CN116240182A - Application of luciferase signal peptide, fusion protein containing luciferase signal peptide and preparation method of fusion protein - Google Patents

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

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CN116240182A
CN116240182A CN202310011211.4A CN202310011211A CN116240182A CN 116240182 A CN116240182 A CN 116240182A CN 202310011211 A CN202310011211 A CN 202310011211A CN 116240182 A CN116240182 A CN 116240182A
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antigen
seq
signal peptide
monkey
dna molecule
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张静静
邢体坤
安文琪
宋路萍
王亚萍
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Hualan Genetic Engineering Co ltd
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Abstract

The invention discloses application of a luciferase signal peptide, fusion protein containing the luciferase signal peptide and a preparation method thereof. The invention provides application of a polypeptide with an 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 the monkey poxvirus A29L antigen in host cells; p2, improving the secretion expression efficiency of the monkey poxvirus A29L antigen in host cells; p3, preparing monkey pox virus A29L antigen secretion protein products. The invention adopts the signal peptide to guide the secretion expression of the monkey pox virus A29L eukaryotic cells, wherein the secretion expression level of the luciferase signal peptide Ga to guide the monkey pox virus A29L antigen is obviously superior to that of albumin signal peptide Al and antibody light chain signal peptide Lc, and the invention is more suitable for large-scale industrialized production and reduces the production cost.

