Method for the production and use of recombinant slo antigens

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to the preparation method and application of recombinant SLO antigen. Background Technology

[0002] Streptolysin O (SLO) is a secretory virulence factor of Group A streptococcal toxins, belonging to the thiol-activated toxin group. Its molecular weight is 63.64 kDa, composed of 571 amino acids. Generally, SLO antibodies can be detected in patients infected with Group A streptococci about two weeks after infection. Furthermore, the SLO antibody level gradually increases with the duration of infection, reaching its peak in the fourth week post-infection. For example, in active rheumatic fever and acute glomerulonephritis, SLO antibodies show a significant increase after infection, with titers reaching 1:400. Therefore, the gradual increase in SLO antibody levels is of significant clinical diagnostic value. Extracting SLO protein from natural hemolytic streptococcal strains has disadvantages such as low yield, high operational risks, and difficulty in industrial production. While the recombinant protein method for preparing SLO antigen solves the problem of source shortage in natural purification, the resulting product has low yield, poor stability, and low activity.

[0003] Therefore, there is still a need in this field to develop a method for preparing recombinant SLO antigen with high yield, good stability, and high activity. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing recombinant SLO antigen.

[0005] Another object of the present invention is to provide a polynucleotide sequence encoding the SLO antigen.

[0006] Another object of the present invention is to provide a vector adapted to a polynucleotide sequence encoding the SLO antigen.

[0007] Another object of the present invention is to provide a kit containing a polynucleotide sequence encoding the SLO antigen.

[0008] To address the aforementioned technical problems, in a first aspect, the present invention provides a polynucleotide encoding an SLO antigen, wherein the polynucleotide is codon-optimized and selected from any of the following:

[0009] (i) A polynucleotide having the sequence shown in SEQ ID NO.2;

[0010] (ii) a polynucleotide having greater than 95% homology to the sequence shown in SEQ ID NO.2; and

[0011] (iii) A polynucleotide having a sequence complementary to the polynucleotide sequence described in (i) or (ii).

[0012] In a second aspect, the present invention provides an expression vector comprising the polynucleotide provided in the first aspect of the present invention.

[0013] In some preferred embodiments, the expression vector is an Escherichia coli expression vector, more preferably pET-28a(+).

[0014] In a third aspect, the present invention provides a host cell comprising the expression vector provided in the second aspect of the present invention; or

[0015] The host cell genome integrates polynucleotides as provided in the first aspect of the present invention.

[0016] In some preferred embodiments, the host cell is *Escherichia coli*.

[0017] In some preferred embodiments, the host cell is Escherichia coli Rosetta (DE3) strain.

[0018] A fourth aspect of the present invention provides a method for preparing SLO antigen, the method comprising the steps of: culturing the host cells described in the third aspect of the present invention to express the target protein; and

[0019] The target protein is isolated to obtain the SLO antigen;

[0020] The target protein has an amino acid sequence as shown in SEQ ID NO:1.

[0021] In some preferred embodiments, the host cell is obtained by transforming Escherichia coli with a plasmid containing the polynucleotide described in the first aspect of the invention.

[0022] In some preferred embodiments, the host cells are cultured in SB, TB, or SOC media. In a more preferred embodiment, to obtain high levels of soluble expression, the host cells are cultured in TB media.

[0023] In some preferred embodiments, the host cells are cultured in an oscillating environment.

[0024] In some preferred embodiments, the host cells are cultured at a temperature of 16 to 19°C; or at a temperature of 36 to 38°C.

[0025] In some preferred embodiments, culturing the host cells in TB medium at a temperature of 16 to 19°C can yield higher expression of the soluble target protein.

[0026] In some preferred embodiments, the culture medium used to culture the host cells contains a kanamycin resistance gene.

[0027] In some preferred embodiments, IPTG is used to induce the expression of the target protein when culturing the host cells.

[0028] In some preferred embodiments, the host cells are cultured until the OD600 is between 0.6 and 0.8, and then induced with IPTG to express the target protein.

[0029] In some preferred embodiments, the step of isolating the target protein includes:

[0030] The supernatant of the lysed target protein is passed through a chromatography column for elution, and the eluent is collected.

