Method for the preparation and use of recombinant jo-1 antigen

Abstract

The application discloses a preparation method and application of a recombinant JO-1 antigen. In the application, a JO-1 antigen preparation method based on genetic engineering technology is developed, a polynucleotide coding the JO-1 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; the method is further optimized in culture conditions such as culture medium and culture temperature, high-proportion soluble protein expression is realized, and the obtained antigen has high activity; the JO-1 antigen prepared by the method has an activity which is significantly improved by nearly 3 times compared with that of a commercial antigen, and the light emission value detected by a chemiluminescence method can reach 9.73 million.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to the preparation method and application of recombinant JO-1 antigen. Background Technology

[0002] The Jo-1 antigen is essentially histyl-tRNA synthetase (HisRS), which catalyzes the binding of histidine to the corresponding tRNA His during protein synthesis to generate histyl-tRNA. Histyl-tRNA synthetase is an antigen of a class of autoantibodies that are hallmarks of connective tissue diseases (autoimmune diseases) such as polymyositis (PM) and dermatomyositis (DM), clinically referred to as Jo-1.

[0003] Anti-Jo-1 antibody is a marker antibody for polymyositis / dermatomyositis (PM / DM), with a positive rate of approximately 25% in PM and 7.1% in dermatomyositis (DM). In PM / DM patients with concurrent interstitial lung disease, the positive rate is as high as 60%. Because the serum of patients with polymyositis and dermatomyositis contains specific anti-Jo-1 antibodies, Jo-1 can be used for clinical differentiation and diagnosis of these two conditions.

[0004] Commonly used detection methods for anti-Jo-1 antigen include enzyme-linked immunosorbent assay (ELISA) and Western blotting. Currently, most domestically produced detection kits are imported or assembled using imported raw materials, making them expensive and significantly limiting the promotion and application of Jo-1 protein detection. Therefore, there is an urgent need to independently develop Jo-1 antigen as a detection raw material to break free from the current dependence on imports.

[0005] The Jo-1 antigen has a complex spatial structure, making in vitro recombinant expression of soluble proteins difficult. Some domestic reports on the extraction of Jo-1 antigen show that natural extraction and purification methods suffer from problems such as difficulty in sourcing, cumbersome extraction steps, and unstable quality and activity. Although recombinant Jo-1 antigen has solved some of the problems of natural purification, it still suffers from low yield, poor stability, and low product activity. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing recombinant JO-1 antigen.

[0007] Another object of the present invention is to provide a polynucleotide sequence encoding the JO-1 antigen.

[0008] Another object of the present invention is to provide a vector adapted to a polynucleotide sequence encoding the JO-1 antigen.

[0009] Another object of the present invention is to provide a kit containing a polynucleotide sequence encoding the JO-1 antigen.

[0010] To address the aforementioned technical problems, in a first aspect, the present invention provides a polynucleotide encoding the JO-1 antigen, wherein the polynucleotide is codon-optimized and selected from any of the following:

[0011] (i) A polynucleotide having the sequence shown in SEQ ID NO.2;

[0012] (ii) a polynucleotide having greater than 95% homology to the sequence shown in SEQ ID NO.2; and

[0013] (iii) A polynucleotide having a sequence complementary to the polynucleotide sequence described in (i) or (ii).

[0014] In a second aspect, the present invention provides an expression vector comprising the polynucleotide provided in the first aspect of the present invention.

[0015] In some preferred embodiments, the expression vector includes a polynucleotide sequence expressing a His×6 tag; more preferably, in the expression vector, the 5' end of the polynucleotide is linked to a polynucleotide sequence expressing a His×6 tag.

[0016] In some preferred embodiments, the expression vector is an Escherichia coli expression vector, more preferably pET-28a(+).

[0017] 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

[0018] The host cell genome integrates polynucleotides as provided in the first aspect of the present invention.

[0019] In some preferred embodiments, the host cell is *Escherichia coli*.

[0020] In some preferred embodiments, the host cell is Escherichia coli Rosetta (DE3) strain.

[0021] A fourth aspect of the present invention provides a method for preparing JO-1 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

[0022] The target protein is isolated to obtain the JO-1 antigen;

[0023] The target protein has an amino acid sequence as shown in SEQ ID NO:1.

