CN116120467A

CN116120467A - Fusion protein containing monkey pox virus E8L antigen and preparation method thereof

Info

Publication number: CN116120467A
Application number: CN202310010685.7A
Authority: CN
Inventors: 邢体坤; 张静静; 安文琪; 宋路萍
Original assignee: Hualan Genetic Engineering Co ltd
Current assignee: Hualan Genetic Engineering Co ltd
Priority date: 2023-01-05
Filing date: 2023-01-05
Publication date: 2023-05-16

Abstract

The invention discloses a fusion protein containing a monkey pox virus E8L antigen and a preparation method thereof. The invention relates to the technical field of biology, in particular to a fusion protein containing a monkey pox virus E8L antigen and a preparation method thereof. The fusion protein provided by the invention is obtained by fusing polypeptide with the amino acid sequence of SEQ ID No.1 to the N end of a monkey poxvirus E8L antigen, and the amino acid sequence of the fusion protein is SEQ ID No.5, the 1 st-291 th positions of SEQ ID No.5 or the 1 st-296 th positions of SEQ ID No.5. The invention adopts the signal peptide to guide the secretion expression of the monkey pox virus E8L eukaryotic cells, wherein the albumin signal peptide Al guides the secretion expression level of the monkey pox virus E8L antigen to be obviously superior to that of luciferase signal peptide Ga and antibody light chain signal peptide Lc, is more suitable for large-scale industrial production, and reduces the production cost.

Description

Fusion protein containing monkey pox virus E8L antigen and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method of a monkey pox virus E8L antigen.

Background

The basic infection form of monkey poxvirus is Mature Virions (MV), and there is another form: the mature virions are surrounded by a lipid membrane derived from the endoplasmic reticulum membrane (extracellular enveloped, EV). Wherein, the E8L antigen is membrane protein of mature virus particles of the monkey pox virus, and is also one of important immune targets. Thus, the preparation of the E8L protein is a key step in the development of key products for monkey pox detection reagents or vaccines.

Disclosure of Invention

The main problem to be solved by the present invention is how to prepare the monkey poxvirus E8L antigen.

In order to solve the problems, the invention provides an albumin signal peptide (signal peptide Al, SEQ ID No. 1), wherein the signal peptide Al can guide monkey pox virus E8L antigen to be secreted into culture supernatant, high-purity antigen can be obtained through nickel column affinity chromatography, and the secretion and expression yield of the albumin signal peptide is obviously higher than that of luciferase signal peptide (signal peptide Ga) and antibody light chain signal peptide (signal peptide Lc).

The present invention first provides a fusion protein.

The fusion protein provided by the invention is obtained by fusing a polypeptide with an amino acid sequence of SEQ ID No.1 to the N end of a monkey poxvirus E8L antigen.

Further, the amino acid sequence of the fusion protein is SEQ ID No.5, positions 1-291 of SEQ ID No.5 or positions 1-296 of SEQ ID No.5.

The 1 st to 16 th positions of SEQ ID No.5 are signal peptide Al (namely SEQ ID No. 1), the 18 th to 291 th positions are the monkey poxvirus E8L antigen, the 292 nd to 296 th positions are Linker joints, and the 297 th to 302 th positions are histidine tags.

The invention also provides a nucleic acid molecule encoding the fusion protein.

Further, the nucleic acid molecule sequentially comprises a coding gene of the polypeptide and a coding gene of the monkey pox virus E8L antigen from the 5 'end to the 3' end.

Further, the encoding gene of the polypeptide may be any of the following:

(a1) A DNA molecule with the nucleotide sequence of the coding strand being SEQ ID No. 2;

(a2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (a 1) and which encodes said polypeptide;

(a3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in (a 1) or (a 2) and encoding the polypeptide.

Further, the encoding gene of the monkey poxvirus E8L antigen may be any one of the following:

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

(b2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (b 1) and which encodes the same protein;

(b3) A DNA molecule having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the DNA sequence defined in (b 1) or (b 2) and encoding the same protein.

More specifically, the nucleic acid molecule may be the nucleotide sequence of the coding strand SEQ ID No.8, SEQ ID No.8 at positions 1-888 or SEQ ID No.8 at positions 1-903.

