Preparation method of mouse interleukin 23 heterodimer

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a preparation method of a mouse interleukin 23 heterodimer, which comprises the following steps: S1, constructing a double-vector expression system which is composed of a first expression vector and a second expression vector, the first expression vector comprises a first purification tag, a first flexible connecting peptide and a p19 gene, and the second expression vector comprises a second purification tag; the second expression vector comprises a second purification tag, a second flexible connecting peptide and a p40 gene, and the first purification tag and the second purification tag are different; and S2, co-transforming the dual-vector expression system into eukaryotic host cells, culturing, collecting a cell culture solution, and carrying out affinity purification to obtain the mouse interleukin 23 heterodimer. A p19 subunit and a p40 subunit are expressed through a double-vector expression system respectively, different purification tags are arranged at two N ends, the subunits and the purification tags are connected through flexible connecting peptides, and high-purity and high-activity mouse IL-23 can be obtained through two-step affinity purification.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and in particular to a method for preparing mouse interleukin 23 heterodimer. Background Art

[0002] Interleukin-23 (IL-23 or IL23 for short) is a heterodimeric cytokine. IL-23 is composed of IL-23A (p19) subunit and IL-12B (p40) subunit connected by disulfide bonds to form an active complex. Among them, the p19 subunit is unique to IL23, and the p40 subunit is shared with IL-12. The subunits do not have biological functions when they exist alone, and they can only exert biological functions when they are connected to form dimers.

[0003] IL-23 mainly exerts its biological functions by binding to specific receptors. The IL-23 receptor (IL-23R) is composed of two subunits, IL-12Rβ1 and IL-23R. p19 and p40 interact with IL-23R and IL-12Rβ1, respectively. The activation of these receptors further activates the JAK / STAT signaling pathway, including JAK2 and TYK2, which are associated with the intracellular domains of the receptor subunits and initiate specific signal associations to regulate the activity of immune cells and inflammatory responses. IL-23 plays an important role in both innate immunity and adaptive immunity. It mainly acts on memory CD4+T cells, promotes their proliferation, and induces the production of interferon-γ to enhance immune responses. IL23 can also activate CD4 + Th17 cells have pathogenic functions and stimulate other effector cells, including γδT cells, NKTs, "natural" Th17 cells, Tc17 cells, MAITs and ILC3s, which together produce cytokines such as IL-17A, IL-17F, IL-22, GM-CSF, IL-6 and TNF-α, which play an important role in the pathogenesis of inflammatory diseases, autoimmune diseases, as well as anti-tumor and anti-infection. IL-23's regulation of autoreactive Th17 cells plays a key role in the occurrence and development of chronic autoimmune diseases and is involved in many immune-mediated inflammatory diseases, including psoriasis, psoriatic arthritis and inflammatory bowel disease. IL-23 has also been shown to have pro-inflammatory activity that promotes tumors, and blockade of IL-23 can make tumors susceptible to infiltration of IL-12-induced cytotoxic T cells.

[0004] In vitro recombinant expression is a common method for obtaining a large amount of IL-23 heterodimeric protein. In the prior art, most recombinant heterodimeric IL23 proteins are constructed with a single tag on one subunit, transformed into an expression host for expression, and then purified by a series of purification methods such as affinity purification, ion exchange, and molecular sieves to obtain heterodimeric IL23 proteins. This process is usually time-consuming and labor-intensive, and the removal effect of homodimers and monomers cannot be guaranteed, thereby affecting the activity and purity of the final recombinant protein. Therefore, it is of great significance to optimize the in vitro expression method of IL-23 to effectively form heterodimers and obtain IL-23 proteins with high purity and good biological activity. Summary of the invention

[0005] In view of the problems of low expression amount and / or poor biological activity of interleukin 23 (IL-23) heterodimers in the prior art, the present invention provides a method for preparing mouse IL-23 heterodimers based on a dual-vector expression system, wherein the p19 and p40 subunits of IL-23 are respectively expressed by the dual-vector expression system, and different purification tags are set at the N-termini of the two subunits, and the subunits and the purification tags are connected by a flexible connecting peptide, and introduced into eukaryotic host cells for expression, and high-purity and high-activity recombinant heterodimeric mouse IL-23 can be obtained through two-step affinity purification, which greatly reduces the purification steps, saves the time cost and material cost of purification, and provides a reliable way for the efficient preparation of high-purity and high-activity IL-23 protein.

[0006] The present invention is specifically implemented through the following technical solutions:

[0007] The present invention provides a method for preparing a mouse interleukin-23 heterodimer, comprising the following steps:

[0008] S1. Constructing a dual-vector expression system, the dual-vector expression system is composed of a first expression vector and a second expression vector, the first expression vector comprises a p19 fusion gene, the p19 fusion gene comprises a first purification tag, a first flexible connecting peptide and a p19 gene from upstream to downstream, the second expression vector comprises a p40 fusion gene, the p40 fusion gene comprises a second purification tag, a second flexible connecting peptide and a p40 gene from upstream to downstream, and the first purification tag and the second purification tag are different;

[0009] S2. Co-transform eukaryotic host cells with the dual vector expression system, collect the cell culture fluid after cultivation, and sequentially perform affinity purification through the affinity chromatography matrix of the first purification tag and the second purification tag to obtain mouse interleukin 23 heterodimer.