[0031] In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column.

[0032] A fifth aspect of the present invention provides a kit comprising: a polynucleotide as provided in the first aspect of the present invention; or

[0033] Such as the expression vector provided in the second aspect of the present invention; or

[0034] The host cell as described in the third aspect of the present invention; or

[0035] Or the SLO antigen prepared by the method according to the fourth aspect of the present invention.

[0036] Compared with the prior art, the present invention has at least the following advantages:

[0037] (1) This invention develops a method for preparing SLO antigen based on genetic engineering technology. The polynucleotide encoding SLO antigen, which has been optimized by synonym codon preference, is introduced into a vector to successfully construct a prokaryotic expression plasmid, thereby increasing the expression level of the target protein.

[0038] (2) This invention develops a method for preparing SLO antigen based on genetic engineering technology. After optimizing culture conditions such as culture medium and culture temperature, a high proportion of soluble protein expression is achieved, and the resulting antigen has high activity.

[0039] (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.

[0040] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0041] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0042] Figure 1 This is an image showing the SDS-PAGE identification results of the SLO antigen prepared according to the embodiments of the present invention.

[0043] Figure 2 This is an electrophoresis image of SLO antigen induced at 18°C ​​according to an embodiment of the present invention.

[0044] Figure 3 This is an electrophoresis image of SLO antigen induced at 25°C according to an embodiment of the present invention. Detailed Implementation

[0045] Through extensive and in-depth research, the inventors developed an SLO antigen expression system based on a prokaryotic expression system. Furthermore, through synonymous codon preference optimization, they obtained a polynucleotide sequence encoding the SLO antigen that can express the target protein in large quantities in the E. coli expression system. The expressed target protein exhibits high activity and good stability.

[0046] The inventors further optimized the SLO antigen expression conditions, including the culture medium and culture temperature, to obtain a method for highly efficient soluble expression of the SLO antigen. In a more preferred embodiment of the invention, host cells containing the polynucleotide encoding the SLO antigen of the present invention are cultured in TB medium at a temperature of 18°C.

[0047] Obtain the nucleic acid sequence related to the target gene / protein.

[0048] In this invention, the full-length nucleotide sequence or fragments of the target protein or its elements can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers are designed based on publicly available nucleotide sequences, especially open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art are used as templates to amplify the relevant sequences. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0049] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0050] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0051] The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the gene of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0052] In one embodiment of the present invention, the amino acid sequence (SEQ ID NO: 2) of the target protein is analyzed using the NCBI database to obtain the target gene sequence information. For example, the amino acid sequence of the Arthrobacter spheroidae SLO antigen is analyzed using the NCBI database to obtain the target gene sequence encoding it.

[0053] Synonymous codon preference optimization

[0054] To overcome the potential problem of reduced yield when expressing heterologous proteins in *E. coli*, this invention relates to a polynucleotide sequence optimized by synonymous codon preference. The obtained target gene sequence is optimized by synonymous codon preference. The optimized target gene sequence (SEQ ID NO:2) can express the same amino acid sequence as the target protein, but the stability and efficiency of the expression process are improved, and the final target protein maintains high activity.

[0055] The present invention also relates to polynucleotides with greater than 95% homology to the sequence shown in SEQ ID NO:2; and polynucleotides complementary to the sequence shown in SEQ ID NO:2.

[0056] Vector of the target gene

[0057] This invention also relates to vectors comprising the polynucleotides of this invention. In this invention, "vector" refers to a linear or circular DNA molecule containing a fragment encoding a target protein, said target protein being operatively 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 optional markers, enhancers, polyadenylation signals, vectors, etc. The vector fragment may be derived from a host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector may be any expression vector, either synthetic or readily manipulated with recombinant DNA, and the choice of vector generally depends on the host cell to which the vector is to be introduced. Thus, the vector may be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be a vector that integrates into the host cell genome upon introduction into a host cell and replicates along with the chromosome into which it is integrated. In one embodiment, the vector of this invention is an expression vector. In one embodiment of this invention, pET-28a(+) is selected as the vector to obtain more efficient expression.