The 1 st to 6 th sites of SEQ ID No.8 are HindIII recognition sites, the 7 th to 15 th sites are Kozak sequences, the 16 th to 63 th sites are albumin signal peptide Al coding genes, the 64 th to 888 th sites are E8L antigen coding genes, the 889 th to 903 th sites are connecting peptide (Linker) genes, the 904 th to 921 th sites are histidine tag genes, the 922 th to 924 th sites are stop codons, and the 925 th to 932 th sites are PacI recognition sites. The recombinant expression plasmid pCGS3-Al-E8L contains the coding gene of recombinant protein Ga-E8L, the coding sequence of the coding gene of the recombinant protein Al-E8L is the 16 th-924 th site of SEQ ID No.7, and the coding amino acid sequence of the coding gene of the recombinant protein Al-E8L is the protein of sequence 5.

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 BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of nucleotide sequences can be searched for by using blastp as a program, setting the aspect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, perresidue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then the value (%) of the identity can be obtained.

In the above nucleic acid molecule or encoding gene, the stringent conditions may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

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

Wherein the expression cassette refers to DNA capable of expressing the fusion protein in a host cell, and the DNA not only comprises a promoter for promoting the transcription of a target gene, but also comprises a terminator for stopping the transcription of the target gene. Further, the expression cassette may also include an enhancer sequence.

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 (SEQ ID No.2, which is the coding gene of signal peptide Al at positions 16-63 of SEQ ID No. 8) into the multiple cloning site (e.g., hindIII and PacI) of pCGS3 vector. Accordingly, the transgenic cell line is obtained by introducing the recombinant plasmid into an Expi293F cell.

The invention also provides application of the polypeptide with the amino acid sequence of SEQ ID No.1 or related biological materials thereof in any one of the following:

p1, improving the secretion expression yield of monkey pox virus E8L antigen in host cells;

p2, improving the secretion expression efficiency of monkey pox virus E8L antigen in host cells;

p3, preparing monkey pox virus E8L antigen secretion protein products;

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

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 (SEQ ID No.2, which is the coding gene of signal peptide Al at positions 16-63 of SEQ ID No. 8) into the multiple cloning site (e.g., hindIII and PacI) of pCGS3 vector.

Further, the host cell may be a eukaryotic host cell, such as: HEK293 cells, CHO cells, yeast cells, insect cells, and the like.

In a specific embodiment of the invention, the host cell is specifically an Expi293F cell.

Further, the coding gene may be any of the following:

(a2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (a 1) and which encodes a polypeptide represented by SEQ ID No. 1;

In the above proteins, homology refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

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

The invention also provides the application of the fusion protein or the nucleic acid molecule or the expression cassette, the recombinant vector, the recombinant microorganism or the transgenic cell line in any one of the following:

application of P3 in preparing monkey pox virus E8L antigen secretion protein product.

The amino acid sequence of the monkey poxvirus E8L antigen secretion protein is SEQ ID No.5.

Wherein the host cell is a eukaryotic host cell.

Further, the eukaryotic host cell may be HEK293 cells, CHO cells, yeast cells, insect cells, and the like.

Methods for preparing monkey poxvirus E8L antigen-secreting proteins are also within the scope of the claimed invention.

The method for preparing the monkey pox virus E8L antigen secretion protein can comprise the following steps:

(A1) Introducing the nucleic acid molecule described above into a host cell to obtain a recombinant cell;

(A2) Culturing the recombinant cells, and obtaining monkey pox virus E8L antigen secretion proteins from the culture supernatant.

Wherein the nucleic acid molecule may be introduced into the host cell by means of a recombinant vector as described above.

In step (A1), the host cell is a eukaryotic host cell. Such as: HEK293 cells, CHO cells, yeast cells, insect cells, and the like.

In the step (A2), the culture is stopped when the cell viability is reduced to 65% -75%.

In step (A2), monkey poxvirus E8L antigen-secreting protein is obtained from the culture supernatant according to a method comprising the steps of: the culture was collected and centrifuged at 3500g for 30min, and the supernatant was collected for ultrafiltration concentration and nickel column purification.