[0010] Furthermore, the first purification tag and the second purification tag are selected from one of 6×His, 8×His, Trx, Flag, strep, HA, GFP, cMyc, and mFC.

[0011] Furthermore, the first purification tag is a 6×His tag, and the second purification tag is a strep tag.

[0012] Furthermore, the affinity purification comprises the following steps: S21, centrifuging the cell culture fluid, taking the supernatant and mixing it with the balanced Strep tag affinity chromatography matrix, and incubating it at 15-20rpm and 4°C for 3h; S22, flowing through the cell culture fluid, and eluting the Strep tag affinity chromatography matrix with 10-15 column volumes of a first binding buffer, wherein the first binding buffer (pH7.4±0.1) is PBS; S23, eluting the Strep tag affinity chromatography matrix with a first elution buffer, and collecting the first eluate, wherein the first elution buffer (pH7.4±0.1) is a 5mM D-biotin PBS; S24, mixing the first eluent with the balanced His tag affinity chromatography matrix, incubating at 15-20rpm and 4°C for 3h; S25, flowing through the first eluent, eluting the His tag affinity chromatography matrix with 10-15 column volumes of a second binding buffer, the formula of the second binding buffer (pH8.0±0.1) is: 250mM NaCl, 20mM Tris, 10mM imidazole and 10% glycerol; S26, eluting the His tag affinity chromatography matrix with a second elution buffer (pH8.0±0.1), collecting the second eluent, the formula of the second elution buffer is: 250mM NaCl, 20mM Tris, 10mM-500mM imidazole and 10% glycerol.

[0013] Furthermore, the first flexible connecting peptide and the second flexible connecting peptide are selected from one or more of (GGGGS)n, (GGGS)n, (GGGS)nGGSA, wherein n=2, 3, 4, 5 or 6.

[0014] Furthermore, the first flexible connecting peptide is (GGGS)3, and the second flexible connecting peptide is (GGGS)2GGSA.

[0015] Furthermore, the nucleic acid sequence of the p19 fusion gene is shown as SEQ ID NO.2, and / or the nucleic acid sequence of the p40 fusion gene is shown as SEQ ID NO.4.

[0016] Furthermore, the original vectors of the first expression vector and the second expression vector are eukaryotic expression vectors pCDNA3.1.

[0017] Furthermore, constructing the first expression vector and the second expression vector includes the following steps: synthesizing the p19 fusion gene and the p40 fusion gene by total gene synthesis, and inserting the p19 fusion gene and the p40 fusion gene into the downstream of the signal peptide of the vector pCDNA3.1 by homologous recombination, respectively, to obtain the first expression vector and the second expression vector.

[0018] Furthermore, the eukaryotic host cell is a HEK293F cell. After the first expression vector and the second expression vector are co-transfected into the HEK293F cell, the HEK293F cell is cultured at 37°C, 8% CO2, 95% humidity and 125 rpm for 16-22 hours, and then 293-ProFeed with a volume ratio of 5% and glucose with a final concentration of 4 g / L are added to the cell culture medium, and the culture is continued until the cell viability is less than 70%, and the cell culture medium is harvested.

[0019] The advantages and positive effects of the present invention are:

[0020] The present invention expresses the p19 and p40 subunits of interleukin 23 respectively through a dual-vector expression system, and sets different purification tags at the N-termini of the p19 and p40 subunits, and connects the subunit protein and the purification tag through a flexible connecting peptide to ensure the structure and function of the purification tag. After the dual-vector expression system of the present invention is introduced into eukaryotic host cells for expression, it can be secreted in the cell culture fluid to form an active heterodimer, and a high-purity, high-activity recombinant heterodimer mouse IL-23 can be obtained through two-step affinity purification, which greatly reduces the purification steps, saves the time cost and material cost of purification, and provides a reliable way for the efficient preparation of high-purity, high-activity IL-23 protein. According to testing, the expression amount of the IL-23 heterodimer of the present invention can reach 1.05 mg / 100 mL cell culture fluid, the protein purity can reach more than 95%, and the obtained IL-23 heterodimer can effectively stimulate spleen cells to secrete IL-17A, and has good biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 This is a polyacrylamide gel electrophoresis diagram of the IL-23 heterodimer expressed and purified in Example 1 of the present invention;

[0023] Figure 2 This is a polyacrylamide gel electrophoresis diagram of the IL-23 heterodimer expressed and purified in Comparative Example 1 of the present invention;

[0024] Figure 3 This is a polyacrylamide gel electrophoresis diagram of the IL-23 heterodimer expressed and purified in Comparative Example 2 of the present invention;

[0025] Figure 4 This is a curve diagram of the effect example of the present invention, which shows that mouse spleen cells are induced to secrete IL-17A after being treated with different concentrations of IL-23 heterodimers. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0028] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained according to the reported significant figures and by conventional rounding methods.