[0058] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the protein of the present invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Exemplarily, the vector DNA molecule is cleaved into linear molecules that can be linked to a foreign gene using a DNA endonuclease, and then a codon-optimized target gene fragment is ligated into the vector. The insertion of the foreign DNA fragment can be achieved by using sticky-end ligation at a single restriction enzyme site, directed cloning of double-restricted fragments, sticky-end ligation at different restriction enzyme sites, blunt-end ligation, artificial adapter ligation, or ligation to oligonucleotide ends.

[0059] Transform host cells with a vector containing the target gene

[0060] This invention also relates to host cells genetically engineered using the vector or fusion protein coding sequence of this invention. A vector containing a codon-optimized target gene can be inserted into a host cell by known methods, transfected, or otherwise transformed to obtain a transformant containing the codon-optimized target gene of this invention and capable of expressing the target protein. In this invention, "host cell" refers to a cell incorporating exogenous polynucleotides and / or a vector. The host cell can be a eukaryotic or prokaryotic host cell, preferably a bacterium, and more preferably *Escherichia coli* Rosetta(DE3) strain.

[0061] Methods for preparing target proteins

[0062] This invention also relates to a method for preparing the target protein, which can be used to express or produce recombinant proteins using the polynucleotide sequence of this invention. Generally, the method includes the following steps:

[0063] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding the protein of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0064] (2) Host cells cultured in a suitable culture medium;

[0065] (3) Isolate and purify proteins from culture media or cells.

[0066] In step (1), the recombinant expression vector containing the polynucleotide is transformed or transduced into a suitable host cell by conventional techniques known to those skilled in the art. When the host is Escherichia coli, heat shock and electroconversion methods can be used.

[0067] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media, preferably SB, TB, or SOC media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period. In a preferred embodiment of this invention, to promote the expression of the target protein and increase the expression level of soluble proteins, host cells are cultured in TB media containing a kanamycin resistance gene.

[0068] To further promote the soluble expression of the target protein, in a preferred embodiment of the present invention, host cells are cultured to OD0.05. 600 After reaching a concentration between 0.6 and 0.8, induction was performed using IPTG, and the mixture was further cultured at 17 to 19°C for approximately 8 to 12 hours.

[0069] The proteins described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods. In one embodiment of the invention, affinity chromatography is used to molecularly target the protein.

[0070] In this invention, any exemplary or illustrative terminology (e.g., “”) used with respect to certain embodiments herein is merely for the purpose of better presenting the invention and does not limit the scope of the invention as otherwise claimed. No terminology herein should be construed as indicating an element not described in the claims that is indispensable to the implementation of this invention.

[0071] If the definition or use of a term in a cited reference is inconsistent with or inconsistent with the definition of a term described herein, the definition of the term described herein shall be used instead of the definition of the term in the cited reference.

[0072] The various terms used herein are as follows. If a term used in the claims is not defined below, the broadest definition of that term given by a person skilled in the art should be given, as reflected in the publication printed at the time of application or in the published patent.

[0073] As used herein, the term "isolated" refers to a nucleic acid or polypeptide isolated from at least one other component (e.g., a nucleic acid or polypeptide) present in its natural source. In one embodiment, the nucleic acid or polypeptide is found to be present only (if any) in a solvent, buffer, ion, or other component normally present in its solution. The terms "isolated" and "purified" do not include nucleic acids or polypeptides present in their natural source.

[0074] As used herein, the terms "polynucleotide" and "polynucleotide sequence" can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding.

[0075] This invention also relates to variants of the aforementioned polynucleotides that encode protein fragments, analogs, and derivatives having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. 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 the substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the encoded polypeptide.

[0076] As used in this article, the term "codon optimization" refers to the method of improving gene synthesis efficiency by avoiding the use of low-utilization or rare codons based on the differences in codon utilization exhibited by the actual organisms performing protein expression or production (including E. coli, yeast, mammalian blood cells, plant cells, insect cells, etc.).

