CN115820677A - Preparation method and application of recombinant SLO antigen - Google Patents

Abstract

The application discloses a preparation method and application of a recombinant SLO antigen. In the application, an SLO antigen preparation method based on a genetic engineering technology is developed, the polynucleotide for coding the SLO antigen optimized by the preference of the synonymous codon is introduced into a vector, a prokaryotic expression plasmid is successfully constructed, and the expression quantity of a target protein is improved; and the expression of high proportion soluble protein is realized through the optimization of culture conditions such as culture medium, culture temperature and the like, and the activity of the obtained antigen is high.

Description

Preparation method and application of recombinant SLO antigen

Technical Field

The invention relates to the field of genetic engineering, in particular to a preparation method and application of a recombinant SLO antigen.

Background

Streptolysin O (Streptolysin-O or SLO for short) is a secretory virulence factor of A-group streptococcal toxin and belongs to a thiol activated toxin group. The molecular weight of the amino acid is 63.64kDA, and the amino acid is composed of 571 amino acids. In general, SLO antibodies are detected in group A streptococcal infected patients at about 2 weeks post infection. And as the infection time increased, the SLO antibody content gradually increased, reaching a peak at 4 weeks after infection. In the case of active rheumatic fever and acute glomerulonephritis, the SLO antibody is obviously increased after infection, and the titer of the SLO antibody can reach 1:400. therefore, the gradual increase of the content of the SLO antibody has important significance for clinical diagnosis. The SLO protein extracted from the natural streptococcus hemolyticus strain has the defects of low yield, high operation risk, difficulty in industrial production and the like. Although the problem of source shortage in natural purification is solved by applying a recombinant protein method to prepare the SLO antigen, the obtained product has low yield, poor stability and low activity.

Therefore, there is still a need in the art to develop a method for preparing recombinant SLO antigens with high yield, good stability and high activity.

Disclosure of Invention

The invention aims to provide a preparation method of a recombinant SLO antigen.

It is another object of the present invention to provide a polynucleotide sequence encoding an SLO antigen.

It is another object of the invention to provide a vector that is adapted to a polynucleotide sequence encoding an SLO antigen.

It is another object of the present invention to provide a kit comprising a polynucleotide sequence encoding an SLO antigen.

In order to solve the above technical problems, in a first aspect of the present invention, there is provided a polynucleotide encoding an SLO antigen, the polynucleotide being codon-optimized and selected from any one of:

(i) A polynucleotide having a sequence shown as SEQ ID NO. 2;

(ii) A polynucleotide having a homology of more than 95% with the sequence shown in SEQ ID NO. 2; and

(iii) (iii) a polynucleotide having a sequence complementary to the polynucleotide sequence described in (i) or (ii).

In a second aspect of the invention, there is provided an expression vector comprising a polynucleotide as provided in the first aspect of the invention.

In some preferred embodiments, the expression vector is an E.coli expression vector, more preferably pET-28a (+).

In a third aspect of the invention, there is provided a host cell comprising an expression vector as provided in the second aspect of the invention; or

The host cell has integrated into its genome a polynucleotide as provided in the first aspect of the invention.

In some preferred embodiments, the host cell is E.coli (Escherichia coli).

In some preferred embodiments, the host cell is the E.coli Rosetta (DE 3) strain.

The fourth aspect of the present invention provides a method for producing an SLO antigen, the method comprising the steps of: culturing the host cell of the third aspect of the invention to express the protein of interest; and

separating the target protein to obtain the SLO antigen;

wherein the target protein has an amino acid sequence shown as SEQ ID NO. 1.

In some preferred embodiments, the host cell is obtained by transformation of E.coli with a plasmid comprising a polynucleotide according to the first aspect of the invention.

In some preferred embodiments, the host cell is cultured in SB, TB or SOC media. In order to obtain a high amount of soluble expression, in a more preferred embodiment, the host cell is cultured in TB medium.

In some preferred embodiments, the host cell is cultured in a shaking environment.

In some preferred embodiments, the host cell is cultured at a temperature of 16 to 19 ℃; or culturing the host cell at a temperature of 36 to 38 ℃.

