plasmids, pCGS3-Lc-E8L, pCGS3-Ga-E8L, and pCGS3-Al-E8L, were obtained.

[0097] The results of the restriction enzyme digestion identification of the above three recombinant expression plasmids are as Figure 1 shown. The first lane is the double digestion identification of the pCGS3-Lc-E8L expression plasmid, the second lane is the double digestion identification of the pCGS3-Ga-E8L expression plasmid, and the third lane is the double digestion identification of the pCGS3-Al-E8L expression plasmid. After digestion, the size of the vector fragment is approximately 7100 bp, and the size of the target gene is approximately 940 bp; the sizes of the digestion bands are consistent with the expected values.

[0098] Example 2. Protein transient expression study

[0099] A. Cell transfection experiment of Expi293F

[0100] I. Host cell culture

[0101] Take the seed bank cells of the host cell Expi293F (Expi293F TM Cells) from the liquid nitrogen tank, quickly thaw them in a 37°C water bath, and aseptically transfer the thawed cell suspension to a 125 mL medicine bottle containing 30 mL of preheated complete growth medium. The shaking culture conditions are: 37°C, 8% CO2, 120 rpm, amplitude 25 mm, humidity ≥ 80%. After 15 - 30 min, take the cell suspension to detect the cell density and viability.

[0102] Wait until the cell viability recovers to more than 90% and the cell density reaches 3 - 5×10 6 cells / mL, and inoculate and amplify at 0.3 - 0.5×10 6 cells / mL.

[0103] II. Cell transfection

[0104] The recombinant vectors pCGS3-Lc-E8L, pCGS3-Ga-E8L, pCGS3-Al-E8L constructed in Example 1 and the pCGS3 vector were separately transfected into host cells.

[0105] 1. One day before transfection

[0106] 24 h before transfection, reseed the cells at 2.5 - 3×10 6 cells / mL and culture for 24 h.

[0107] 2. On the day of transfection

[0108] (1) The cell density should reach 4.5 - 5.5×10 6 cells / mL and the viability should be ≥95%. Dilute the cells with freshly pre-warmed complete growth medium to 3×10 6 cells / mL.

[0109] (2) Prepare the transfection reagent and DNA complex

[0110] 1) DNA dilution

[0111] Dilute the plasmids (pCGS3-Lc-E8L, pCGS3-Ga-E8L, and pCGS3-Al-E8L constructed in Example 1) to 1 μg / μL with sterile water. According to the amount of 1 μg plasmid for 1 mL cell transfection, take the amount of plasmid required for transfecting 50 mL cells, that is, add 50 μL plasmid to 3 mL Opti-MEMTM I Reduced Serum medium for standby.

[0112] 2) Transfection reagent dilution

[0113] Gently invert and mix the transfection reagent ExpiFectamine293 TM Reagent up and down gently before use. Take the amount of transfection reagent required for transfecting 50 mL cells, that is, 160 μL ExpiFectamine293 TM Reagent and gently invert and mix it in 2.8 mL Opti-MEMTM I Reduced Serum medium, and let it stand at room temperature for 5 min.

[0114] 3) Add the diluted transfection reagent to the plasmid and gently invert and mix. React at room temperature for 10 - 20 min. Slowly add the mixed transfection reagent and DNA complex to the cell culture. Culture under the conditions of 37 °C, 8% CO2, 120 rpm, amplitude 25 mm, and humidity ≥80%.

[0115] 3. On the first day after transfection

[0116] At 18 - 22 h after transfection, the enhancer was added according to the amount of 50 mL of transfected cells. That is, 300 μL of ExpiFectamineTM 293 Transfection Enhancer 1 was mixed with 3 mL of ExpiFectamineTM 293 Transfection Enhancer 2 and slowly added to the cell culture to obtain pCGS3 - Al - E8L transfected cells, pCGS3 - Ga - E8L transfected cells, pCGS3 - Lc - E8L transfected cells, and pCGS3 vector transfected cells respectively.

[0117] 4. Collection of culture supernatant

[0118] The cell viability was monitored daily after transfection. When the viability decreased to 65% - 75% on the 4th day, the culture was terminated. The culture was collected and centrifuged at 3500 g for 30 min to collect the supernatant, obtaining the culture supernatants of pCGS3 - Lc - E8L transfected cells, pCGS3 - Ga - E8L transfected cells, pCGS3 - Al - E8L transfected cells, and pCGS3 vector transfected cells respectively.

[0119] B. SDS protein electrophoresis and gray - scale analysis

[0120] The above - mentioned supernatant was subjected to protein electrophoresis analysis. The gray - scale analysis of the SDS protein electrophoresis map 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; use the rectangular tool to select the lane → Analyze → Gel → Select First Lane to determine the analysis lane, and repeat to select multiple lanes for simultaneous analysis; Analyze → Gel → Plot Lane to generate the peak area; use the linear tool to select the peak map corresponding to the target band, and use the Wand tool to calculate the area of the corresponding peak map, then the percentage of the target protein in the total protein can be obtained.