[0077] As used herein, the terms “homology” and “identity” are used interchangeably and refer to the percentage of identical (i.e., same) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by arranging the nucleotide or amino acid sequences of the polynucleotide or polypeptide, scoring the number of positions in the arranged polynucleotide or polypeptide containing the same nucleotide or amino acid residues, and comparing this to the number of positions in the arranged polynucleotide or polypeptide containing different nucleotide or amino acid residues. Polynucleotides can differ at one position, for example, by containing different nucleotides (i.e., substitutions or variations) or by the deletion of nucleotides (i.e., the insertion or deletion of one or two nucleotides in the polynucleotide). Polypeptides can differ at one position, for example, by containing amino acids (i.e., substitutions or variations) or by the deletion of amino acids (i.e., the insertion of one or two amino acids in the polypeptide or the deletion of amino acids). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percentage identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

[0078] As used herein, the terms “sequence complement” and “reverse sequence complement” are used interchangeably and refer to a sequence that is in the opposite direction to the original polynucleotide sequence and is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, then its reverse complementary sequence is GTTCAT.

[0079] 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. Post-expression can be harvested, i.e., the host cell or the expressed product can be recovered.

[0080] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0081] Unless otherwise specified, the 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 pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0082] Example 1: Construction of SLO plasmid and transfection of host cells

[0083] (1) The human SLO amino acid sequence SEQ ID NO:1 was obtained and analyzed to obtain the gene sequence. Synonymous codon bias optimization was performed on the gene sequence to obtain several gene sequences optimized by synonymous codon bias. After E. coli synonymous codon bias optimization, the gene sequences were ligated into the vector pET-28a(+) to synthesize recombinant expression plasmids.

[0084] (2) Introduction of recombinant plasmids into host Escherichia coli

[0085] Take 1 μL of the expression plasmid prepared in step (1), add it to 30 μL of competent E. coli Rosetta (DE3) cells under ice bath conditions, incubate on ice for 20 min, heat shock for 90 s, immediately place on ice for 2 min, add 400 μL of antibiotic-free SOC medium, and incubate at 37°C and 220 rpm for 50 min with shaking. Take 100 μL of bacterial culture and spread it evenly on an LB agar plate containing 100 μg / mL kanamycin resistance, and incubate overnight at 37°C.

[0086] SEQ ID NO:1

[0087] MSNKKTFKKYSRVAGLLTAALIIGNLVTANAESNKQNTASTETTTTSEQPKPESSELTIEKAGQKMDDMLNSNDMIKLAP

[0088] KEMPLESAEKEEKKSEDKKKSEEDHTEEINDKIYSLNYNELEVLAKNGETIENFVPKEGVKKADKFIVIERKKKNINTTP

[0089] VDISIIDSVTDRTYPAALQLANKGFTENKPDAVVTKRNPQKIHIDLPGMGDKATVEVNDPTYANVSTAIDNLVNQWHDNY

[0090] SGGNTLPARTQYTESMVYSKSQIEAALNVNSKILDGTLGIDFKSISKGEKKVMIAAYKQIFYTVSANLPNNPADVFDKSV

[0091] TFKDLQRKGVSNEAPPLFVSNVAYGRTVFVKLETSSKSNDVEAAFSAALKGTDVKTNGKYSDILENSSFTAVVLGGDAAE

[0092] HNKVVTKDFDVIRNVIKDNATFSRKNPAYPISYTSVFLKNNKIAGVNNRTEYVETTSTEYTSGKINLSHQGAYVAQYEIL

[0093] WDEINYDDKGKEVITKRRWDNNWYSKTSPFSTVIPLGANSRNIRIMARECTGLAWEWWRKVIDERDVKLSKEINVNISGS

[0094] TLSPYGSITYK

[0095] Examples of partially optimized codons are as follows:

[0096] Optimized codon 1 (SEQ ID NO:2)