In some more preferred embodiments, higher soluble protein expression of interest is obtained by culturing the host cell in TB medium at a temperature of 16 to 19 ℃.

In some preferred embodiments, the host cell is cultured in a medium containing the kanamycin resistance gene.

In some preferred embodiments, the host cell is cultured and induced with IPTG to express the protein of interest.

In some preferred embodiments, the host cell is cultured to an OD600 of 0.6 to 0.8, followed by induction with IPTG to express the protein of interest.

In some preferred embodiments, the step of isolating the protein of interest comprises:

and (3) passing the crushed target protein supernatant through a chromatographic column, eluting, and collecting the eluent.

In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column.

In a fifth aspect, the present invention provides a kit comprising: a polynucleotide as provided in the first aspect of the invention; or

An expression vector as provided in the second aspect of the invention; or

A host cell according to the third aspect of the invention; or

Or an SLO antigen prepared by the method according to the fourth aspect of the present invention.

Compared with the prior art, the invention has at least the following advantages:

(1) The invention develops an SLO antigen preparation method based on genetic engineering technology, and introduces polynucleotide which is optimized by the bias of synonymous codon and used for coding SLO antigen into a vector, successfully constructs prokaryotic expression plasmid, and improves the expression quantity of target protein;

(2) The invention develops an SLO antigen preparation method based on the genetic engineering technology, realizes the expression of high proportion soluble protein through the optimization of culture conditions such as culture medium, culture temperature and the like, and the obtained antigen has high activity;

(4) The SLO antigen prepared by the SLO antigen preparation method provided in the preferred embodiment of the present invention can obtain a standard curve with an R value greater than 0.99 in chemiluminescence detection, and has good linearity.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

One or more embodiments are illustrated by the corresponding figures in the drawings, which are not meant to be limiting.

FIG. 1 is a diagram showing the results of SDS-PAGE identification of SLO antigens prepared in examples according to the present invention;

FIG. 2 is an electrophoresis chart of SLO antigen expression induced at 18 ℃ in the example according to the present invention.

FIG. 3 is an electrophoretogram of SLO antigen expressed by induction at 25 ℃ in an example according to the present invention.

Detailed Description

The inventor develops an SLO antigen expression system based on a prokaryotic expression system through extensive and intensive research, and obtains a polynucleotide sequence for coding an SLO antigen capable of expressing a large amount of target proteins in an escherichia coli expression system through preference optimization of synonymous codons, wherein the expressed target proteins have high activity and good stability.

The inventor further obtains a method for efficiently and soluble expressing the SLO antigen by optimizing the expression condition of the SLO antigen, including optimizing a culture medium and a culture temperature. In a more preferred embodiment of the invention, a host cell comprising a polynucleotide encoding an SLO antigen of the invention is cultured with TB medium at a temperature of 18 ℃.

Obtaining target gene/obtaining target protein related nucleic acid sequence

The full-length nucleotide sequence or a fragment thereof of the target protein or an element thereof of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

In one embodiment of the present invention, the amino acid sequence (SEQ ID NO: 2) of the target protein is analyzed by the NCBI database to obtain the target gene sequence information. The amino acid sequence of the SLO antigen of the arthrobacter globiformis is analyzed, for example, by NCBI database, and the target gene sequence encoding the SLO antigen is obtained.

Optimization of synonymous codon preference

To overcome the potential problem of reduced yield when expressing heterologous proteins in E.coli, the present invention relates to polynucleotide sequences optimized for synonymous codon bias. The obtained target gene sequence is optimized in the bias of the synonymous codon, the target gene sequence (SEQ ID NO: 2) optimized in the bias of the synonymous codon can express the amino acid sequence which is the same as the target protein, but the stability and the efficiency of the expression process are improved, and finally the obtained target protein maintains higher activity.

The invention also relates to a polynucleotide having a homology of more than 95% with the sequence shown in SEQ ID NO. 2; and a polynucleotide complementary to the sequence shown in SEQ ID NO. 2.