Description

Application of luciferase signal peptide, fusion protein containing luciferase signal peptide and preparation method of fusion protein
Technical Field
The invention relates to the field of biotechnology, in particular to application of luciferase signal peptide, fusion protein containing the luciferase signal peptide and a preparation method thereof.
Background
In 1958 it was found that a poxvirus caused a disease similar to that of human smallpox in experimental macaques, and was therefore called a monkey poxvirus. In 1970, a monkey pox virus was first found in a 9 year old boy of congo, with symptoms of blisters or pustules, with weaker infectivity than smallpox. Thereafter, monkey pox cases were continuously found in congo, middle and western alives. In 2022, month 5, a new round of "monkey pox" epidemic was first discovered in the uk, and has been found in multiple countries, such as the uk, portugal, spanish, australia, germany, france. At present, the epidemic situation of the 'monkey pox' virus still tends to spread, and therefore, the epidemic situation is concerned by the health supervision departments of a plurality of countries.
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 A29L antigen is membrane protein of mature virus particles of the monkey pox virus, and is also one of important immune targets. Therefore, how to prepare A29L protein is a urgent problem to be solved in the research and development of key products of monkey pox virus detection reagents or vaccines.
Disclosure of Invention
The main problem to be solved by the present invention is how to prepare the monkey poxvirus a29L antigen.
In order to solve the problems, the invention provides a luciferase signal peptide (signal peptide Ga, SEQ ID No. 1), wherein the signal peptide Ga can guide monkey poxvirus A29L 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 Yu Bai protein signal peptide (signal peptide Al) and antibody light chain signal peptide (signal peptide Lc) are remarkably high.
The invention firstly 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:
use of P1 to increase secretory expression yield of monkey poxvirus a29L antigen in a host cell;
p2, improving the secretion expression efficiency of monkey pox virus A29L antigen in host cells;
p3, preparing monkey pox virus A29L 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.7 (the coding gene of the signal peptide Ga at the 16 th-66 th positions of SEQ ID No.5, namely SEQ ID No. 2) into the multiple cloning site (such as HindIII and PacI) of the 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:
(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 a polypeptide represented by SEQ ID No. 1;
(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.
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 a fusion protein.
The fusion protein provided by the invention is a protein obtained by fusing the polypeptide shown in SEQ ID No.1 to the N end of the monkey pox virus A29L antigen shown in SEQ ID No. 3.
Further, the amino acid sequence of the fusion protein is SEQ ID No.5, positions 1-127 of SEQ ID No.5 or positions 1-132 of SEQ ID No.5.
The 1 st to 17 th positions of SEQ ID No.5 are signal peptide Ga (namely SEQ ID No. 1), the 18 th to 127 th positions are the monkey pox virus A29L antigen, the 128 th to 132 th positions are Linker joints, and the 133 th to 138 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 A29L antigen from the 5 'end to the 3' end.
Further, the encoding gene of the polypeptide may be any one 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 a29L 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.7, positions 1-396 of SEQ ID No.7 or positions 1-411 of SEQ ID No. 7.
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 396 th sites are genes encoding A29L antigen, the 397 th to 411 th sites are connecting peptide (Linker) genes, the 412 th to 429 th sites are histidine tag genes, the 430 th to 432 th sites are stop codons, and the 433 th to 440 th sites are PacI recognition sites. The recombinant expression plasmid pCGS3-Ga-A29L contains the coding gene of recombinant protein Ga-A29L, the coding sequence of the coding gene of the recombinant protein Ga-A29L is the 16 th position to 432 th position of SEQ ID No.7, and the coding amino acid sequence of the coding gene of the recombinant protein Ga-A29L 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, per residue 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 ℃, at 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: hybridization at 50℃in a mixed solution of 7% SDS, 0.5M Na3PO4 and 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.7 (the coding gene of the signal peptide Ga at the 16 th-66 th positions of SEQ ID No.5, namely 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 an Expi293F cell.
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:
use of P1 to increase secretory expression yield of monkey poxvirus a29L antigen in a host cell;
p2, improving the secretion expression efficiency of monkey pox virus A29L antigen in host cells;
application of P3 in preparing monkey pox virus A29L antigen secretion protein product.
The amino acid sequence of the monkey poxvirus A29L 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.
In a specific embodiment of the invention, the host cell is specifically an Expi293F cell.
Methods for preparing monkey poxvirus A29L antigen-secreting proteins are also within the scope of the claimed invention.
The method for preparing the monkey pox virus A29L 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 A29L 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 a specific embodiment of the invention, the host cell is specifically an Expi293F cell.
In the step (A2), the culture is stopped when the cell viability is reduced to 65% -75%.
In step (A2), monkey poxvirus a29L antigen secretion 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, luciferase signal peptide Ga (SEQ ID No. 1) and antibody signal peptide Lc to guide monkey pox virus A29L antigen eukaryotic cell secretion expression. The study proves that: the monkey poxvirus A29L antigen secretion expression quantity of the luciferase signal peptide Ga experimental group is obviously superior to that of an albumin signal peptide Al 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 A29L 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-Al-A29L (Al is an albumin signal peptide), 2 is pCGS3-Ga-A29L (Ga is a luciferase signal peptide), and 3 is pCGS3-Lc-A29L (Lc is an antibody light chain signal peptide).
FIG. 2 is an SDS-PAGE identification of monkey poxvirus A29L antigen cell secretion supernatants. Wherein 1 is albumin signal peptide Al secretion supernatant (pCGS 3-Al-A29L transfected cell culture supernatant), 2 is luciferase signal peptide Ga secretion supernatant (pCGS 3-Ga-A29L transfected cell culture supernatant), 3 is antibody light chain signal peptide Lc secretion supernatant (pCGS 3-Lc-A29L 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 monkey poxvirus A29L antigen cells. Wherein 1 is albumin signal peptide Al secretion supernatant (pCGS 3-Al-A29L transfected cell culture supernatant), 2 is luciferase signal peptide Ga secretion supernatant (pCGS 3-Ga-A29L transfected cell culture supernatant), 3 is antibody light chain signal peptide Lc secretion supernatant (pCGS 3-Lc-A29L 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 A29L antigen protein. Wherein 1 is an albumin signal peptide Al secretion expression purification sample, 2 is a luciferase signal peptide Ga secretion expression purification sample, and 3 is an antibody light chain signal peptide Lc secretion 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;
Figure BDA0004038604510000051
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;
cell counter: roche Co;
ultra-clean bench: suzhou Antai air technologies Co., ltd;
electric heating constant temperature water bath kettle: fisher Scientific company;
CO 2 constant temperature shaking table: CRYSTAL Co;
ext> HYGext> -ext> Aext> fullext> constantext> temperatureext> shakeext> flaskext> cabinetext>:ext> Experiment equipment factory in Taicang city;
DYY-6C type electrophoresis apparatus: six instrument factories in Beijing;
DYCP-31DN horizontal electrophoresis tank: six instrument factories in Beijing;
micropipettes: eppendorf Inc.
Example 1 construction of recombinant expression plasmid