[0097] ATGTCAAATAAAAAAACATTTAAAAAGTATTCTCGTGTTGCTGGCCTGTTGACTGCGGCTTTGATCATCGGCAATTTGGT

[0098] TACTGCCAACGCGGAATCTAACAAACAGAATACCGCTAGCACGGAAACCACGACTACCTCTGAACAGCCGAAGCCGGAAT

[0099] CCAGCGAGCTGACCATTGAGAAAGCAGGTCAAAAAATGGATGATATGCTGAACAGCAACGACATGATCAAACTGGCACCG

[0100] AAAGAGATGCCGCTGGAAAGCGCCGAGAAGGAAGAGAAGAAGTCGGAGGATAAGAAAAAAAGCGAAGAAGACCACACCGA

[0101] GGAGATCAACGACAAAATCTACTCGCTGAATTACAATGAACTGGAAGTGTTGGCGAAAAACGGCGAAACGATTGAGAACT

[0102] TTGTACCGAAAGAGGGTGTTAAGAAAGCGGACAAGTTTATTGTTATCGAGCGCAAAAAGAAGAACATTAACACCACTCCG

[0103] GTTGATATCTCCATTATCGACAGCGTTACCGATCGTACGTACCCGGCTGCGCTCCAACTGGCAAATAAAGGTTTTACCGA

[0104] GAATAAGCCGGATGCGGTCGTGACGAAACGTAACCCACAGAAAATCCACATTGACCTGCCGGGTATGGGTGATAAGGCGA

[0105] CCGTGGAAGTCAACGATCCGACCTACGCGAATGTAAGCACCGCAATTGACAACTTAGTTAACCAGTGGCATGATAACTAC

[0106] TCTGGTGGCAACACCCTGCCGGCGCGTACCCAGTATACCGAAAGCATGGTTTATTCTAAATCGCAAATTGAGGCGGCGCT

[0107] GAATGTGAACTCCAAGATTCTTGATGGTACACTGGGTATTGACTTCAAAAGCATTAGCAAAGGTGAAAAAAAGGTCATGA

[0108] TCGCTGCGTACAAACAAATCTTTTATACCGTTAGCGCCAACCTCCCAAATAACCCGGCGGACGTGTTCGATAAGTCCGTG

[0109] ACCTTCAAAGACCTGCAACGTAAGGGCGTTAGCAACGAGGCGCCTCCGTTGTTTGTTTCGAACGTTGCATACGGCAGAAC

[0110] GGTGTTCGTGAAGCTGGAAACCAGCAGCAAGTCCAACGACGTGGAAGCGGCGTTTAGCGCAGCGCTTAAGGGAACCGATG

[0111] TGAAGACCAATGGTAAATATTCGGATATCTTGGAGAACTCCAGCTTCACGGCGGTGGTGCTGGGTGGTGACGCCGCGGAG

[0112] CACAACAAAGTTGTCACCAAGGACTTCGACGTCATCCGCAATGTCATTAAAGACAACGCCACCTTCTCTAGAAAGAACCC

[0113] GGCGTACCCGATTAGCTATACCTCCGTGTTCCTGAAGAACAACAAGATCGCAGGCGTTAACAACCGTACCGAGTATGTTG

[0114] AGACGACCAGCACTGAGTACACGAGCGGCAAAATCAACTTAAGCCATCAGGGTGCTTACGTTGCTCAGTATGAAATCCTG

[0115] TGGGATGAAATTAATTACGATGACAAAGGCAAAGAGGTGATCACCAAGCGCCGTTGGGACAATAATTGGTATAGCAAAAC

[0116] CTCACCGTTTAGCACCGTAATCCCGCTGGGTGCCAACTCGCGCAACATTCGTATTATGGCACGTGAGTGCACCGGTTTGG

[0117] CCTGGGAATGGTGGCGTAAAGTGATTGACGAACGCGACGTGAAGCTGTCCAAGGAGATCAATGTGAATATCTCCGGCTCC

[0118] ACGCTGTCTCCGTATGGCAGCATTACCTACAAA

[0119] Optimized codon 2 (SEQ ID NO:3)