Vector of target gene

The present invention also relates to vectors comprising the polynucleotides of the invention. By "vector" in the present invention is meant a linear or circular DNA molecule comprising a fragment encoding a protein of interest operably linked to other fragments that provide for its transcription. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, vectors, and the like. Vector fragments may be derived from a host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector may be any expression vector which is synthetic or conveniently subjected to recombinant DNA procedures, and the choice of vector will usually depend on the host cell into which the vector is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. In one embodiment, the vector of the invention is an expression vector. In one embodiment of the present invention, pET-28a (+) is selected as a vector for more efficient expression.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences encoding the proteins of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Illustratively, vector DNA molecules are cleaved into linear molecules that can be ligated to foreign genes using endonucleases, and then codon-optimized gene segments of interest are ligated to the vector, optionally with cohesive-end ligation at a single cleavage site, directional cloning of double cleavage fragments, cohesive-end ligation at different restriction cleavage sites, blunt-end ligation, artificial adaptor ligation, or ligation to the ends of oligonucleotides to achieve insertion of the foreign DNA fragments.

Vector transformation host cell containing target gene

The invention also relates to genetically engineered host cells that have been engineered with the vector or fusion protein coding sequences of the invention. The vector containing the codon-optimized gene of interest can be inserted, transfected or otherwise transformed into a host cell by known methods to obtain a transformant containing the codon-optimized gene of interest of the present invention and capable of expressing the protein of interest. A "host cell" in the present invention is a cell into which an exogenous polynucleotide and/or vector has been introduced. The host cell may be a eukaryotic host cell or a prokaryotic host cell, the host cell preferably being a bacterium, and preferably being E.coli, more preferably E.coli ROSETTA (DE 3) species (Escherichia coli Rosetta (DE 3) strain).

Method for producing target protein

The present invention also relates to a method for preparing a protein of interest, which can express or produce a recombinant protein using the polynucleotide sequence of the present invention. Generally, the following steps are performed:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) A host cell cultured in a suitable medium;

(3) Separating and purifying protein from culture medium or cell.

Wherein, the transformation or transduction of a suitable host cell with the recombinant expression vector containing the polynucleotide of step (1) can be carried out by a conventional technique well known to those skilled in the art, and when the host is Escherichia coli, a heat shock method, an electrical transformation method, or the like can be used.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media, preferably SB, TB or SOC media, depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time. In order to promote the expression of the target protein and increase the expression level of the soluble protein, the present invention provides a preferred embodiment, wherein the host cell is cultured in TB medium, and the medium contains kanamycin resistance gene.

To further facilitate soluble expression of the protein of interest, in a preferred embodiment of the invention, the host cell is cultured to OD ₆₀₀ After between 0.6 and 0.8 induction with IPTG was carried out and cultivation was continued at 17 to 19 ℃ for about 8 to 12 hours.

The protein in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof. In one embodiment of the invention, the protein of interest is molecularly imprinted using affinity chromatography.

In the present disclosure, any exemplary or exemplary language (e.g., ") provided with respect to certain embodiments herein is used merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

If a definition or use of a term in a cited document is inconsistent or inconsistent with the definition of the term described herein, the definition of the term described herein applies and the definition of the term in the cited document does not apply.

Various terms used herein are as follows. If a term used in a claim is not defined below, the broadest definition persons in the art have given that term as reflected in a printed publication or issued patent at the time of filing.

As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from at least one other component (e.g., nucleic acid or polypeptide) with which the nucleic acid or polypeptide is present in its natural source. In one embodiment, the nucleic acid or polypeptide is found in the presence of only, if any, solvents, buffers, ions or other components that are normally present in solution. The terms "isolated" and "purified" do not include nucleic acids or polypeptides that are present in their natural source.

As used herein, the terms "polynucleotide" and "polynucleotide sequence" may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The invention also relates to variants of the above polynucleotides which encode protein fragments, analogues and derivatives having the same amino acid sequence as the invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.