[0024] 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.

[0025] In some preferred embodiments, the host cells are cultured in SB, TB, SOC, or LB media. In a more preferred embodiment, to obtain high levels of soluble expression, the host cells are cultured in TB media.

[0026] In some preferred embodiments, the host cells are cultured in an oscillating environment.

[0027] 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.

[0028] 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.

[0029] In some preferred embodiments, the culture medium used to culture the host cells contains a kanamycin resistance gene.

[0030] In some preferred embodiments, IPTG is used to induce the expression of the target protein when culturing the host cells.

[0031] 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.

[0032] In some preferred embodiments, the step of isolating the target protein includes:

[0033] The supernatant of the lysed target protein is passed through a chromatography column for elution, and the eluent is collected.

[0034] In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column.

[0035] A fifth aspect of the present invention provides a kit comprising: a polynucleotide as provided in the first aspect of the present invention; or

[0036] Such as the expression vector provided in the second aspect of the present invention; or

[0037] The host cell as described in the third aspect of the present invention; or

[0038] Or the JO-1 antigen prepared by the method according to the fourth aspect of the present invention.

[0039] Compared with the prior art, the present invention has at least the following advantages:

[0040] (1) This invention develops a method for preparing JO-1 antigen based on genetic engineering technology. The polynucleotide encoding JO-1 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.

[0041] (2) This invention develops a method for preparing JO-1 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.

[0042] (3) The JO-1 antigen preparation method provided in this invention only requires the connection of His×6 tag to the N-terminus, without the need to add large molecular weight MBP tag or other tags, and can express soluble protein in large quantities. There is no need to remove the tag afterward, the purification process is simple, and the purity of the target protein obtained after chromatography is as high as 95% or more.

[0043] (4) The JO-1 antigen prepared by the method provided in this invention has a significantly higher activity than commercially available antigens by nearly 3 times, and the luminescence value detected by chemiluminescence method can reach up to 9.73 million.

[0044] 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

[0045] 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.

[0046] Figure 1 This is an SDS-PAGE identification result of the JO-1 antigen prepared at 18°C ​​according to an embodiment of the present invention;

[0047] Figure 2 This is an SDS-PAGE identification result of the JO-1 antigen prepared at 37°C according to an embodiment of the present invention;

[0048] Figure 3 This is an electrophoresis diagram of the JO-1 antigen according to an embodiment of the present invention. Detailed Implementation

[0049] Through extensive and in-depth research, the inventors developed a JO-1 expression system based on a prokaryotic expression system. Furthermore, through synonymous codon preference optimization, they obtained a polynucleotide sequence encoding the JO-1 antigen that can express the target protein in large quantities in the E. coli expression system. The expressed target protein has high activity and good stability.

[0050] This invention achieves highly soluble expression without the need for adding large molecular weight MBP tags or other tags through the synergistic optimization of synonymous codon preference, culture medium and culture conditions. The purification process is simple and suitable for industrial application.

[0051] Obtain the nucleic acid sequence related to the target gene / protein.

[0052] 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 can be 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 can be 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.

[0053] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0054] 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.

[0055] 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.

[0056] In one embodiment of the present invention, the amino acid sequence (SEQ ID NO: 1) of the target protein is analyzed using the NCBI database to obtain the target gene sequence information.

[0057] Synonymous codon preference optimization

[0058] 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.

[0059] 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.

[0060] Vector of the target gene

[0061] 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.

[0062] 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.

[0063] In one embodiment of the present invention, the vector further comprises a polynucleotide sequence expressing a His×6 tag, preferably, the polynucleotide sequence expressing a His×6 tag is linked to the 5' end (N-terminus) of the target gene sequence.

[0064] Transform host cells with a vector containing the target gene

[0065] 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.

[0066] Methods for preparing target proteins

[0067] 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:

[0068] (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;

[0069] (2) Host cells cultured in a suitable culture medium;

[0070] (3) Isolate and purify proteins from culture media or cells.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.).