[0120] ATGTCCAACAAGAAAACCTTCAAAAAATACAGCCGTGTTGCAGGTCTGCTGACTGCAGCACTGATCATCGGTAACCTGGT

[0121] TACCGCTAACGCAGAATCTAACAAACAGAACACCGCGAGCACTGAAACCACTACTACTTCCGAACAGCCGAAACCGGAAT

[0122] CTTCCGAACTGACCATCGAAAAGGCAGGCCAGAAGATGGACGACATGCTGAATAGCAACGACATGATTAAACTGGCACCG

[0123] AAAGAAATGCCACTGGAGTCCGCTGAAAAAGAAGAAAAAAAATCCGAAGATAAAAAGAAAAGCGAAGAAGATCATACCGA

[0124] AGAAATCAACGACAAAATTTATTCTCTGAACTATAACGAACTGGAAGTTCTGGCAAAGAACGGCGAGACCATCGAAAACT

[0125] TTGTCCCGAAAGAAGGTGTAAAGAAAGCTGACAAATTCATCGTTATCGAGCGCAAAAAAAAAAATATCAACACCACCCCG

[0126] GTCGATATTTCTATCATCGACTCTGTTACTGACCGTACCTACCCGGCGGCACTGCAGCTGGCAAACAAAGGTTTCACCGA

[0127] AAACAAACCGGATGCGGTTGTTACCAAACGTAACCCGCAGAAAATTCATATCGACCTGCCGGGCATGGGCGATAAAGCTA

[0128] CCGTTGAAGTGAACGATCCTACCTACGCGAACGTTTCTACTGCTATCGACAACCTGGTAAACCAGTGGCATGACAACTAT

[0129] TCCGGTGGTAACACGCTGCCGGCGCGTACCCAGTACACCGAATCCATGGTGTACTCCAAAAGCCAGATCGAAGCTGCGCT

[0130] GAACGTTAATAGCAAGATTCTGGACGGTACTCTGGGTATCGACTTTAAATCCATCTCCAAAGGCGAAAAAAAAGTTATGA

[0131] TTGCGGCTTACAAACAAATCTTCTATACCGTTAGCGCGAACCTGCCGAATAACCCGGCGGACGTTTTCGACAAAAGCGTT

[0132] ACCTTTAAGGACCTGCAACGTAAAGGTGTATCTAACGAAGCCCCACCGCTGTTCGTATCTAACGTGGCTTACGGCCGCAC

[0133] TGTGTTCGTGAAACTGGAAACCTCTTCCAAGAGCAACGATGTCGAAGCTGCATTCTCTGCAGCTCTGAAGGGCACTGATG

[0134] TGAAGACCAACGGTAAATACTCCGACATTCTGGAAAATTCCTCTTTCACCGCCGTTGTCCTGGGTGGTGACGCCGCAGAA

[0135] CACAACAAAGTTGTTACTAAAGATTTCGACGTTATTCGTAACGTTATCAAAGACAATGCTACCTTCTCCCGCAAAAACCC

[0136] AGCATATCCGATCTCCTACACCAGCGTGTTCCTGAAAAACAACAAAATCGCTGGCGTCAACAACCGTACCGAGTACGTAG

[0137] AAACGACCTCCACCGAATATACTTCTGGTAAAATTAACCTGTCCCACCAGGGCGCGTATGTGGCACAGTACGAAATCCTG

[0138] TGGGACGAGATTAACTATGATGATAAAGGCAAAGAAGTTATTACTAAACGTCGTTGGGACAACAATTGGTACTCCAAGAC

[0139] CTCCCCGTTCTCTACCGTGATCCCACTGGGTGCTAACTCCCGTAACATCCGTATTATGGCTCGTGAATGTACCGGTCTGG

[0140] CTTGGGAATGGTGGCGTAAAGTTATCGACGAACGTGACGTTAAGCTGTCTAAGGAGATTAATGTCAACATCTCTGGCTCT

[0141] ACCCTGTCTCCGTACGGTTCTATTACCTATAAA

[0142] Optimized codon 3 (SEQ ID NO:4)