As used herein, the term "codon optimization" refers to a means of increasing the efficiency of gene synthesis by avoiding the use of poorly available or rare codons based on differences in codon usage exhibited by the organism actually making the protein expression or production (including E.coli, yeast, mammalian blood cells, plant cells, insect cells, etc.).

As used herein, the terms "homology" and "identity" are used interchangeably to refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by the following methods. The nucleotide or amino acid sequences of a polynucleotide or polypeptide are aligned, the number of positions in the aligned polynucleotide or polypeptide containing the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotide or polypeptide containing a different nucleotide or amino acid residue. Polynucleotides may differ at one position, for example, by comprising different nucleotides (i.e., substitutions or variations) or deletions of nucleotides (i.e., insertions or deletions of one or two nucleotides in a polynucleotide). Polypeptides may differ at one position, for example, by containing amino acids (i.e., substitutions or variations) or amino acid deletions (i.e., amino acids inserted into one or both polypeptides or amino acid deletions). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

As used herein, the terms "sequence complementary" and "reverse sequence complementary" are used interchangeably to refer to a sequence that is in the opposite direction to, and complementary to, the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, its reverse complement is GTTCAT.

As used herein, the term "expression" includes any step involved in the production of a polypeptide in a host cell, including but not limited to transcription, translation, post-translational modification, and secretion. After expression, the host cell or the expression product can be harvested, i.e.recovered.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the present invention is further described below with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and it is to be noted that the terms used herein are merely for describing particular embodiments and are not intended to limit example embodiments of the present application.

Example 1 construction of SLO plasmid and transfection of host cells

(1) Obtaining a human SLO amino acid sequence SEQ ID NO 1, analyzing the human SLO amino acid sequence to obtain a gene sequence, and carrying out synonymous codon preference optimization on the gene sequence to obtain a plurality of gene sequences subjected to the synonymous codon preference optimization. After optimization of Escherichia coli synonymous codon preference, the plasmids are respectively connected with vectors pET-28a (+) to synthesize recombinant expression plasmids.

(2) Introduction of recombinant plasmid into host Escherichia coli

mu.L of the expression plasmid prepared in the step (1) was taken, added to 30. Mu.L of Escherichia coli competent Rosetta (DE 3) under ice bath conditions, placed in ice bath for 20min, heat-shocked for 90s, immediately placed on ice for 2min, added to 400. Mu.L of SOC medium containing no antibiotic, and subjected to shake culture at 37 ℃ and 220rpm for 50min. 100 μ L of the suspension was spread evenly on LB plates containing 100 μ g/mL kanamycin resistance and cultured overnight in a 37 ℃ incubator.

SEQ ID NO:1

MSNKKTFKKYSRVAGLLTAALIIGNLVTANAESNKQNTASTETTTTSEQPKPESSELTIEKAGQKMDDMLNSNDMIKLAP

KEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNELEVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTP

VDISIIDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGMGDKATVEVNDPTYANVSTAIDNLVNQWHDNY

SGGNTLPARTQYTESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAYKQIFYTVSANLPNNPADVFDKSV

TFKDLQRKGVSNEAPPLFVSNVAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILENSSFTAVVLGGDAAE

HNKVVTKDFDVIRNVIKDNATFSRKNPAYPISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAYVAQYEIL

WDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLGANSRNIRIMARECTGLAWEWWRKVIDERDVKLSKEINVNISGS

TLSPYGSITYK

Partially optimized codons are exemplified below:

optimized codon 1 (SEQ ID NO: 2)