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] Example 1: Construction of the Jo-1 protein plasmid JO-1 and transfection of host cells

[0087] (1) Obtain the human JO-1 amino acid sequence SEQ ID NO:1, analyze it to obtain the gene sequence, and perform synonym codon preference optimization on the gene sequence to obtain the gene sequence SEQ ID NO:2. After E. coli synonym codon preference optimization, the ligation vector is pET-28a(+), with an N-terminal fusion expression (His)6 tag, and a recombinant expression plasmid is synthesized and named vector A.

[0088] Using the same method, the gene sequence encoding human JO-1 was optimized for E. coli synonymous codon bias, resulting in several optimized codon sequences, such as SEQ ID NO:3-5. These optimized codon sequences were then ligated into the pET-28a(+) vector, with an N-terminal fusion expression (His)6 tag, and recombinant expression plasmids were synthesized. The vector containing the codon shown in SEQ ID NO:3 was named vector B, the vector containing the codon shown in SEQ ID NO:4 was named vector C, and the vector containing the codon shown in SEQ ID NO:5 was named vector D.

[0089] (2) Introduction of recombinant plasmids into host Escherichia coli

[0090] 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.

[0091] SEQ ID NO:1

[0092] AERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVII

[0093] RCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNP

[0094] AMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKL

[0095] DKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFD

[0096] LSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALE

[0097] EKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSV

[0098] TSREEVDVRREDLVEEIKRRTGQPLCIC

[0099] SEQ ID NO:2

[0100] GCTGAAAGGGCGGCATTAGAAGAGCTAGTAAAACTGCAAGGTGAACGTGTTCGTGGTTTGAAGCAACAAAAAGCTTCTGC

[0101] TGAGCTGATTGAAGAGGAGGTGGCCAAACTGTTGAAACTGAAGGCGCAGCTGGGTCCGGATGAAAGCAAACAGAAATTCG

[0102] TGCTGAAGACTCCGAAGGGCACCCGTGATTACAGCCCACGTCAGATGGCGGTTCGTGAGAAGGTCTTCGACGTTATCATC

[0103] AGATGCTTTAAACGCCACGGCGCAGAGGTGATCGACACCCCTGTTTTTGAACTCAAAGAAACGCTGATGGGTAAATATGG

[0104] TGAGGATAGCAAACTTATCTACGACCTCAAGGACCAGGGTGGCGAGCTTCTGTCCCTGCGTTACGACCTGACCGTTCCGT

[0105] TTGCGCGTTACCTGGCCATGAATAAACTGACGAACATTAAACGTTACCACATTGCAAAAGTTTATCGTCGTGATAACCCG