The invention adopts albumin signal peptide Al (SEQ ID No. 1), luciferase signal peptide Ga and antibody signal peptide Lc to guide monkey pox virus E8L antigen eukaryotic cell secretion expression. The study proves that: the monkey poxvirus E8L antigen secretion expression quantity of the albumin signal peptide Al experimental group is obviously superior to that of the luciferase signal peptide Ga and antibody light chain signal peptide Lc experimental group, is more suitable for large-scale industrial production, and reduces the production cost. The invention adopts eukaryotic cell secretion expression mode to prepare E8L protein, and has the following advantages: 1) The secreted supernatant protein has low background and is easy to purify; 2) Soluble expression, avoiding inclusion body formation; 3) Signal peptidase cleaves precisely, with no redundant Met residues at the N-terminus, resulting in a protein sequence that meets expectations. The invention is suitable for the application of antigen preparation in vaccine development and the like.

Drawings

FIG. 1 is a diagram of the construction of a restriction enzyme map for recombinant expression plasmids. Wherein 1 is pCGS3-Lc-E8L (Lc is an antibody light chain signal peptide), 2 is pCGS3-Ga-E8L (Ga is a luciferase signal peptide), and 3 is pCGS3-Al-E8L (Al is an albumin signal peptide).

FIG. 2 is an SDS-PAGE identification of monkey poxvirus E8L antigen cell secretion supernatants. Wherein 1 is antibody light chain signal peptide Lc secretion supernatant (pCGS 3-Lc-E8L transfected cell culture supernatant), 2 is luciferase signal peptide Ga secretion supernatant (pCGS 3-Ga-E8L transfected cell culture supernatant), 3 is albumin signal peptide Al secretion supernatant (pCGS 3-Al-E8L transfected cell culture supernatant), 4 is a negative group without transfected expression plasmid (pCGS 3 vector transfected cell culture supernatant).

FIG. 3 is a graph showing the analysis of ash removal on secretion of E8L antigen cells of monkey poxvirus. Wherein 1 is antibody light chain signal peptide Lc secretion supernatant (pCGS 3-Lc-E8L transfected cell culture supernatant), 2 is luciferase signal peptide Ga secretion supernatant (pCGS 3-Ga-E8L transfected cell culture supernatant), 3 is albumin signal peptide Al secretion supernatant (pCGS 3-Al-E8L transfected cell culture supernatant), 4 is a negative group without transfected expression plasmid (pCGS 3 vector transfected cell culture supernatant).

FIG. 4 shows SDS-PAGE purification identification of monkey poxvirus E8L antigen protein. Wherein 1 is an antibody light chain signal peptide Lc secretory expression purification sample, 2 is a luciferase signal peptide Ga secretory expression purification sample, and 3 is an albumin signal peptide Al secretory expression purification sample.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The quantitative experiments in the following examples were performed in triplicate unless otherwise indicated.

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

pCGS3 vector: merck company;

HindIII endonuclease: NEB company;

PacI endonuclease: NEB company;

GXL Premix: TAKARA Co;

DNA Ligation Kit Ver.2.1: TAKARA Co;

Expi293F ^TM cells: thermo Fisher company;

Expi293 ^TM expression Medium: thermo Fisher company;

ExpiFectamine ^TM 293Transfection Kit: thermo Fisher company;

Opti-MEM ^TM i Reduced Serum Medium: thermo Fisher company;

Ni-NTA protein purification kit: bioengineering (Shanghai) stock Co.Ltd;

amicon Ultra-15 centrifugal filtration device: millipore Co;

amicon Ultra-0.5 centrifugal filtration device: millipore Co;

PBS ph7.4 (1×): gibco company;

gel imaging system: protein Simple company.

Example 1 construction of recombinant expression plasmid

The invention selects the E8L antigen (NCBI accession number is URK 20542.1) of the new monkey poxvirus (NCBI genome accession number is ON 563414.3) at the 1 st-275 th position (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 are fused to the 5' end of the coding gene of the monkey pox virus E8L antigen protein, the corresponding fragment names are Lc-E8L, ga-E8L and Al-E8L respectively, and cloned to a pCGS3 vector to construct eukaryotic recombinant expression plasmids pCGS3-Lc-E8L, pCGS3-Ga-E8L and pCGS3-Al-E8L.