ATGTCAAATAAAAAAACATTTAAAAAGTATTCTCGTGTTGCTGGCCTGTTGACTGCGGCTTTGATCATCGGCAATTTGGT

TACTGCCAACGCGGAATCTAACAAACAGAATACCGCTAGCACGGAAACCACGACTACCTCTGAACAGCCGAAGCCGGAAT

CCAGCGAGCTGACCATTGAGAAAGCAGGTCAAAAAATGGATGATATGCTGAACAGCAACGACATGATCAAACTGGCACCG

AAAGAGATGCCGCTGGAAAGCGCCGAGAAGGAAGAGAAGAAGTCGGAGGATAAGAAAAAAAGCGAAGAAGACCACACCGA

GGAGATCAACGACAAAATCTACTCGCTGAATTACAATGAACTGGAAGTGTTGGCGAAAAACGGCGAAACGATTGAGAACT

TTGTACCGAAAGAGGGTGTTAAGAAAGCGGACAAGTTTATTGTTATCGAGCGCAAAAAGAAGAACATTAACACCACTCCG

GTTGATATCTCCATTATCGACAGCGTTACCGATCGTACGTACCCGGCTGCGCTCCAACTGGCAAATAAAGGTTTTACCGA

GAATAAGCCGGATGCGGTCGTGACGAAACGTAACCCACAGAAAATCCACATTGACCTGCCGGGTATGGGTGATAAGGCGA

CCGTGGAAGTCAACGATCCGACCTACGCGAATGTAAGCACCGCAATTGACAACTTAGTTAACCAGTGGCATGATAACTAC

TCTGGTGGCAACACCCTGCCGGCGCGTACCCAGTATACCGAAAGCATGGTTTATTCTAAATCGCAAATTGAGGCGGCGCT

GAATGTGAACTCCAAGATTCTTGATGGTACACTGGGTATTGACTTCAAAAGCATTAGCAAAGGTGAAAAAAAGGTCATGA

TCGCTGCGTACAAACAAATCTTTTATACCGTTAGCGCCAACCTCCCAAATAACCCGGCGGACGTGTTCGATAAGTCCGTG

ACCTTCAAAGACCTGCAACGTAAGGGCGTTAGCAACGAGGCGCCTCCGTTGTTTGTTTCGAACGTTGCATACGGCAGAAC

GGTGTTCGTGAAGCTGGAAACCAGCAGCAAGTCCAACGACGTGGAAGCGGCGTTTAGCGCAGCGCTTAAGGGAACCGATG

TGAAGACCAATGGTAAATATTCGGATATCTTGGAGAACTCCAGCTTCACGGCGGTGGTGCTGGGTGGTGACGCCGCGGAG

CACAACAAAGTTGTCACCAAGGACTTCGACGTCATCCGCAATGTCATTAAAGACAACGCCACCTTCTCTAGAAAGAACCC

GGCGTACCCGATTAGCTATACCTCCGTGTTCCTGAAGAACAACAAGATCGCAGGCGTTAACAACCGTACCGAGTATGTTG

AGACGACCAGCACTGAGTACACGAGCGGCAAAATCAACTTAAGCCATCAGGGTGCTTACGTTGCTCAGTATGAAATCCTG

TGGGATGAAATTAATTACGATGACAAAGGCAAAGAGGTGATCACCAAGCGCCGTTGGGACAATAATTGGTATAGCAAAAC

CTCACCGTTTAGCACCGTAATCCCGCTGGGTGCCAACTCGCGCAACATTCGTATTATGGCACGTGAGTGCACCGGTTTGG

CCTGGGAATGGTGGCGTAAAGTGATTGACGAACGCGACGTGAAGCTGTCCAAGGAGATCAATGTGAATATCTCCGGCTCC

ACGCTGTCTCCGTATGGCAGCATTACCTACAAA

Optimized codon 2 (SEQ ID NO: 3)