[0106] GCGATGACCCGTGGCCGTTATCGCGAATTTTATCAGTGTGATTTCGACATCGCCGGTAATTTCGACCCGATGATTCCGGA

[0107] CGCGGAGTGCCTGAAGATCATGTGTGAAATCTTGTCTTCCTTACAGATTGGCGACTTCCTGGTGAAGGTGAACGATCGTC

[0108] GCATCCTGGATGGCATGTTCGCCATCTGCGGTGTGAGCGATAGCAAGTTCCGCACCATTTGCTCCAGCGTTGATAAATTG

[0109] GACAAGGTCTCCTGGGAAGAGGTTAAAAACGAAATGGTTGGCGAGAAGGGTCTGGCACCAGAAGTTGCGGATCGTATTGG

[0110] AGACTACGTGCAGCAGCATGGTGGTGTATCCCTGGTGGAACAATTGTTGCAAGATCCGAAACTGTCGCAAAACAAACAAG

[0111] CGCTGGAAGGTTTAGGTGATCTGAAGTTGTTATTCGAATATCTGACTCTTTTTGGTATCGATGATAAGATCAGCTTTGAC

[0112] CTGTCGCTGGCGCGTGGTCTGGACTACTATACCGGTGTTATTTATGAAGCGGTTCTGCTGCAGACCCCGGCGCAGGCGGG

[0113] CGAGGAGCCGCTGGGCGTGGGTAGCGTTGCGGCAGGCGGTCGTTACGACGGCCTGGTCGGTATGTTTGACCCGAAGGGTC

[0114] GCAAAGTGCCGTGTGTGGGCCTGTCTATTGGCGTGGAGCGCATCTTCAGCATTGTTGAGCAGCGTCTGGAGGCTCTGGAG

[0115] GAGAAAATCCGCACCACCGAAACCCAGGTTCTGGTGGCGTCTGCTCAAAAGAAATTGTTGGAGGAGCGCCTCAAGTTGGT

[0116] GAGCGAACTGTGGGATGCAGGCATAAAAGCCGAACTACTGTATAAAAAGAACCCGAAACTACTGAATCAGTTGCAATACT

[0117] GCGAAGAGGCTGGCATCCCGCTGGTCGCGATTATTGGCGAGCAAGAACTCAAGGATGGCGTCATCAAGCTGAGAAGCGTT

[0118] ACCAGCCGTGAAGAGGTTGACGTGCGCCGTGAGGACCTCGTCGAGGAGATCAAGCGCAGAACGGGTCAGCCGCTGTGCAT

[0119] TTGC

[0120] SEQ ID NO:3

[0121] GCAGAACGTGCAGCGCTGGAAGAACTGGTAAAACTGCAGGGTGAACGTGTTCGTGGCCTGAAACAGCAGAAAGCGAGCGC

[0122] GGAACTGATCGAAGAAGAGGTTGCGAAACTGCTGAAGCTGAAAGCTCAACTGGGTCCGGATGAAAGCAAACAGAAATTTG

[0123] TTCTGAAAACGCCAAAAGGTACCCGCGATTACTCTCCGCGTCAGATGGCCGTCCGTGAAAAGGTGTTTGATGTGATCATT

[0124] CGCTGCTTCAAGCGTCACGGTGCTGAAGTTATCGATACCCCTGTTTTCGAGCTGAAAGAAACCCTGATGGGTAAATACGG

[0125] TGAAGATAGCAAGCTGATCTACGACCTGAAAGACCAAGGTGGTGAACTGCTGTCCCTGCGTTACGATCTGACCGTACCTT

[0126] TCGCTCGTTACCTGGCGATGAACAAACTGACCAACATCAAACGTTACCACATCGCGAAGGTTTACCGTCGTGATAATCCA

[0127] GCAATGACCCGTGGCCGTTACCGCGAATTTTACCAGTGCGACTTCGATATCGCAGGTAACTTCGACCCGATGATCCCGGA

[0128] CGCGGAATGTCTGAAAATCATGTGCGAAATCCTGAGCTCCCTGCAGATCGGCGACTTCCTGGTAAAAGTAAACGATCGTC

[0129] GTATCCTGGACGGCATGTTTGCAATTTGTGGTGTGTCCGATTCCAAATTCCGCACTATCTGCTCTTCTGTTGATAAACTG

[0130] GACAAAGTCTCTTGGGAGGAAGTTAAAAACGAAATGGTAGGTGAAAAAGGCCTGGCCCCGGAAGTTGCTGATCGTATCGG

[0131] TGATTATGTGCAGCAGCATGGCGGCGTTAGCCTGGTTGAACAGCTGCTGCAGGATCCGAAACTGTCTCAGAACAAACAGG

[0132] CACTGGAAGGTCTGGGTGATCTGAAACTGCTGTTTGAATATCTGACCCTGTTCGGTATTGACGATAAAATCAGCTTCGAC