[0029] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those of ordinary skill in the art.

[0030] The meaning of the terms "include", "comprising", "containing", "having" and the like is non-restrictive, i.e., other steps and other ingredients that do not affect the result may be added. The term "and / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" will be regarded as including the following situations: (i) A, (ii) B, and (iii) A and B. The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or indicating or implying relative order.

[0031] The term "gene" is the entire nucleotide sequence required to produce a polypeptide chain or functional RNA. Therefore, gene expression includes transcription and the stable accumulation of coding RNA (mRNA) or functional RNA derived from the gene, and can also refer to the translation of mRNA into polypeptide or protein.

[0032] The term "heterologous" or "foreign" refers to a source different from the native (original) organism, such as an organism from another species. "Heterologous gene" or "foreign gene" refers to a gene that does not naturally exist in the host organism and is introduced into the host organism by gene transfer, such as the p19 gene and the p40 gene in the present invention that do not naturally exist in E. coli and HEK293F cells.

[0033] The terms "chimeric gene" or "fusion gene" have the same meaning and refer to any gene that is not a native gene and contains regulatory sequences and coding sequences that do not exist together in nature. Thus, a fusion gene may include regulatory sequences and coding sequences that originate from different organisms, or regulatory sequences and coding sequences that originate from the same organism but are arranged in a manner different from that found in nature.

[0034] The term "chimeric protein" or "fusion protein" is used in its conventional sense to refer to a protein composed of multiple polypeptides that are not associated in their natural state. A fusion protein can be a combination of two, three or more different proteins, including a combination of polypeptides derived from different organisms, or a combination of polypeptides derived from the same organism but arranged in a manner different from that found in nature.

[0035] The term "vector" refers to a self-replicating DNA molecule that transfers an exogenous target gene into a host organism, and is often in the form of a circular double-stranded DNA molecule. A vector containing an exogenous gene is a recombinant vector. Typical vectors include plasmids, viruses, bacteriophages, cosmids, and minichromosomes. Plasmids are the most common form of vectors, and therefore, in the context of the present invention, plasmids and vectors can be used interchangeably.

[0036] The term "expression vector" allows the exogenous target gene inserted into the vector to be expressed in a host organism, and contains regulatory elements expressed in a specified host organism, such as a promoter and / or an enhancer. The expression vector is introduced into a suitable host organism to enable it to express the inserted exogenous target gene (such as the p19 fusion gene and the p40 fusion gene of the present invention). This is a well-known technology in the art and will not be described in detail here.

[0037] The term "introduction" refers to the transfer of foreign genes into a host organism, such as a cell, resulting in stable genetic inheritance. The introduced gene can be in the form of a plasmid retained in the host organism, or it can be integrated into the host organism genome. Host organisms containing introduced genes are called "transgenic" or "recombinant" or "transformed" organisms or "engineered" organisms. The introduction of expression vectors into host organisms can be carried out using transformation or transfection techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA are harvested after the exponential growth phase and treated with CaCl2 or MgCl2; transformation methods such as microinjection, electroporation or liposome packaging can also be used. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging or gene gun bombardment can be used to achieve gene introduction.

[0038] The term "operably linked" refers to the connection of multiple nucleic acid fragments in a functional relationship. When a nucleic acid forms a functional relationship with another nucleic acid sequence, it is "operably linked". For example, if a promoter or other transcription regulatory sequence affects the transcription of a coding sequence (or gene), it is operably linked to a coding sequence, and the promoter can be connected in a way that induces transcription of the gene. Operably linked means that the nucleotide sequences being linked may be continuous or discontinuous.

[0039] Unless otherwise indicated, similar terms such as "nucleotide", "nucleic acid", "nucleic acid molecule" and "nucleic acid fragment" are interchangeable in the context of the present invention. The terms "peptide", "polypeptide" and "protein" are interchangeable in the context of the present invention. The term "sequence" can refer to polynucleotides, nucleic acids, nucleic acid molecules, peptides, polypeptides and proteins, depending on the context used. The terms "gene", "polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid molecule" used in the present invention refer to polymeric forms of nucleotides of any length, which can be ribonucleotides or deoxyribonucleotides, and the term only refers to the primary structure of the molecule.

[0040] The terms "IL-23", "IL-23 heterodimer", "IL-23 protein", "IL-23 protein complex" or "p19 / p40 heterodimer" and similar expressions have the same meaning and are used interchangeably, referring to a protein complex in the form of a heterodimer formed by IL-23A subunit (also known as p19 subunit) and IL-12B subunit (also known as p40 subunit) through disulfide bonds.