ATGTCCAACAAGAAAACCTTCAAAAAATACAGCCGTGTTGCAGGTCTGCTGACTGCAGCACTGATCATCGGTAACCTGGT

TACCGCTAACGCAGAATCTAACAAACAGAACACCGCGAGCACTGAAACCACTACTACTTCCGAACAGCCGAAACCGGAAT

CTTCCGAACTGACCATCGAAAAGGCAGGCCAGAAGATGGACGACATGCTGAATAGCAACGACATGATTAAACTGGCACCG

AAAGAAATGCCACTGGAGTCCGCTGAAAAAGAAGAAAAAAAATCCGAAGATAAAAAGAAAAGCGAAGAAGATCATACCGA

AGAAATCAACGACAAAATTTATTCTCTGAACTATAACGAACTGGAAGTTCTGGCAAAGAACGGCGAGACCATCGAAAACT

TTGTCCCGAAAGAAGGTGTAAAGAAAGCTGACAAATTCATCGTTATCGAGCGCAAAAAAAAAAATATCAACACCACCCCG

GTCGATATTTCTATCATCGACTCTGTTACTGACCGTACCTACCCGGCGGCACTGCAGCTGGCAAACAAAGGTTTCACCGA

AAACAAACCGGATGCGGTTGTTACCAAACGTAACCCGCAGAAAATTCATATCGACCTGCCGGGCATGGGCGATAAAGCTA

CCGTTGAAGTGAACGATCCTACCTACGCGAACGTTTCTACTGCTATCGACAACCTGGTAAACCAGTGGCATGACAACTAT

TCCGGTGGTAACACGCTGCCGGCGCGTACCCAGTACACCGAATCCATGGTGTACTCCAAAAGCCAGATCGAAGCTGCGCT

GAACGTTAATAGCAAGATTCTGGACGGTACTCTGGGTATCGACTTTAAATCCATCTCCAAAGGCGAAAAAAAAGTTATGA

TTGCGGCTTACAAACAAATCTTCTATACCGTTAGCGCGAACCTGCCGAATAACCCGGCGGACGTTTTCGACAAAAGCGTT

ACCTTTAAGGACCTGCAACGTAAAGGTGTATCTAACGAAGCCCCACCGCTGTTCGTATCTAACGTGGCTTACGGCCGCAC

TGTGTTCGTGAAACTGGAAACCTCTTCCAAGAGCAACGATGTCGAAGCTGCATTCTCTGCAGCTCTGAAGGGCACTGATG

TGAAGACCAACGGTAAATACTCCGACATTCTGGAAAATTCCTCTTTCACCGCCGTTGTCCTGGGTGGTGACGCCGCAGAA

CACAACAAAGTTGTTACTAAAGATTTCGACGTTATTCGTAACGTTATCAAAGACAATGCTACCTTCTCCCGCAAAAACCC

AGCATATCCGATCTCCTACACCAGCGTGTTCCTGAAAAACAACAAAATCGCTGGCGTCAACAACCGTACCGAGTACGTAG

AAACGACCTCCACCGAATATACTTCTGGTAAAATTAACCTGTCCCACCAGGGCGCGTATGTGGCACAGTACGAAATCCTG

TGGGACGAGATTAACTATGATGATAAAGGCAAAGAAGTTATTACTAAACGTCGTTGGGACAACAATTGGTACTCCAAGAC

CTCCCCGTTCTCTACCGTGATCCCACTGGGTGCTAACTCCCGTAACATCCGTATTATGGCTCGTGAATGTACCGGTCTGG

CTTGGGAATGGTGGCGTAAAGTTATCGACGAACGTGACGTTAAGCTGTCTAAGGAGATTAATGTCAACATCTCTGGCTCT

ACCCTGTCTCCGTACGGTTCTATTACCTATAAA

Optimized codon 3 (SEQ ID NO: 4)