[0133] CTGTCCCTGGCTCGCGGTCTGGATTATTACACTGGCGTGATCTACGAAGCTGTTCTGCTGCAGACCCCGGCTCAAGCGGG

[0134] TGAAGAACCACTGGGTGTAGGTTCCGTTGCGGCGGGTGGCCGTTATGACGGTCTGGTTGGTATGTTCGATCCGAAAGGTC

[0135] GTAAGGTTCCGTGCGTTGGTCTGTCTATCGGCGTAGAGCGTATTTTTTCCATCGTGGAACAACGTTCTGGAAGCGCTGGAA

[0136] GAGAAAATCCGTACTACCGAAACCCAGGTTCTGGTCGCCTCCGCACAAAAAAAACTGCTGGAAGAACGTCTGAAGCTGGT

[0137] TTCTGAACTGTGGGACGCGGGTATTAAGGCTGAGCTGCTGTACAAGAAAAACCCGAAGCTGCTGAACCAGCTGCAGTACT

[0138] GCGAGGAAGCGGGTATCCCGCTGGTTGCAATCATCGGTGAACAGGAACTGAAAGACGGCGTTATCAAACTGCGCTCCGTT

[0139] ACGTCTCGCGAAGAAGTGGACGTTCGTCGCGAGGATCTGGTGGAAGAAATCAAACGTCGTACCGGTCAACCGCTGTGCAT

[0140] CTGT

[0141] SEQ ID NO:4

[0142] GCTGAAAGGGCGGCATTAGAAGAGCTAGTAAAGCTTCAAGGTGAACGTGTTCGTGGTCTGAAGCAACAAAAGGCCTCTGC

[0143] GGAACTGATTGAAGAGGAGGTGGCGAAACTGCTGAAGCTGAAGGCGCAGCTGGGTCCGGACGAGAGCAAACAGAAGTTCG

[0144] TGCTGAAAACCCCAAAAGGCACCCGTGACTATAGCCCGCGTCAGATGGCAGTGCGTGAGAAAGTGTTCGACGTCATCATC

[0145] CGCTGCTTTAAACGCCACGGCGCAGAAGTGATCGATACCCCGGTGTTTGAACTGAAAGAAACGTTGATGGGCAAATATGG

[0146] TGAGGACTCCAAGCTGATTTACGACCTGAAGGACCAGGGCGGTGAGCTCCTGTCTTTACGCTACGACCTGACCGTTCCAT

[0147] TTGCGCGTTACCTGGCTATGAACAAACTGACGAACATTAAGCGTTACCATATCGCGAAAGTCTATCGTCGTGATAATCCG

[0148] GCTATGACCAGGGGCAGATACCGCGAGTTCTATCAGTGCGATTTCGATATTGCGGGTAATTTTGACCCTATGATTCCGGA

[0149] TGCCGAATGCCTGAAGATCATGTGTGAAATCCTGTCCTCCCTGCAGATTGGCGACTTCCTGGTTAAAGTGAACGATCGTC

[0150] GCATTCTGGACGGCATGTTCGCGATCTGTGGTGTAAGCGATAGTAAGTTTCGTACCATTTGCAGCAGCGTTGATAAATTA

[0151] GATAAGGTCAGCTGGGAAGAGGTGAAAAACGAAATGGTTGGTGAGAAGGGCCTGGCTCCGGAAGTGGCCGATCGTATCGG

[0152] CGATTACGTGCAGCAGCACGGTGGCGTTTCCTTGGTCGAGCAGCTGTTACAAGATCCGAAGTTGTCGCAAAACAAGCAAG

[0153] CACTGGAAGGTCTGGGCGATTTGAAGCTTCTGTTTGAATATCTCACTCTGTTCGGCATTGACGACAAAATCTCCTTTGAC

[0154] CTATCGCTGGCTCGTGGTTTGGACTACTATACCGGTGTTATCTATGAAGCGGTGCTGCTGCAGACCCCGGCACAAGCGGG

[0155] TGAGGAACCGCTGGGTGTTGGTAGCGTTGCCGCAGGCGGTCGTTACGACGGCTTGGTGGGAATGTTCGACCCGAAAGGTC

[0156] GTAAAGTTCCGTGTGTTGGCCTGTCAATTGGTGTGGAGCGCATTTTCAGCATTGTGGAGCAACGTTTGGAGGCGCTGGAA

[0157] GAAAAAATCCGTACGACAGAAACCCAGGTTCTGGTGGCCAGCGCGCAGAAAAAATTGCTGGAGGAGCGCCTTAAGTTGGT

[0158] TAGCGAACTGTGGGATGCTGGCATCAAGGCGGAGCTGCTTTATAAAAAGAACCCGAAATTGTTGAATCAGCTGCAATACT

[0159] GCGAAGAGGCAGGGATTCCGCTGGTCGCGATTATCGGTGAGCAAGAACTGAAGGACGGTGTGATCAAGCTGCGTAGCGTT

[0160] ACCTCTCGTGAAGAGGTGGATGTTAGACGGGAGGACTTGGTTGAGGAGATCAAACGCCGCACTGGCCAGCCGCTCTGCAT