[0143] ATGTCTAACAAAAAAACCTTCAAAAAATACTCTCGCGTAGCAGGTCTGCTGACCGCCGCACTGATCATCGGTAACCTGGT

[0144] CACCGCTAACGCTGAATCTAATAAACAGAACACGGCTTCCACGGAAACCACCACGACGTCTGAACAACCGAAACCGGAAT

[0145] CTAGCGAACTGACTATCGAAAAAGCTGGCCAAAAAATGGATGATATGCTGAACTCCAATGACATGATTAAACTGGCTCCG

[0146] AAAGAAATGCCTCTGGAATCTGCGGAAAAAGAGGAAAAAAAAAGCGAAGATAAAAAAAAAAGCGAGGAAGACCACACCGA

[0147] AGAAATCAACGATAAAATCTATTCTCTGAACTACAACGAACTGGAGGTTCTGGCTAAAAACGGTGAGACCATTGAAAACT

[0148] TCGTGCCGAAAGAAGGCGTAAAAAAAGCCGACAAATTCATCGTTATTGAACGTAAAAAAAAAAACATCAACACCACCCCG

[0149] GTGGACATCTCTATCATTGACTCTGTAACCGACCGTACCTACCCGGCAGCTCTGCAGCTGGCCAACAAGGGTTTCACCGA

[0150] AAACAAACCGGACGCTGTTGTGACCAAACGCAACCCGCAGAAAATTCACATCGACCTGCCAGGCATGGGTGATAAGGCGA

[0151] CCGTCGAAGTGAACGATCCAACTTACGCTAACGTTTCCACCGCAATTGATAACCTGGTTAACCAGTGGCACGACAACTAC

[0152] TCTGGTGGTAACACCCTGCCAGCGCGCACTCAGTACACCGAGTCTATGGTATACAGCAAATCCCAGATCGAGGCAGCGCT

[0153] GAACGTAAACTCTAAAATCCTGGATGGTACTCTGGGTATCGATTTCAAAAGCATTAGCAAAGGTGAGAAAAAAGTGATGA

[0154] TTGCGGCTTACAAACAGATCTTCTACACCGTGTCTGCTAATCTGCCGAACAACCCAGCTGATGTCTTCGACAAATCCGTA

[0155] ACGTTCAAAGATCTGCAACGTAAAGGCGTCTCTAACGAAGCTCCGCCGCTGTTCGTCAGCAACGTAGCATACGGCCGTAC

[0156] CGTGTTTGTAAAACTGGAAACTTCCTCCAAAAGCAACGACGTTGAGGCGGCTTTCTCTGCAGCACTGAAGGGTACCGATG

[0157] TTAAAACCAACGGCAAATATTCTGACATCCTGGAGAACTCTTCCTTCACTGCTGTTGTGCTGGGCGGTGATGCTGCGGAA

[0158] CACAACAAAGTGGTGACCAAAGACTTCGATGTAATCCGTAACGTGATCAAAGACAACGCTACTTTCTCTCGTAAAAACCC

[0159] GGCTTATCCGATCAGCTATACGAGCGTTTTCCTGAAGAACAACAAGATTGCAGGCGTTAACAACCGTACTGAGTACGTAG

[0160] AAACCACTTCTACCGAATACACCTCTGGTAAAATCAACCTGAGCCACCAGGGTGCTTACGTGGCACAGTACGAAATTCTG

[0161] TGGGATGAGATCAACTATGACGACAAAGGCAAAGAAGTGATTACCAAACGCCGTTGGGATAATAACTGGTATTCTAAGAC

[0162] TTCTCCGTTCAGCACTGTGATCCCGCTGGGCGCGAATTCCCGCAACATCCGCATCATGGCGCGTGAATGTACTGGCCTGG

[0163] CGTGGGAATGGTGGCGTAAAGTTATCGACGAACGCGATGTTAAACTGTCCAAAGAAATCAACGTGAATATTTCCGGTTCT

[0164] ACCCTGTCCCCGTACGGCAGCATTACCTATAAA

[0165] Example 2: Expression of the target gene

[0166] Single clones prepared in Example 1 were aseptically inoculated into TB medium containing 100 μg / mL kanamycin resistance and cultured at 37°C with shaking at 220 rpm until the OD600 reached between 0.6 and 0.8. Induction was then performed with 0.1 mM IPTG, followed by overnight shaking incubation at 18°C ​​and 37°C, respectively. Equal volumes of bacterial suspensions from the three different media were ultrasonically disrupted and analyzed by SDS-PAGE. The results are shown in [Figure showing results]. Figure 1 .

[0167] like Figure 1 As shown, columns 1-2: supernatant and precipitate from fragmented expression of optimized codon 1; columns 3-4: supernatant and precipitate from fragmented expression of optimized codon 2; columns 5-6: supernatant and precipitate from fragmented expression of optimized codon 3. The results show that optimized codon 1 resulted in a higher proportion of the target protein expressed in the supernatant, and the expression level was significantly higher than that of other optimized codons.