[0041] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0042] The embodiment of the present invention provides a method for preparing a mouse interleukin 23 (IL-23) heterodimer, comprising the following steps:

[0043] S1. Constructing a dual-vector expression system, the dual-vector expression system is composed of a first expression vector and a second expression vector, the first expression vector comprises a p19 fusion gene, the p19 fusion gene comprises a first purification tag, a first flexible connecting peptide and a p19 gene from upstream to downstream, the second expression vector comprises a p40 fusion gene, the p40 fusion gene comprises a second purification tag, a second flexible connecting peptide and a p40 gene from upstream to downstream, and the first purification tag and the second purification tag are different;

[0044] S2. Co-transform eukaryotic host cells with the dual vector expression system, collect the cell culture fluid after cultivation, and sequentially perform affinity purification through the affinity chromatography matrix of the first purification tag and the second purification tag to obtain mouse interleukin 23 heterodimer.

[0045] Interleukin 23 (IL-23) is a heterodimeric cytokine, which is formed by connecting its unique p19 subunit and the p40 subunit shared with IL-12 through a disulfide bond. The present invention expresses the p19 subunit and the p40 subunit respectively through a dual expression vector, and connects different purification tags to the N-termini of the two subunits, that is, a first purification tag is set upstream of the p19 subunit gene, and a second purification tag is set upstream of the p40 subunit gene, and the first purification tag and the second purification tag are different. By setting different purification tags, affinity purification can be performed by the first purification tag and the second purification tag in sequence, thereby effectively separating the IL-23 heterodimer from the cell culture medium. Specifically, first, purification is performed by the first purification tag, thereby separating the p19 subunit protein with the first purification tag and the IL-23 heterodimer, and then purification is performed by the second purification tag, and the IL-23 heterodimer is further separated to obtain a high-purity, high-concentration heterodimer. Those skilled in the art will appreciate that the second purification tag may be used for purification first, and then the first purification tag may be used for purification.

[0046] However, the present invention has found in experiments that when a purification tag is directly added, due to the influence of protein folding and heterodimer formation, the purification tag is usually unable to be effectively displayed on the outside of the protein, resulting in the inability to efficiently bind to the affinity chromatography matrix, thereby affecting the purification of the target protein, and the target protein obtained is usually low in yield and poor in biological activity. In order to facilitate the full display of the purification tag, the present invention adds a flexible connecting peptide between the purification tag and the subunit, that is, a first flexible connecting peptide is set between the p19 subunit gene and the first purification tag, and a second flexible connecting peptide is set between the p40 subunit gene and the second purification tag. The first flexible connecting peptide and the second flexible connecting peptide can be the same or different. The p19, p40 subunits and the tag protein are connected and fused by a flexible connecting peptide, avoiding the interference of the p19, p40 subunit domain folding on the tag structure, which is conducive to the complete exposure of the purification tag, and then can be directly subjected to two-step affinity purification after expression in eukaryotic host cells such as HEK293F cells to obtain high-purity recombinant heterodimer mouse IL23, which greatly reduces the purification steps and saves the time cost and material cost of purification. In addition, the expression of the protein is increased by adding a flexible connecting peptide, which is beneficial to promote the formation of active heterodimers, and is beneficial to the protein maintaining its natural conformation, thereby improving the biological activity of the IL-23 heterodimer. The dual-vector expression system of the present invention is transfected into eukaryotic host cells for expression. After detection, the expression of the heterodimer can reach 1.05 mg / 100 mL of cell culture fluid, and the protein purity can reach more than 95%. Moreover, the in vitro stimulation of spleen cells to secrete IL-17A experiments verifies that it has good biological activity, providing a reliable way for the efficient preparation of high-purity, high-activity IL-23 protein.

[0047] The purification tag is selected from one or more of 6×His, 8×His, Trx, Flag, strep, HA, GFP, cMyc, and mFC. Those skilled in the art can set it specifically according to the purification requirements to adapt to different purification systems and equipment, and the present invention is not limited to this.

[0048] In a specific embodiment, the first purification tag is a 6×His tag, and the second purification tag is a strep tag. Through the above settings, the N-terminus of the secreted p19 subunit protein carries a 6×His tag, and the N-terminus of the p40 subunit protein carries a strep tag, thereby obtaining the target protein IL-23 complex through His tag affinity purification and strep tag affinity purification.

[0049] The flexible connecting peptide described above is selected from one or more of (GGGGS)n, (GGGS)n, (GGGS)nGGSA, n=2-6, for example, (GGGGS)3, (GGGGS)4, (GGGS)3, (GGGS)4, (GGGS)2GGSA. Those skilled in the art can set the first flexible connecting peptide and the second flexible connecting peptide according to needs.

[0050] In a specific embodiment, the first flexible connecting peptide is (GGGS)3, and the second flexible connecting peptide is (GGGS)2GGSA; in this case, the strep tag at the N-terminus of the p40 subunit protein can be set to a double strep tag, which contains the (GGGS)2GGSA sequence, thereby eliminating the need to repeatedly set the second flexible connecting peptide.