[0161] CTGC

[0162] SEQ ID NO:5

[0163] GCTGAACGTGCGGCACTGGAAGAACTGGTTAAACTGCAGGGTGAACGTGTGCGTGGTCTGAAACAGCAGAAAGCGTCTGC

[0164] AGAGCTGATCGAAGAAGAAGTGGCTAAACTGCTGAAACTGAAAGCACAGCTGGGTCCGGACGAATCTAAACAGAAATTCG

[0165] TTCTGAAAACCCCGAAAGGTACCCGTGACTACTCTCCGCGCCAGATGGCGGTTCGCGAAAAAGTCTTCGATGTGATCATC

[0166] CGTTGTTTCAAACGTCACGGCGCAGAGGTAATCGACACCCCGGTCTTCGAACTGAAGGAGACCCTGATGGGCAAATACGG

[0167] CGAAGACTCTAAACTGATCTATGACCTGAAGGATCAGGGTGGCGAGCTGCTGTCTCTGCGCTACGACCTGACCGTTCCAT

[0168] TCGCGCGTTATCTGGCTATGAACAAACTGACCAACATCAAACGCTACCACATCGCTAAAGTCTACCGCCGTGATAACCCG

[0169] GCTATGACTCGCGGTCGTTACCGTGAATTCTATCAATGCGACTTCGACATCGCGGGTAACTTTGATCCGATGATTCCGGA

[0170] CGCCGAATGCCTGAAAATCATGTGCGAAATCCTGTCCAGCCTGCAGATTGGCGATTTCCTGGTTAAAGTTAACGATCGTC

[0171] GTATCCTGGACGGTATGTTCGCTATTTGTGGTGTATCCGATTCTAAGTTTCGTACCATCTGTTCTAGCGTAGACAAACTG

[0172] GATAAGGTGTCCTGGGAAGAAGTGAAGAACGAAATGGTTGGCGAAAAAGGTCTGGCTCCGGAAGTTGCTGATCGTATCGG

[0173] TGATTATGTGCAGCAGCACGGTGGCGTATCTCTGGTCGAACAACTGCTGCAGGACCCAAAGCTGTCCCAGAACAAACAGG

[0174] CACTGGAGGGTCTGGGTGATCTGAAACTGCTGTTCGAGTACCTGACCCTGTTCGGCATCGACGACAAAATTTCCTTTGAC

[0175] CTGTCCCTGGCCCGTGGCCTGGATTATTACACGGGTGTCATCTACGAAGCAGTGCTGCTGCAGACCCCGGCACAAGCAGG

[0176] TGAAGAACCTCTGGGCGTGGGCTCTGTTGCAGCAGGTGGTCGTTACGATGGCCTGGTTGGTATGTTCGATCCGAAAGGTC

[0177] GCAAAGTTCCTTGTGTTGGTCTGTCTATTGGCGTTGAGCGCATCTTTTCTATCGTTGAACAGCGTCTGGAAGCGCTGGAA

[0178] GAAAAAATCCGCACTACCGAAACGCAGGTGCTGGTGGCTAGCGCCCAGAAAAAACTGCTGGAAGAACGTCTGAAACTGGT

[0179] ATCTGAACTGTGGGACGCGGGCATCAAAGCGGAACTGCTGTACAAGAAAAACCCGAAACTGCTGAACCAGCTGCAGTACT

[0180] GCGAAGAAGCTGGTATCCCACTGGTGGCCATCATTGGTGAACAGGAACTGAAAGATGGTGTCATCAAACTGCGCTCTGTA

[0181] ACGTCCCGTGAAGAAGTTGATGTTCGTCGTGAAGATCTGGTGGAAGAGATCAAACGTCGTACCGGTCAACCGCTGTGCAT

[0182] TTGT

[0183] Example 2: Expression of the target gene

[0184] Single clones prepared in Example 1 were aseptically inoculated into TB culture medium containing 100 μg / mL kanamycin 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, and the clones were incubated overnight at 18°C ​​and 37°C with shaking, respectively. Equal volumes of bacterial suspensions with different carriers were ultrasonically disrupted and analyzed by SDS-PAGE. The results are shown in [Figure showing results]. Figure 1 and Figure 2 .