The recombinant expression plasmid pCGS3-Lc-E8L is a recombinant expression vector obtained by replacing a fragment between the restriction endonuclease HindIII recognition site and the restriction endonuclease PacI recognition site of the pCGS3 vector with a DNA molecule whose nucleotide sequence is SEQ ID No.6, and keeping the other nucleotides of the pCGS3 vector unchanged. The 1 st to 6 th sites of SEQ ID No.6 are HindIII recognition sites, the 7 th to 15 th sites are Kozak sequences, the 16 th to 75 th sites are signal peptide Lc coding genes, the 76 th to 900 th sites are E8L antigen coding genes, the 901 st to 915 th sites are connecting peptide (Linker) genes, the 916 th to 933 th sites are histidine tag genes, the 934 th to 936 th sites are stop codons, and the 937 th to 944 th sites are PacI recognition sites. The recombinant expression plasmid pCGS3-Lc-E8L contains the coding gene of recombinant protein Lc-E8L, the coding sequence of the coding gene of the recombinant protein Lc-E8L is the 16 th-936 th site of SEQ ID No.6, and the coded recombinant protein Lc-E8L is a protein fused by Lc signal peptide and the 17 th-302 th site of sequence 5 and consists of 306 amino acids.

The recombinant expression plasmid pCGS3-Ga-E8L replaces the fragment between the restriction endonuclease HindIII recognition site and the restriction endonuclease PacI recognition site of the pCGS3 vector with a DNA molecule with the nucleotide sequence of SEQ ID No.7, and the recombinant expression vector is obtained by keeping the other nucleotides of the pCGS3 vector unchanged. The 1 st to 6 th sites of SEQ ID No.7 are HindIII recognition sites, the 7 th to 15 th sites are Kozak sequences, the 16 th to 66 th sites are genes encoding luciferase signal peptide Ga, the 67 th to 891 th sites are E8L antigen encoding genes, the 892 th to 906 th sites are connecting peptide (Linker) genes, the 907 th to 924 th sites are histidine tag genes, the 925 th to 927 th sites are stop codons, and the 928 th to 935 th sites are PacI recognition sites. The recombinant expression plasmid pCGS3-Ga-E8L contains the coding gene of recombinant protein Ga-E8L, the coding sequence of the coding gene of the recombinant protein Ga-E8L is the 16 th-927 th position of SEQ ID No.7, and the coded recombinant protein Ga-E8L is the protein fused by Ga signal peptide and the 17 th-302 th position of sequence 5 and consists of 303 amino acids.

The recombinant expression plasmid pCGS3-Al-E8L replaces the fragment between the restriction endonuclease HindIII recognition site and the restriction endonuclease PacI recognition site of the pCGS3 vector with a DNA molecule with the nucleotide sequence of SEQ ID No.8, and the recombinant expression vector is obtained by keeping other nucleotides of the pCGS3 vector unchanged. The 1 st to 6 th sites of SEQ ID No.8 are HindIII recognition sites, the 7 th to 15 th sites are Kozak sequences, the 16 th to 63 th sites are albumin signal peptide Al coding genes, the 64 th to 888 th sites are E8L antigen coding genes, the 889 th to 903 th sites are connecting peptide (Linker) genes, the 904 th to 921 th sites are histidine tag genes, the 922 th to 924 th sites are stop codons, and the 925 th to 932 th sites are PacI recognition sites. The recombinant expression plasmid pCGS3-Al-E8L contains the coding gene of recombinant protein Al-E8L, the coding sequence of the coding gene of the recombinant protein Al-E8L is the 16 th-924 th site of SEQ ID No.8, and the coding gene of the recombinant protein Al-E8L codes for the protein of amino acid sequence 5.