[0051] When the first purification tag is a 6×His tag and the first flexible connecting peptide is (GGGS)3, the nucleic acid sequence of the p19 fusion gene is shown in SEQ ID NO.2; when the second purification tag is a double strep tag and the second flexible connecting peptide is (GGGS)2GGSA, the nucleic acid sequence of the p40 fusion gene is shown in SEQ ID NO.4.

[0052] Optionally, the original vectors of the first expression vector and the second expression vector are eukaryotic expression vector pCDNA3.1. Selecting the same original vector is conducive to maintaining substantially the same expression rate of the p19 and p40 subunits on different vectors, thereby promoting the formation of heterodimers.

[0053] Specifically, constructing the first expression vector and the second expression vector includes the following steps: synthesizing the p19 fusion gene (e.g., SEQ ID NO.2) and the p40 fusion gene (e.g., SEQ ID NO.4) by whole gene synthesis, inserting the aforementioned target genes into the space downstream of the signal peptide of the vector pCDNA3.1 by homologous recombination, and then transforming them into competent Escherichia coli cells, performing positive clone screening, and obtaining the first expression vector and the second expression vector.

[0054] The eukaryotic host cell is selected according to the type of expression vector, and the present invention preferably uses HEK293F cells. After obtaining the HEK293F cells co-transfected with the first expression vector and the second expression vector as described above, they are cultured under suitable conditions, for example, at 37°C, 8% CO2, 95% humidity and 125 rpm for 16-22 hours, and then 293-ProFeed with a volume ratio of 5% and glucose with a final concentration of 4 g / L are added to the cell culture fluid to induce the co-expression of p19 and p40 subunits and form heterodimers. When the cell viability is less than 70%, the cell culture fluid is harvested, and then affinity purification is performed in sequence through the purification tags contained in each subunit protein to obtain high-purity and high-concentration IL-23 heterodimers.

[0055] When the first purification tag is a 6×His tag and the second purification tag is a Strep tag, purification is first performed using a Strep tag affinity chromatography matrix, and then purification is performed using a His tag affinity chromatography matrix, specifically comprising the following steps: S21, centrifuging the cell culture fluid, taking the supernatant and mixing it with the balanced Strep tag affinity chromatography matrix, and incubating at 15-20 rpm and 4° C. for 3 h; S22, flowing through the cell culture fluid, and eluting the Strep tag affinity chromatography matrix with 10-15 column volumes of the first binding buffer; S23, eluting with the first elution buffer The Strep tag affinity chromatography matrix is ​​mixed with the first eluate; S24, the first eluate is mixed with the balanced His tag affinity chromatography matrix, and incubated at 15-20rpm and 4°C for 3h; S25, the first eluate is passed through, and the His tag affinity chromatography matrix is ​​eluted with 10-15 column volumes of the second binding buffer; S26, the His tag affinity chromatography matrix is ​​eluted with the second elution buffer, and the second eluate is collected; wherein the first binding buffer (pH7.4±0.1) is PBS, and the first elution buffer (pH7.4±0.1) is a 5mM The formula of D-biotin in PBS (pH7.4) is as follows: 250 mM NaCl, 20 mM Tris, 10 mM imidazole and 10% glycerol; the formula of the second elution buffer (pH8.0±0.1) is as follows: 250 mM NaCl, 20 mM Tris, 10 mM-500 mM imidazole and 10% glycerol.

[0056] It should be noted that in the context of the present invention, upstream refers to the 5' end of the gene or the N-terminus of the protein, and downstream refers to the 3' end of the gene or the C-terminus of the protein, and from upstream to downstream is from 5' end to 3' end or N-terminus to C-terminus.

[0057] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or usually under the conditions recommended by the manufacturer.

[0058] Example 1

[0059] 1. Expression vector construction

[0060] IL-23 (interleukin 23, IL23) is a heterodimeric cytokine composed of the IL-23A subunit (also known as the p19 subunit, unique to IL-23) and the IL-12B subunit (also known as the p40 subunit, shared with IL-12) linked by disulfide bonds. In the embodiment of the present invention, a (GGGS) 3 flexible connecting peptide and a 6×His purification tag (6×His-(GGGS) 3, the amino acid sequence from the N-C terminus is: HHHHHHGGGSGGGSGGGS) are added to the N-terminus of the IL-23A (p19) subunit gene of mouse IL23 to form a p19 fusion gene, and the p19 fusion gene is inserted into the expression vector pCDNA3.1 to construct a first expression vector named pCDNA3.1-6×His-(GGGS) 3-p19; a double strep purification tag (strep-(GGGS) 2GGSA-strep, the amino acid sequence from the N-C terminus is: WSHPQFEKGGGSGGGSGGSAWSHPQFEK) to form a p40 fusion gene, and the p40 fusion gene is inserted into the expression vector pCDNA3.1 to construct a second expression vector, named pCDNA3.1-strep-(GGGS)2GGSA-strep-p40; then the first expression vector and the second expression vector are co-transformed into eukaryotic host cells, and after culture, the expressed p19 fusion protein and p40 fusion protein carry a 6×His purification tag and a strep purification tag, respectively, and the heterodimer formed after the two are combined will have a double tag. Therefore, the IL23 heterodimer with high purity and good biological activity can be obtained by two affinity purifications using the His and strep double tags.