[0185] like Figure 1 As shown, columns 1-2: Vector A, supernatant and precipitate from expression disruption after incubation at 18℃;

[0186] Columns 3-4: Vector B, cultured at 18℃, final expression fragmentation supernatant and precipitate;

[0187] Columns 5-6: Vector C, supernatant and precipitate from expression disruption in TB medium at 18℃;

[0188] Columns 7-8: Vector D, TB medium at 18℃, the supernatant and precipitate of the final induced expression.

[0189] like Figure 2 As shown, columns 1-2: Supernatant and precipitate of vector A expressed in TB medium at 37℃;

[0190] Columns 3-4: Carrier B, supernatant and precipitate from 37℃ incubation;

[0191] Columns 5-6: Vector C, expressed at 37℃, supernatant and precipitate after expression disruption;

[0192] 7-8 columns: Vector D, cultured at 37℃ to express the fragmented supernatant and precipitate.

[0193] The predicted molecular weight of the target protein is 59.9 kDa. Experimental results show that... Figure 1The target protein expressed in columns 1-2 of the 18℃ TB medium was mainly expressed in the supernatant and the expression level was relatively high. The target protein expressed in other vectors was mostly precipitated and had less protein in the supernatant.

[0194] Figure 1 and Figure 2 In contrast, 18°C ​​is more conducive to the expression of soluble target proteins.

[0195] Example 3: Purification of the expression product

[0196] 1.5 L of bacterial culture (carrier A) was induced and cultured in TB medium at 18℃ in a shake flask. The wet weight of the bacterial cells was 23.64 g after centrifugation. Approximately 4 g of bacterial cells were weighed and resuspended in 20 ml of Lysis Buffer on ice. After sonication and centrifugation 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 filtered bacterial culture. The filtered culture was then subjected to Ni-column affinity chromatography. The protein eluted with 50 mM Tris-HCl, 50 mM NaCl, and 200 mM imidazole at pH 7.0 was the target protein. The electrophoresis image is shown below. Figure 3 As shown.

[0197] The target protein expression level was calculated to be 199.8 mg / L in the fermentation broth, with a purity of 95%.

[0198] Example 4: Chemiluminescence method for identifying the activity of the target protein

[0199] (1) Preparation of antigen-antibody sandwich complex

[0200] The sample expressed in 18℃ TB medium (vector A) was mixed with magnetic beads coated with Jo-1 monoclonal antibody. After washing, another Jo-1 monoclonal antibody labeled with acrylonitrile salt was added and reacted under incubation conditions. The antibody captured the antigen in the sample, forming an antigen-antibody sandwich complex. A commercially available natural Jo-1 antigen was used as a positive control to compare the detection activity and linearity of the antigen.

[0201] (2) Detection reading

[0202] After incubation, a magnetic field is applied to precipitate the material. The supernatant is removed, and the precipitated complex is washed with a washing solution. The waste liquid is then dried to remove any substances not bound to the magnetic particles. The reaction vessel is then transferred to the measurement chamber. The instrument automatically pumps in two excitation solutions, causing the complex to generate a chemiluminescent signal, and the luminescence intensity is measured.

[0203] The Jo-1 antigen expressed and purified in TB medium at 18℃ was detected by chemiluminescence immunoassay, and the results are recorded in Table 1.

[0204] According to Table 1, the linear regression equation for the Jo-1 antigen prepared in this invention is: y = 2E + 0.8x + 410511, R 2=0.9906; The linear regression equation for commercially available natural Jo-1 is: y = 5E + 0.7x - 16701, R 2 =0.9948, both have good linearity (R²). 2 >0.99).

[0205] The Jo-1 antigen prepared by this invention can reach a maximum luminescence value of 9.73 million, while the highest luminescence value of natural Jo-1 can reach 3.61 million, indicating that the Jo-1 antigen recombinantly expressed by this invention has significantly better activity than commercially available antigens.

[0206] Table 1

[0207]

[0208] 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 the JO-1 antigen, characterized in that, The polynucleotide is codon-optimized and is a polynucleotide with 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 includes a polynucleotide sequence expressing a His×6 tag.

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

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

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

7. A method for preparing JO-1 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 JO-1 antigen.

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

9. The method according to claim 7, characterized in that, The host cells were cultured at a temperature of 16 to 19°C.

10. The method according to claim 7, characterized in that, When culturing the host cells, the target protein is expressed by IPTG induction.

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 to 5; or The host cell as described in claim 6.

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