The monkey poxvirus was selected from the 2022 latest monkey poxviruses (NCBI genome accession No. ON 563414.3), wherein the NCBI accession No. for the a29L antigen is URK20577.1. The coding genes of albumin signal peptide Al, luciferase signal peptide Ga and antibody light chain signal peptide Lc are fused to the 5' end of the coding gene of monkey pox virus A29L antigen protein, the corresponding fragment names are respectively Al-A29L, ga-A29L and Lc-A29L, and cloned to a pCGS3 vector to construct eukaryotic recombinant expression plasmids, and the specific operation procedures are described below.
Al-A29L gene synthesis: the carbon end of albumin signal peptide Al fuses monkey pox antigen A29L (the amino acid sequence of monkey pox antigen A29L is SEQ ID No.3, the coding sequence of the coding gene is SEQ ID No. 4), the coding gene is subjected to codon optimization, the Al-A29L gene (the nucleotide sequence is SEQ ID No. 6) is synthesized, and the delivery synthetic plasmid of the biological organism is pUC57-Al-A29L (containing the Al-A29L gene).
pUC57-Al-A29L was used as a template, using a polymerase
Figure BDA0004038604510000061
GXL Premix (TAKARA Co.) amplified Ga-A29L and Lc-A29L fragments of interest. The method comprises the following steps:
1) Ga-A29L gene fragment amplification: amplifying the fragment A by using the primer 1 and the primer 5, wherein the fragment A is used as a second round of template, and performing second round of amplification by using the primer 2 and the primer 5 to obtain a fragment B, namely a Ga-A29L target fragment;
2) Lc-A29L gene fragment amplification: and (3) 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 Lc-A29L target fragment.
The primer sequences used for the PCR amplification were as follows:
primer 1:5'-TGTTTGCTCTGATTTGTATTGCCGTGGCTGAGGCCATGGATGGCACCCTGTTTCC-3';
primer 2:5'-CCCAAGCTTGCCGCCACCATGGGGGTGAAGGTGTTGTTTGCTCTGATTTGTATTG-3';
primer 3:5'-CTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAGATGCATGGATGGCACCCTGTTTCC-3';
primer 4:5'-CCCAAGCTTGCCGCCACCATGAGCGTGCCAACCCAGGTGCTGGGCCTGCTGCTGCTGTG-3';
primer 5:5'-CCTTAATTAATCAGTGGTGATGGTGGTGGTGAGAG-3'.
The pCGS3 vector was digested with HindIII (NEB) and PacI (NEB) to obtain a vector fragment, and the total synthetic gene pUC57-Al-A29L was digested with HindIII and PacI to obtain an amplified fragment Ga-A29L and Lc-A29L. Ligation was performed using DNA Ligation Kit Ver.2.1 (TAKARA Co.), transformation, plasmid extraction, and identification. Three recombinant expression plasmids pCGS3-Al-A29L, pCGS3-Ga-A29L and pCGS3-Ga-A29L 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-Al-A29L expression plasmid, lane 2 is the double restriction enzyme digestion identification of the pCGS3-Ga-A29L expression plasmid, and lane 3 is the double restriction enzyme digestion identification of the pCGS3-Lc-A29L expression plasmid. The size of the vector fragment after enzyme digestion is about 7100bp, and the size of the target gene is about 440bp; the size of the enzyme cutting strip meets the expectations.
Structural description of recombinant expression plasmid pCGS 3-Al-A29L: the fragment between the restriction endonuclease HindIII recognition site and the restriction endonuclease PacI recognition site of the pCGS3 vector was replaced with a DNA molecule whose nucleotide sequence is SEQ ID No.6, and the other nucleotides of the pCGS3 vector were kept unchanged to obtain a recombinant expression vector. 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 63 th sites are albumin signal peptide Al coding genes, the 64 th to 393 th sites are A29L antigen coding genes, the 394 th to 408 th sites are connecting peptide (Linker) genes, the 409 th to 426 th sites are histidine tag genes, the 427 th to 429 th sites are stop codons, and the 429 th to 437 th sites are PacI recognition sites. The recombinant expression plasmid pCGS3-Al-A29L contains the coding gene of recombinant protein Al-A29L, the coding sequence of the coding gene of the recombinant protein Al-A29L is the 16 th bit to 429 th bit of SEQ ID No.6, and the coding recombinant protein Al-A29L is a protein fused by Al signal peptide and the 18 th bit to 138 th bit of sequence 5 and consists of 137 amino acids.
Structural description of recombinant expression plasmid pCGS 3-Ga-A29L: the fragment between the restriction endonuclease HindIII recognition site and the restriction endonuclease PacI recognition site of the pCGS3 vector was replaced with a DNA molecule whose nucleotide sequence is SEQ ID No.7, and the other nucleotides of the pCGS3 vector were kept unchanged to obtain a recombinant expression vector. 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 396 th sites are genes encoding A29L antigen, the 397 th to 411 th sites are connecting peptide (Linker) genes, the 412 th to 429 th sites are histidine tag genes, the 430 th to 432 th sites are stop codons, and the 433 th to 440 th sites are PacI recognition sites. The recombinant expression plasmid pCGS3-Ga-A29L contains the coding gene of recombinant protein Ga-A29L, the coding sequence of the coding gene of the recombinant protein Ga-A29L is the 16 th position to 432 th position of SEQ ID No.7, and the coding amino acid sequence of the coding gene of the recombinant protein Ga-A29L is the protein of sequence 5.
Structural description of recombinant expression plasmid pCGS 3-Lc-A29L: the fragment between the restriction endonuclease HindIII recognition site and the restriction endonuclease PacI recognition site of the pCGS3 vector was replaced with a DNA molecule whose nucleotide sequence is SEQ ID No.8, and the other nucleotides of the pCGS3 vector were kept unchanged to obtain a recombinant expression vector. 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 75 th sites are signal peptide Lc coding genes, the 76 th to 405 th sites are A29L antigen coding genes, the 406 th to 420 th sites are connecting peptide (Linker) genes, the 421 th to 438 th sites are histidine tag genes, the 439 th to 441 th sites are stop codons, and the 442 th to 449 th sites are PacI recognition sites. The recombinant expression plasmid pCGS3-Lc-A29L contains the coding gene of recombinant protein Lc-A29L, the coding sequence of the coding gene of the recombinant protein Lc-A29L is the 16 th-441 th position of SEQ ID No.8, and the coded recombinant protein Lc-A29L is a protein fused by Lc signal peptide and the 18 th-138 th position of sequence 5 and consists of 141 amino acids.
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-Al-A29L, pCGS3-Ga-A29L, pCGS3-Lc-A29L and pCGS3 vectors 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-Al-A29L, pCGS3-Ga-A29L and pCGS3-Lc-A29L 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 ExpiFectamineTM293Transfection Enhancer 1 and 3mL ExpiFectamineTM293Transfection Enhancer 2 were mixed and slowly added 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.
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-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, 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: albumin signal peptide Al secretion supernatant (pCGS 3-Al-A29L transfected cell culture supernatant), luciferase signal peptide Ga secretion supernatant (pCGS 3-Ga-A29L transfected cell culture supernatant), and antibody light chain signal peptide Lc secretion supernatant (pCGS 3-Lc-A29L transfected cell culture supernatant) expressed 2.16%,12.09% and 11.42% of the total protein. It can be seen from the above that: compared with the signal peptide Al and the signal peptide Lc, the luciferase signal peptide guided Ga monkey poxvirus A29L antigen has higher secretion expression quantity.
Example 3 protein purification of A29L antigen
1. Ultrafiltration concentration
The supernatant of the cell culture transfected with pCGS3-Al-A29L, the supernatant of the cell culture transfected with pCGS3-Ga-A29L and the supernatant of the cell culture transfected with pCGS3-Lc-A29L 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 Ga-A29L in the supernatant of the cell culture transfected by the pCGS3-Ga-A29L has the yield of 161.78mg/L, the recombinant protein Lc-A29L in the supernatant of the cell culture transfected by the pCGS3-Lc-A29L has the yield of 142.15mg/L, and the recombinant protein Al-A29L in the supernatant of the cell culture transfected by the pCGS3-Al-A29L has the yield of 38.62mg/L (FIG. 4), wherein the signal peptide Ga has the highest yield after the purification of the experimental group.
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 monkey pox virus A29L 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 (10)