[0121] The results of SDS - PAGE identification and gray - scale analysis showed that in the culture supernatants secreted by the antibody light - chain signal peptide Lc (the culture supernatant of pCGS3 - Lc - E8L transfected cells), the luciferase signal peptide Ga (the culture supernatant of pCGS3 - Ga - E8L transfected cells), and the albumin signal peptide Al (the culture supernatant of pCGS3 - Al - E8L transfected cells), the expression levels of the target proteins accounted for 7.05%, 13.49%, and 16.39% of the total protein ( Figure 2 and Figure 3 ). In summary, it can be seen that compared with the signal peptide Ga and the signal peptide Lc, the monkeypox virus E8L antigen guided by the albumin signal peptide Al has a higher secretion and expression level.

[0122] Example 3: Protein Purification of E8L Antigen

[0123] 1. Ultrafiltration and Concentration

[0124] The culture supernatants of pCGS3-Al-E8L transfected cells, pCGS3-Ga-E8L transfected cells, and pCGS3-Lc-E8L transfected cells in Example 2 were respectively ultrafiltered and concentrated by centrifugation at 4°C using an Amicon Ultra-15 centrifugal filtration device (Millipore) at 6000g for 20 min. Finally, the cell supernatants were concentrated to 20 - 30 mL.

[0125] 2. Nickel Column Purification

[0126] The ultrafiltered and concentrated supernatant obtained in Step 1 was mixed with Binding / Wash Buffer at a volume ratio of 1:1 and allowed to stand for 20 min for sufficient incubation. The column was equilibrated with two column volumes of Binding / Wash Buffer, and the buffer was allowed to flow through the pre-packed column by gravity. The mixture of the ultrafiltered and concentrated supernatant and Binding / Wash Buffer was added to the column and allowed to flow through the pre-packed column by gravity; if there was remaining sample, it could be loaded again and circulated once more, and the flow-through was collected into a centrifuge tube. The column was washed with two column volumes of Binding / Wash Buffer and the flow-through was collected until the absorbance at 280 nm of the flow-through approached the baseline. The histidine-tagged protein on the column was eluted with two column volumes of Elution Buffer, and this step was repeated until the absorbance at 280 nm of the flow-through approached the baseline, and the eluate was collected for purification.

[0127] 3. Ultrafiltration and Buffer Exchange

[0128] The protein solution after nickel column purification was added to an Amicon Ultra-15 centrifugal filtration device (Millipore) and centrifuged in batches at 10000g for 3 min until the solution remained approximately 150 μL. 300 μL of PBS (pH 7.4) was gently added, and centrifuged at 10000g until 150 μL remained, and this was repeated three times. The ultrafiltration tube was eluted with PBS (pH 7.4) to collect the sample, and the final volume was approximately 1 - 2 mL. 5 μL was reserved for protein concentration determination and SDS-PAGE protein electrophoresis detection.

[0129] After identification and analysis by Ni column purification, the yield of the recombinant protein Lc-E8L in the culture supernatant of pCGS3-Lc-E8L transfected cells was 40.15 mg / L, the yield of the recombinant protein Ga-E8L in the culture supernatant of pCGS3-Ga-E8L transfected cells was 65.98 mg / L, and the yield of the recombinant protein Al-E8L in the culture supernatant of pCGS3-Al-E8L transfected cells was 83.81 mg / L( Figure 4), among which, the yield after purification of the signal peptide Ga experimental group is the highest.

[0130] Using aluminum salts; or CpG; or liposomes; or oil adjuvants can produce monkeypox virus immune compositions for preventing monkeypox infection.

[0131] Based on the results of the above embodiments, the present invention uses three signal peptides, namely signal peptide Al, signal peptide Ga and signal peptide Lc, to guide the eukaryotic cell secretory expression of monkeypox virus E8L antigen. The secretory expression level of the luciferase signal peptide Ga of the present invention is significantly better than the other two signal peptides.

[0132] The above has detailed the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although the present invention gives specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses or improvements to the present invention, including those that depart from the scope disclosed in this application and are made with conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A nucleic acid molecule, characterized in that: The nucleic acid molecule is the DNA molecule of SEQ ID No.

8.

2. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of claim 1.

3. Use of the nucleic acid molecule of claim 1 in any of the following: P1. Use in increasing the secretion expression yield of the E8L antigen of monkeypox virus in host cells; P2. Use in increasing the secretion expression efficiency of the E8L antigen of monkeypox virus in host cells; P3. Use in preparing a secreted protein product of the E8L antigen of monkeypox virus; The host cell is Expi293F cell.

4. Use of the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line of claim 2 in any of the following: P1. Use in increasing the secretion expression yield of the E8L antigen of monkeypox virus in host cells; P2. Use in increasing the secretion expression efficiency of the E8L antigen of monkeypox virus in host cells; P3. Use in preparing a secreted protein product of the E8L antigen of monkeypox virus; The host cell is Expi293F cell.

5. A method for preparing a secreted protein of the E8L antigen of monkeypox virus, 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 cell and obtaining the secreted protein of the E8L antigen of monkeypox virus from the culture supernatant; The host cell is Expi293F cell.

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