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

Lc-E8L Gene Synthesis: the carbon end of the antibody light chain signal peptide Lc is fused with monkey pox antigen E8L, and the codon optimization is carried out by the artificial organism, the nucleic acid sequence of the sequence Lc-E8L (SEQ ID No. 6) is synthesized, and the artificial organism delivers the synthetic plasmid pUC57-Lc-E8L.

pUC57-Lc-E8L was used as template, using a polymerase

GXL Premix (TAKARA Co.) amplified Ga-E8L and Al-E8L target fragments:

1) Ga-E8L gene fragment amplification: the primer 1 and the primer 5 are used for amplifying the fragment A, the fragment A is used as a second round of template, and the primer 2 and the primer 5 are used for carrying out second round of amplification to obtain a fragment B, namely the Ga-E8L target fragment;

2) Amplification of the Al-E8L Gene fragment: and amplifying the fragment C by using the primer 3 and the primer 5, wherein the fragment C is used as a second round of template, and the primer 4 and the primer 5 are used for carrying out second round of amplification to obtain a fragment D, namely the Al-E8L target fragment.

The primer sequences used for the PCR amplification were as follows:

primer 1:5'-TGTTTGCTCTGATTTGTATTGCCGTGGCTGAGGCCATGCCTCAGCAGCTGTCTCC-3';

primer 2:5'-CCCAAGCTTGCCGCCACCATGGGGGTGAAGGTGTTGTTTGCTCTGATTTGTATTG-3';

primer 3:5'-TTTATTTCCCTGCTGTTTAGCTCCGCCTATAGTATGCCTCAGCAGCTGTCTCC-3';

primer 4:5'-CCCAAGCTTGCCGCCACCATGAAGTGGGTGACATTTATTTCCCTGCTGTTTAG-3';

primer 5:5'-CCTTAATTAATCAGTGGTGGTGGTGATGGTGGGAG-3'.

The pCGS3 vector is digested with HindIII (NEB) and PacI (NEB) to obtain vector fragment, the total synthetic gene pUC57-Lc-E8L is digested with amplified fragments Ga-E8L and Al-E8L, and the target fragment is obtained by digestion with HindIII and PacI. Ligation was performed using DNA Ligation Kit Ver.2.1 (TAKARA Co.), transformation, plasmid extraction, and identification. Three recombinant expression plasmids pCGS3-Lc-E8L, pCGS3-Ga-E8L and pCGS3-Al-E8L were obtained.

The results of the restriction enzyme digestion identification of the three recombinant expression plasmids are shown in figure 1, wherein lane 1 is the double restriction enzyme digestion identification of the pCGS3-Lc-E8L expression plasmid, lane 2 is the double restriction enzyme digestion identification of the pCGS3-Ga-E8L expression plasmid, and lane 3 is the double restriction enzyme digestion identification of the pCGS3-Al-E8L expression plasmid. The size of the vector fragment after enzyme digestion is about 7100bp, and the size of the target gene is about 940bp; the size of the enzyme cutting strip meets the expectations.

Example 2 transient protein expression Studies

A. Cell Expi293F transfection experiment

1. Host cell culture

Host cell Expi293F (Expi 293F) was taken from liquid nitrogen tank ^TM Cells), rapidly thawing in a water bath at 37 ℃, aseptically transferring the thawed cell suspension to a 125mL vial containing 30mL of pre-warmed complete growth medium, shake culture conditions: 37 ℃,8% CO ₂ 120rpm, amplitude 25mm, humidity more than or equal to 80%. And taking the cell suspension after 15-30min to detect the cell density and the activity.

After the cell activity rate is recovered by more than 90%, the cell density reaches 3-5 multiplied by 10 ⁶ cell/mL, at 0.3-0.5X10 ⁶ cells/mL were used for seed amplification.

2. Cell transfection

The recombinant vectors pCGS3-Lc-E8L, pCGS3-Ga-E8L, pCGS3-Al-E8L and pCGS3 vector constructed in example 1 were transfected into host cells separately, respectively.

1. The day before transfection

Cells were incubated at 2.5-3X 10 at 24h prior to transfection ⁶ The cells/mL were re-inoculated and cultured for 24h.