[0061] When His and Strep purification tags are added to the N-terminus, the present invention connects them to the target proteins p21 and p40 respectively through a flexible connecting peptide ((GGGS)3 or (GGGS)2GGSA), which can solve the problem that when the purification tag is directly connected to the N-terminus of the target protein, the tag cannot be effectively displayed and cannot be combined with the corresponding affinity chromatography filler. In addition, the present invention experimentally verifies that the expression amount of the heterodimer can be increased by adding a flexible connecting peptide.

[0062] The expression vector construction process of this embodiment specifically includes the following steps:

[0063] (1) The p19 fusion gene (see SEQ ID NO. 2) and the p40 fusion gene (see SEQ ID NO. 4) were synthesized by whole gene synthesis. The gene sequences were synthesized by General Biotechnology (Anhui) Co., Ltd. (referred to as "Anhui General"). The relevant sequences are shown in Table 1.

[0064] Table 1 p19 fusion gene and p40 fusion gene and their protein sequence information

[0065]

[0066]

[0067] (2) The synthesized p19 fusion gene and p40 fusion gene were respectively inserted into the expression vector pCDNA3.1, with the insertion site being downstream of the signal peptide of the vector itself, to construct the recombinant expression vectors pCDNA3.1-6×His-(GGGS)3-p19 and pCDNA3.1-strep-(GGGS)2GGSA-strep-p40.

[0068] (3) Add 1 μL of the recombinant expression vectors pCDNA3.1-6×His-(GGGS)3-p19 and pCDNA3.1-strep-(GGGS)2GGSA-strep-p40 obtained in step (2) to 50 μL of pre-cooled DH5ɑ competent cells (from Wuhan Abotek, catalog number CD001), heat shock at 42°C for 90 seconds, and then place in an ice bath for 5 minutes. Add 500 μL of anti-antibody LB medium to the transformation system and resuscitate at 37°C and 220 rpm for 45 minutes. After recovery, centrifuge at 6000 rpm for 1 minute, collect the cells, add PBS to resuspend, and apply the bacterial solution to an LB plate containing ampicillin resistance. Single colonies of positive clones are obtained through resistance screening.

[0069] (4) A single colony was picked and cultured in LB medium overnight. Endotoxin-free plasmid mini-extraction kit (purchased from Beijing Tiangen Biotechnology, catalog number DP118-02) was used to extract the endotoxin-free recombinant expression vectors pCDNA3.1-6×His-(GGGS)3-p19 and pCDNA3.1-strep-(GGGS)2GGSA-strep-p40. The plasmid concentration was 1000 ng / μL.

[0070] 2. Transient transfection of expression vector into eukaryotic host cells

[0071] The prepared endotoxin-free recombinant expression vectors pCDNA3.1-6×His-(GGGS)3-p19 and pCDNA3.1-strep-(GGGS)2GGSA-strep-p40 plasmids were co-transfected into HEK293F cells. The specific process was as follows: (1) Before transfection, HEK293 cells were subcultured until the cell density reached about 3-4×10 6 viable cells / mL, and the cell viability was ≥95%; (2) One day before transfection (day -1), the cells in step (1) were cultured at 1.5-1.8×10 6 (3) On the day of transfection (day 0), data including cell density and viability were obtained. Before transfection, the density of HEK293 cells was adjusted to 3.0×10 6 / mL, the cell volume is 500mL, and the cell viability of the transfected cells is ≥95%; (4) 500μg of recombinant plasmid DNA is added to 25mL Opti-MEM medium, and 1.25mL PEI MAX solution (pH 7.1, purchased from polysciences, trade name PEI MAX-Transfection Grade Linear PolyethylenimineHydrochloride (MW 40000), product number 24765-1) is added to 25mL Opti-MEM medium (purchased from: Aupuma, catalog number: 81075-001), and then the DNA solution and the PEI solution are mixed to obtain a DNA / PEI mixed solution; (5) the DNA / PEI mixed solution of step (4) is added to the HEK293 cell solution of step (3), mixed thoroughly, and incubated at room temperature for about 20 minutes to perform plasmid transfection; (6) the transfected HEK293 cells are placed in a constant temperature shaker at 37°C, 8% CO2, and 95% humidity, and the shaker speed is 125 rpm; (7) 16-22 hours after transfection, 5% (v / v) 293-ProFeed (supplement) (purchased from: Aupuma, catalog number: F081918-001) and a final concentration of 4 g / L glucose are added to the cell culture medium to induce transient expression of the target gene on the recombinant expression vector, and the cells are harvested when the cell viability is less than 70%.