ATGTCTAACAAAAAAACCTTCAAAAAATACTCTCGCGTAGCAGGTCTGCTGACCGCCGCACTGATCATCGGTAACCTGGT

CACCGCTAACGCTGAATCTAATAAACAGAACACGGCTTCCACGGAAACCACCACGACGTCTGAACAACCGAAACCGGAAT

CTAGCGAACTGACTATCGAAAAAGCTGGCCAAAAAATGGATGATATGCTGAACTCCAATGACATGATTAAACTGGCTCCG

AAAGAAATGCCTCTGGAATCTGCGGAAAAAGAGGAAAAAAAAAGCGAAGATAAAAAAAAAAGCGAGGAAGACCACACCGA

AGAAATCAACGATAAAATCTATTCTCTGAACTACAACGAACTGGAGGTTCTGGCTAAAAACGGTGAGACCATTGAAAACT

TCGTGCCGAAAGAAGGCGTAAAAAAAGCCGACAAATTCATCGTTATTGAACGTAAAAAAAAAAACATCAACACCACCCCG

GTGGACATCTCTATCATTGACTCTGTAACCGACCGTACCTACCCGGCAGCTCTGCAGCTGGCCAACAAGGGTTTCACCGA

AAACAAACCGGACGCTGTTGTGACCAAACGCAACCCGCAGAAAATTCACATCGACCTGCCAGGCATGGGTGATAAGGCGA

CCGTCGAAGTGAACGATCCAACTTACGCTAACGTTTCCACCGCAATTGATAACCTGGTTAACCAGTGGCACGACAACTAC

TCTGGTGGTAACACCCTGCCAGCGCGCACTCAGTACACCGAGTCTATGGTATACAGCAAATCCCAGATCGAGGCAGCGCT

GAACGTAAACTCTAAAATCCTGGATGGTACTCTGGGTATCGATTTCAAAAGCATTAGCAAAGGTGAGAAAAAAGTGATGA

TTGCGGCTTACAAACAGATCTTCTACACCGTGTCTGCTAATCTGCCGAACAACCCAGCTGATGTCTTCGACAAATCCGTA

ACGTTCAAAGATCTGCAACGTAAAGGCGTCTCTAACGAAGCTCCGCCGCTGTTCGTCAGCAACGTAGCATACGGCCGTAC

CGTGTTTGTAAAACTGGAAACTTCCTCCAAAAGCAACGACGTTGAGGCGGCTTTCTCTGCAGCACTGAAGGGTACCGATG

TTAAAACCAACGGCAAATATTCTGACATCCTGGAGAACTCTTCCTTCACTGCTGTTGTGCTGGGCGGTGATGCTGCGGAA

CACAACAAAGTGGTGACCAAAGACTTCGATGTAATCCGTAACGTGATCAAAGACAACGCTACTTTCTCTCGTAAAAACCC

GGCTTATCCGATCAGCTATACGAGCGTTTTCCTGAAGAACAACAAGATTGCAGGCGTTAACAACCGTACTGAGTACGTAG

AAACCACTTCTACCGAATACACCTCTGGTAAAATCAACCTGAGCCACCAGGGTGCTTACGTGGCACAGTACGAAATTCTG

TGGGATGAGATCAACTATGACGACAAAGGCAAAGAAGTGATTACCAAACGCCGTTGGGATAATAACTGGTATTCTAAGAC

TTCTCCGTTCAGCACTGTGATCCCGCTGGGCGCGAATTCCCGCAACATCCGCATCATGGCGCGTGAATGTACTGGCCTGG

CGTGGGAATGGTGGCGTAAAGTTATCGACGAACGCGATGTTAAACTGTCCAAAGAAATCAACGTGAATATTTCCGGTTCT

ACCCTGTCCCCGTACGGCAGCATTACCTATAAA

Example 2 expression of the Gene of interest

The monoclonal prepared in step example 1 was picked, aseptically inoculated into 100. Mu.g/mL kanamycin-resistant TB medium, induced at 37 ℃ with shaking at 220rpm until the OD600 was between 0.6 and 0.8 and the final concentration was 0.1mM IPTG, and incubated overnight with shaking at 18 ℃ and 37 ℃ respectively. The same amount of bacterial liquid of three different culture media is taken for ultrasonic disruption and SDS-PAGE identification is carried out, and the identification result is shown in figure 1.

As shown in fig. 1, columns 1-2: optimizing codon 1 to express crushed supernatant and precipitate; 3-4 columns: optimizing codon 2 to express crushed supernatant and precipitate; 5-6 columns: codon 3 was optimized to express the disrupted supernatant, pellet. The results show that: the optimized codon 1 expresses more target protein and is expressed in the supernatant, and the expression amount is obviously higher than that of other optimized codons.