2. Day of transfection

(1) The cell density should reach 4.5-5.5X10 ⁶ The cell/mL has a activity rate of not less than 95%. Cells were diluted to 3X 10 with fresh pre-warmed complete growth medium ⁶ cells/mL。

(2) Preparation of transfection reagent and DNA Complex

1) DNA dilution

Plasmids (pCGS 3-Lc-E8L, pCGS3-Ga-E8L and pCGS3-Al-E8L constructed in example 1) were diluted to 1. Mu.g/. Mu.L with sterile water, and the amount of plasmid required for transfecting 50mL of cells, i.e., 50. Mu.L of plasmid was taken up in 3mL of Opti-MEMTM I Reduced Serum medium for use in an amount of 1. Mu.g of plasmid transfected into 1mL of cells.

2) Transfection reagent dilution

Transfection reagent Expiectamine 293 prior to use ^TM Reagent was gently mixed upside down, and the amount of transfection Reagent required to transfect 50mL of cells was taken, namely 160. Mu.L of Expifectamine293 ^TM Reagent was gently mixed upside down in 2.8mL Opti-MEMTM I Reduced Serum medium and left standing at room temperature for 5min.

3) Adding the diluted transfection reagent into the plasmid, slightly reversing and uniformly mixing, and reacting for 10-20 min at room temperature. The mixed transfection reagent and DNA complex is slowly added to the cell culture. 37 ℃,8% CO ₂ Culturing at 120rpm and amplitude of 25mm under the condition that humidity is more than or equal to 80%.

3. First day after transfection

The enhancer was added 18-22 hours after transfection, in an amount of 50mL cells transfected. That is, 300. Mu. L ExpiFectamineTM 293Transfection Enhancer 1 and 3mL ExpiFectamineTM293 Transfection Enhancer 2 were mixed 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.

4. Culture supernatant collection

Cell viability was monitored daily after transfection, culture was terminated on day 4 when viability was reduced to 65% -75%, and the culture was collected by centrifugation at 3500g for 30min to collect supernatant, and pCGS3-Lc-E8L transfected cell culture supernatant, pCGS3-Ga-E8L transfected cell culture supernatant, pCGS3-Al-E8L transfected cell culture supernatant and pCGS3 vector transfected cell culture supernatant, respectively.

B. SDS protein electrophoresis and gray scale analysis

And carrying out protein electrophoresis analysis on the supernatant, wherein Image J software is adopted for SDS protein electrophoresis Image gray level analysis. The operation steps are that Image- & gt Type- & gt 32-Bit is converted into a gray scale Image; process-Subtract Background-OK to remove background color; rectangular tool selection lane- & gt Analyze- & gt Gel- & gt Select First Lane determination analysis lane, repeated selection of multiple lanes for simultaneous analysis; analyze- & gt Gel- & gt Plot Lane generates peak area; and selecting a corresponding peak diagram of the target band by using a linear tool, and calculating the area of the corresponding peak diagram by using a Wand tool to obtain the percentage of the target protein to the total protein.

SDS-PAGE identification and gray level analysis result prove that: antibody light chain signal peptide Lc secretion supernatant (pCGS 3-Lc-E8L transfected cell culture supernatant), luciferase signal peptide Ga secretion supernatant (pCGS 3-Ga-E8L transfected cell culture supernatant) and albumin signal peptide Al secretion supernatant (pCGS 3-Al-E8L transfected cell culture supernatant) contained the target protein expressed in amounts of 7.05%,13.49% and 16.39% of the total protein (fig. 2 and 3). It can be seen from the above that: the monkey poxvirus E8L antigen guided by the albumin signal peptide Al has a higher secretory expression level than the signal peptide Ga and the signal peptide Lc.

Example 3 protein purification of E8L antigen

1. Ultrafiltration concentration

The supernatant of the transfected cells of pCGS3-Al-E8L, the supernatant of the transfected cells of pCGS3-Ga-E8L and the supernatant of the transfected cells of pCGS3-Lc-E8L of example 2 were subjected to ultrafiltration concentration by centrifugation at 6000g for 20min at 4℃in an Amicon Ultra-15 centrifugal filtration device (Millipore Co.) respectively, and the final cell supernatant was concentrated to 20-30mL.