[0072] 3. IL23 Heterodimer Affinity Purification

[0073] First, Strep tag affinity chromatography matrix was used for purification, and the steps were as follows: (1) the harvested cell culture fluid was centrifuged at 3500rpm for 20min, the supernatant was collected, and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) was added to the final concentration of 0.1mM; (2) a sterilized 15mL purification column tube was taken out, placed in a purification stand, and the column tube was rinsed with endotoxin-free water for 3-5 times; (3) an endotoxin-free Pasteur pipette was used to draw Strep tag affinity chromatography matrix (purchased from Changzhou Tiandi Renhe, catalog number SA092100) and added to the column tube. After the protective solution in the matrix flowed out, the matrix was washed with 10-15 column volumes of endotoxin-free water; (4) the binding buffer (Binding (5) Add the balanced matrix to a 50 mL centrifuge tube containing cell culture supernatant and seal it. Place the 50 mL centrifuge tube on a rotating incubator and incubate at 15-20 rpm and 4°C for 3 h to allow the protein with the Strep tag to bind to the matrix. (6) Perform a flow-through operation on the turbid liquid in step (5) in a purification column to retain the matrix bound to the target protein in the column. (7) Add 10-15 column volumes of Binding Buffer to the column to wash off the impurities with weaker binding ability on the matrix. (8) Add elution buffer. Buffer) was used to wash the column, and the eluate was collected in a 5 mL endotoxin-free centrifuge tube. Each column volume was collected in one centrifuge tube, and the protein concentration in the eluate was verified by polyacrylamide gel electrophoresis (SDS-PAGE); (9) The eluate containing the protein sample in step (8) was collected, and imidazole was added to a final concentration of 10 mM. Wherein, the Binding Buffer was PBS (pH 7.4), and the Elution Buffer was PBS (pH 7.4) containing 5 mM D-biotin.

[0074] Then, His-tag affinity chromatography matrix is ​​used for purification, and the steps are as follows: (10) Take out the sterilized 15 mL purification column tube, place it in the purification stand, and rinse the column tube with endotoxin-free water for 3-5 times; (11) Use an endotoxin-free Pasteur pipette to draw the His-tag affinity chromatography matrix into the column tube, and after the protective solution in the matrix flows out, wash the matrix with 10-15 column volumes of endotoxin-free water; (12) Equilibrate the matrix with binding buffer, and use an endotoxin-free Pasteur pipette to draw 6-10 column volumes of binding buffer. Buffer is added to the column tube containing the matrix; (13) the balanced matrix is ​​added to the 50 mL centrifuge tube containing the protein elution solution of step (9) and the tube is sealed. The 50 mL centrifuge tube is placed on a rotating incubator and incubated at 15-20 rpm and 4°C for 3 h to allow the protein with the His tag to bind to the matrix; (14) the turbid liquid of step (13) is flowed through the purification column tube to retain the matrix bound to the target protein in the column tube; (15) 10-15 times the column volume of Binding Buffer is added to the column tube to wash off the impurities with weaker binding ability on the matrix; (16) Elution buffer is added The column was washed with Binding Buffer, and the imidazole concentration was gradually increased (gradient setting: 10mM, 40mM, 80mM, 250mM, 500mM) for elution. The eluate was collected in a 5mL endotoxin-free centrifuge tube. Each column volume of eluate was collected in one centrifuge tube and sampled for verification of the protein concentration in the eluate by polyacrylamide gel electrophoresis (SDS-PAGE). The formula of Binding Buffer is: 250mM NaCl, 20mM Tris, 10mM imidazole, 10% glycerol, pH 8.0; the formula of Elution Buffer is: 250mM NaCl, 20mMTris, 10mM-500mM imidazole, 10% glycerol, pH 8.0.

[0075] The eluates containing the target protein IL23 heterodimer were combined, the solution was changed and concentrated, and the gel image was re-run to verify the results. Figure 1 Lane R represents denatured sample, lane NR represents non-denatured sample, and the middle lane is a molecular marker. The theoretical size of IL23 heterodimer is 59.97KDa. It can be seen from the figure that there is an obvious 62-67KDa band in the gel image, and there is basically no impurity and dragging phenomenon, indicating that the protein purity is high. The IL23 heterodimer yield calculated based on the concentration determined by BCA is 1.05mg / 100mL, that is, 1.05mg of the target protein can be purified from every 100mL of cell culture medium, and the protein purity is 95%.