[0168] Example 3: Purification of the expression product

[0169] Recombinant strains constructed using optimized codon 1 were selected and cultured in TB medium at 18℃ and 25℃ for induced expression. Bacterial cells were collected and weighed, and resuspended on ice in Lysis Buffer. After sonication, the cells were centrifuged at 20,000 rpm for 30 min at 4℃. The supernatant was collected and filtered through a 0.22 μm syringe filter to obtain the stock solution. The stock solution was passed through a Ni-NTA chromatography column, and the protein eluted with 50 mM Tris-HCl, 50 mM NaCl, and 200 mM imidazole at pH 7.0 was the target protein. Electrophoresis images of the expression and purification at 18℃ and 25℃ are shown below. Figure 2 , 3 As shown, approximately 328 mg of the target protein could be purified per liter of culture medium at 18°C, while approximately 150 mg of the target protein could be purified per liter of culture medium at 25°C. This indicates that the expression level of recombinant SLO protein is higher at 18°C ​​than at 25°C in TB medium.

[0170] Example 4: Chemiluminescence method for identifying the activity of the target protein

[0171] (1) Antigen activity assay

[0172] The antigenic activity of recombinant SLO protein expressed in TB medium at 18℃ and 25℃ was detected using latex immunoturbidimetry. SLO and latex were mixed, incubated for 8 hours, and then dialyzed to remove unlinked antigen. Blocking buffer was added, followed by centrifugation to remove the supernatant, yielding latex microspheres of SLO antigen. Then, PEG-containing PB buffer and SLO monoclonal antibody (abcam) were added, and the analysis was performed at 600 nm using a Hitachi 7180 fully automated biochemical analyzer using a two-point endpoint method.

[0173] As shown in Table 1, the highest absorbance of the expressed protein at 18℃ was 3471, and the linear regression equation was y = 5.4388x + 37.314, R0. 2 =0.9979(R) 2 The linear correlation was >0.95, indicating a high degree of linearity. The highest absorbance of the protein expressed at 25℃ was 665, and the linear regression equation was: y = 1.0498x + 59.371, R0. 2=0.9283(R) 2 <0.95), indicating poor linear correlation. The above results show that within the linear assay range, the recombinant SLO protein expressed at 18℃ exhibits better detection activity and linear correlation than that expressed at 25℃. This indicates that the SLO antigen preparation method in this invention is suitable for use at a temperature below room temperature, around 18℃.

[0174] Table 1

[0175]

[0176] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. An isolated polynucleotide encoding an SLO antigen, characterized in that, The polynucleotide is codon-optimized and has the sequence shown in SEQ ID NO.

2.

2. An expression carrier, characterized in that, The expression vector comprises the polynucleotide as described in claim 1.

3. The expression vector according to claim 2, characterized in that, The expression vector is an Escherichia coli expression vector.

4. The expression vector according to claim 3, characterized in that, The expression vector is pET-28a(+).

5. A host cell, characterized in that, The host cell comprises the expression vector as described in any one of claims 2-4; or The host cell genome contains the polynucleotides as described in claim 1.

6. A method for preparing SLO antigen, characterized in that, The method includes the following steps: Transform host cells using a vector containing the polynucleotide as described in claim 1; The host cells are cultured to express the SLO antigen.

7. The method according to claim 6, characterized in that, The host cells were cultured in SB, TB, or SOC media.

8. The method according to claim 6, characterized in that, When culturing the host cells, the target protein is expressed by IPTG induction.

9. The method according to claim 6, characterized in that, When culturing the host cells, the host cells are cultured at a temperature of 16 to 19°C.

10. The method according to claim 8, characterized in that, When culturing the host cells, they were cultured until the OD600 was between 0.6 and 0.8, and then induced with IPTG to express the target protein.

11. A reagent kit, characterized in that, The kit comprises: the polynucleotide as described in claim 1; or The expression vector as described in any one of claims 2-4; or The host cell as described in claim 5.

Images