2. Nickel column purification

The ultrafiltration concentrated supernatant obtained in the step 1 and Binding/Wash Buffer are mixed according to the volume ratio of 1:1, mixing evenly, standing for 20min and incubating fully. Double column volume Binding/Wash Buffer equilibrates the column and Buffer flows through the pre-packed column by gravity flow. Adding the ultrafiltration concentrated supernatant and Binding/Wash Buffer mixed solution into a column, and passing through a pre-packed column by gravity flow; if the residual sample can be loaded again, the sample is circulated again, and the collected flow-through liquid is filled into a centrifuge tube. The column was washed with a Binding/wash buffer of twice the column volume and the flow-through was collected until the absorbance 280nm of the flow-through was near baseline. The histidine-tagged proteins on the column were eluted by an Elutation Buffer of twice the column volume, and this procedure was repeated until the absorbance of the flow-through solution was 280nm close to baseline, and the eluate was collected for purification.

3. Ultrafiltration replacement

The protein solution after nickel column purification was subjected to an Amicon Ultra-15 centrifugal filtration device (Millipore Co.) and centrifuged at 10000g in batches for 3min until about 150. Mu.L of the solution remained. 300. Mu.L of PBS (pH 7.4) was gently added, and 10000g was centrifuged to 150. Mu.L, and repeated three times. The sample was collected from the PBS (pH 7.4) eluting ultrafiltration tube to a final volume of about 1-2mL, and 5. Mu.L of the sample was used for protein concentration measurement and SDS-PAGE protein electrophoresis detection.

The recombinant protein Lc-E8L yield in the supernatant of the pCGS3-Lc-E8L transfected cell culture was 40.15mg/L, the recombinant protein Ga-E8L yield in the supernatant of the pCGS3-Ga-E8L transfected cell culture was 65.98mg/L, and the recombinant protein Al-E8L yield in the supernatant of the pCGS3-Al-E8L transfected cell culture was 83.81mg/L (FIG. 4), wherein the signal peptide Ga experimental group was the highest yield after purification.

Aluminum salt is adopted; or CpG; or a liposome; or oily adjuvant can be used for producing monkey pox virus immune composition for preventing monkey pox infection.

The results of the above examples are combined, and three signal peptides of the signal peptide Al, the signal peptide Ga and the signal peptide Lc are adopted to guide the secretion and expression of the monkey pox virus E8L antigen eukaryotic cells. The Ga secretion expression level of the luciferase signal peptide is obviously superior to that of other two signal peptides.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. The fusion protein is obtained by fusing a polypeptide with the amino acid sequence of SEQ ID No.1 to the N end of a monkey poxvirus E8L antigen.

2. The fusion protein of claim 2, wherein: the amino acid sequence of the fusion protein is SEQ ID No.5, 1 st to 291 th positions of SEQ ID No.5 or 1 st to 296 th positions of SEQ ID No.5.

3. A nucleic acid molecule encoding the fusion protein of claim 1 or 2.

4. A nucleic acid molecule according to claim 3, wherein: the nucleic acid molecule consists of a coding gene of the polypeptide and a coding gene of the monkey pox virus E8L antigen;

the encoding gene of the polypeptide is any one of the following:

(a3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in (a 1) or (a 2) and encoding the polypeptide;

the encoding gene of the monkey poxvirus E8L antigen is any one of the following:

(b3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in (b 1) or (b 2) and encoding the same protein;

(b4) The nucleic acid molecule is a DNA molecule of which the nucleotide sequence of the coding chain is SEQ ID No.8, positions 1-888 of SEQ ID No.8 or positions 1-903 of SEQ ID No. 8.

5. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of claim 4.

6. Use of the polypeptide of claim 1 or a related biological material thereof in any of the following:

p3, preparing monkey pox virus E8L antigen secretion protein products;

the related biological material is the encoding gene of the polypeptide or an expression cassette or a recombinant vector or a recombinant bacterium or a transgenic cell line containing the encoding gene.

7. The use according to claim 2, characterized in that: the host cell is a eukaryotic cell.

8. Use according to claim 6 or 7, characterized in that: the coding gene is any one of the following:

9. Use of the fusion protein of claim 1 or 2 or the nucleic acid molecule of claim 3 or 4 or the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line of claim 5 in any of the following:

10. A method for preparing a monkey poxvirus E8L antigen secretion protein comprising the steps of:

(A1) Introducing the nucleic acid molecule of claim 3 or 4 into a host cell to obtain a recombinant cell;