Example 3 purification of expression product

Selecting a recombinant strain constructed by the optimized codon 1, adopting a TB culture medium, placing the recombinant strain at 18 ℃ and 25 ℃ for induced expression culture, collecting and weighing thalli, and respectively adding the thalli into lysine Buffer ice for heavy suspension. Centrifuging at 20000rpm for 30min at 4 deg.C after ultrasonication, collecting supernatant, and filtering with 0.22um needle filter to obtain filtered stock solution. And (3) allowing the stock solution to pass through a Ni-NTA chromatographic column, eluting the protein with 50mM Tris-HCl,50mM NaCl and 200mM imidazole at pH 7.0 to obtain the target protein, and eluting to obtain the protein. The electrophoretograms of the 18 ℃ and 25 ℃ expression purification are shown in FIGS. 2 and 3, respectively, wherein 328 mg/L of the culture medium at 18 ℃ can purify about 328 mg of the target protein, and 150 mg/L of the culture medium at 25 ℃ can purify about 150 mg of the target protein. Thus, the expression level of the recombinant SLO protein is higher at 18 ℃ than at 25 ℃ in TB medium.

Example 4 identification of target protein Activity by chemiluminescence

(1) Antigen activity assay

And detecting the antigen activity of the expression recombinant SLO protein in the TB culture medium at 18 ℃ and 25 ℃ by adopting a latex immunoturbidimetry method. The SLO and latex were mixed, mixed well and left for 8 hours, after which the unattached antigen was removed by dialysis. Adding a sealing liquid, sealing, centrifuging to remove the supernatant, and preparing the SLO antigen latex microspheres. Then adding PB buffer solution containing PEG and SLO monoclonal antibody (abcam), and adopting Hitachi 7180 full-automatic biochemical instrument to perform determination under the wavelength of 600nm, wherein the analysis method is a two-point end point method.

As shown in Table 1, the maximum absorbance of the expressed protein at 18 ℃ is 3471, the linear regression equation is y =5.4388x +37.314, R ² ＝0.9979(R ² Greater than 0.95), the linear correlation is very high; the highest absorbance of the protein expressed by the protein at 25 ℃ is 665, the linear regression equation is y =1.0498x +59.371, R ² ＝0.9283(R ² < 0.95), the linear correlation is poor. From the above results, it was found that the detection activity and linear correlation of the recombinant SLO protein expressed at 18 ℃ were better than those at 25 ℃ in the linear measurement range. The method for producing the SLO antigen of the present invention is suitably carried out at a temperature of 18 ℃ which is lower than room temperature.

TABLE 1

It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples for carrying out the invention, and that various changes in form and details may be made therein without departing from the spirit and scope of the invention in practice.

Claims (10)

1. An isolated polynucleotide encoding an SLO antigen, wherein the polynucleotide is codon optimized and the polynucleotide is selected from any one of:

(i) A polynucleotide having a sequence shown as SEQ ID NO. 2;

(ii) A polynucleotide having a homology of more than 95% with the sequence shown in SEQ ID NO. 2; and

(iii) (iii) a polynucleotide having a sequence complementary to the polynucleotide sequence described in (i) or (ii).

2. An expression vector comprising the polynucleotide of claim 1.

3. The expression vector according to claim 2, characterized in that it is an E.coli expression vector, preferably pET-28a (+).

4. A host cell comprising the expression vector of any one of claims 2 or 3; or

The host cell having integrated into its genome the polynucleotide of claim 1.

5. A method of producing an SLO antigen, comprising the steps of:

transforming a host cell with a vector comprising the polynucleotide of claim 1;

culturing the host cell to express the SLO antigen.

6. The method of claim 5, wherein the host cell is cultured in SB, TB or SOC media;

and/or, when the host cell is cultured, the culture medium contains a kanamycin resistance gene.

7. The method of claim 5, wherein the host cell is cultured and induced to express the protein of interest by IPTG.

8. The method according to claim 5, wherein the host cell is cultured at a temperature of 16 to 19 ℃.

9. The method of claim 7, wherein the host cell is cultured to an OD600 of 0.6 to 0.8, followed by induction with IPTG to express the protein of interest.

10. A kit, comprising: the polynucleotide of claim 1; or

The expression vector of any one of claims 2 or 3; or

The host cell of claim 4; or

An SLO antigen produced by the method of any one of claims 6 to 9.

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