1. Use of a polypeptide having the amino acid sequence SEQ ID No.1 or a related biological material thereof in any of the following:
use of P1 to increase secretory expression yield of monkey poxvirus a29L antigen in a host cell;
p2, improving the secretion expression efficiency of monkey pox virus A29L antigen in host cells;
p3, preparing monkey pox virus A29L 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.
2. The use according to claim 1, characterized in that: the host cell is a eukaryotic cell.
3. Use according to claim 1 or 2, characterized in that: the coding gene is any one 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 a polypeptide represented by SEQ ID No. 1;
(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.
4. A fusion protein obtained by fusing the polypeptide of claim 1 to the N-terminus of the A29L antigen of the monkey poxvirus.
5. The fusion protein of claim 4, wherein: the amino acid sequence of the fusion protein is SEQ ID No.5, 1 st to 127 th positions of SEQ ID No.5 or 1 st to 132 th positions of SEQ ID No.5.
6. A nucleic acid molecule encoding the fusion protein of claim 4 or 5.
7. The nucleic acid molecule of claim 6, wherein: the nucleic acid molecule consists of a coding gene of the polypeptide and a coding gene of the monkey pox virus A29L antigen;
the encoding gene of the polypeptide is any one 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;
the encoding gene of the monkey poxvirus A29L antigen is 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 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.7, positions 1-396 or positions 1-411 of SEQ ID No.7 or the like.
8. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of claim 7.
9. Use of the fusion protein of claim 4 or 5 or the nucleic acid molecule of claim 6 or 7 or the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line of claim 8 in any of the following:
use of P1 to increase secretory expression yield of monkey poxvirus a29L antigen in a host cell;
p2, improving the secretion expression efficiency of monkey pox virus A29L antigen in host cells;
application of P3 in preparing monkey pox virus A29L antigen secretion protein product.
10. A method for preparing a monkey poxvirus a29L antigen secretion protein comprising the steps of:
(A1) Introducing the nucleic acid molecule of claim 6 or 7 into a host cell to obtain a recombinant cell;
(A2) Culturing the recombinant cells, and obtaining monkey pox virus A29L antigen secretion proteins from the culture supernatant.
CN202310011211.4A 2023-01-05 2023-01-05 Application of luciferase signal peptide, fusion protein containing luciferase signal peptide and preparation method of fusion protein Pending CN116240182A (en)

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