[0076] Comparative Example

[0077] Comparative Example 1

[0078] A (GGGS)3 flexible connecting peptide and a 6×His purification tag were added to the N-terminus of the IL-23A (p19) subunit gene of mouse IL23 to obtain a recombinant expression vector pCDNA3.1-6×His-(GGGS)3-p19; no tag was constructed at the N-terminus of the IL-12B (p40) subunit gene, and the p40 gene was directly inserted into the expression vector pCDNA3.1 to obtain a recombinant expression vector pCDNA3.1-p40; the recombinant expression vectors pCDNA3.1-6×His-(GGGS)3-p19 and pCDNA3.1-p40 were first transformed into DH5α competent cells to screen positive clones, and then pCDNA3.1-6×His-(GGGS)3-p19 and pCDNA3.1-p40 of the positive clones were extracted and co-transformed into HEK293F cells, and after culture, the cell culture fluid was collected.

[0079] The harvested cell culture medium was centrifuged at 3500 rpm for 20 min, the supernatant was collected, AEBSF was added to a final concentration of 0.1 mM, imidazole was added to a final concentration of 10 mM, and purification was performed using a His affinity chromatography matrix. The eluate containing the target protein was collected and dialyzed into a buffer solution (pH 7.5) containing 20 mM HEPES and 50 mM NaCl for desalting.

[0080] The protein solution after dialysis and desalination was subjected to ion exchange chromatography, and the steps were as follows: (1) Take out the sterilized 15 mL purification column tube, place it in the purification bracket, and rinse the column tube with endotoxin-free water for 3-5 times; (2) Use an endotoxin-free Pasteur pipette to absorb the ion exchange matrix (purchased from Bogelon, product number AI0022), and after the protective solution in the matrix has flowed out, wash the matrix with 10-15 column volumes of endotoxin-free water; (3) Equilibrate the matrix with binding buffer, and use an endotoxin-free Pasteur pipette to absorb 6-10 column volumes of binding buffer. Buffer is added to the column tube containing the matrix; (4) the balanced matrix is ​​added to the 50mL centrifuge tube containing the protein solution and sealed, and the 50mL centrifuge tube is placed on a rotating incubator and incubated at 15-20rpm and 4°C for 40min; (5) the turbid liquid in step (4) is flowed through in the purification column tube to retain the matrix bound to the target protein in the column tube; (6) 10-15 times the column volume of Binding Buffer is added to the column tube to wash off the impurities with weaker binding ability on the matrix; (7) Elution Buffer is added to wash the column, and the salt concentration is gradually increased (100-500mM) for elution, and the eluate is collected in a 5mL endotoxin-free centrifuge tube, and each column volume is collected in one centrifuge tube, and a sample is taken to verify the protein concentration in the eluate using SDS-PAGE. The formula of Binding Buffer is: 20mM HEPES and 50mM NaCl, pH7.5; the formula of Elution Buffer is: 20mM HEPES, 100-500mM NaCl, pH7.5.

[0081] Collect the eluate containing the target protein in step (8) and concentrate it. Use a Superdex 75pg column (purchased from Cytiva, catalog number 28989334) to pass through a molecular sieve in a PBS environment, and collect the target protein according to the peak graph (start collecting when the A280 absorbance value rises to 5mAU, and stop collecting when it drops to 5mAU). After changing the solution and concentrating, re-run the gel image for verification. The results are shown in Figure 2 Lane R represents a denatured sample, lane NR represents a non-denatured sample, and the middle lane is a molecular marker. The yield of the obtained IL23 heterodimer is 0.35 mg / 100 mL, and the protein purity is 90%.

[0082] Comparative Example 2

[0083] The IL-23A (p19) subunit gene and the IL-12B (p40) subunit gene of mouse IL23 were connected by a (GGGS)4 flexible linker peptide, and a (GGGS)3 flexible linker peptide and a 6×His purification tag were added to the N-terminus to obtain the target gene 6×His-(GGGS)3-p19-(GGGS)4-p40. The 6×His-(GGGS)3-p19-(GGGS)4-p40 gene was inserted into the expression vector pCDNA3.1 to obtain The recombinant expression vector pCDNA3.1-6×His-(GGGS)3-p19-(GGGS)4-p40 was first transformed into DH5α competent cells to screen positive clones, and then His-(GGGS)3-p19-(GGGS)4-p40 of the positive clones was extracted to transform HEK293F cells. After culturing, the cell culture fluid was collected.

[0084] The harvested cell culture fluid was centrifuged at 3500rpm for 20min, the supernatant was collected, AEBSF was added to a final concentration of 0.1mM, imidazole was added to a final concentration of 10mM, and His tag affinity chromatography matrix was used for purification. The eluate containing the target protein was collected and combined, the solution was changed and concentrated, and the gel image was re-run for verification. The results are shown in Figure 3 Lane R represents a denatured sample, lane NR represents a non-denatured sample, and the middle lane is a molecular marker. The yield of the obtained IL23 heterodimer is 0.83 mg / 100 mL, and the protein purity is 95%.

[0085] Comparative Example 3

[0086] A 6×His purification tag was added to the N-terminus of the IL-23A (p19) subunit gene of mouse IL23 to obtain the recombinant expression vector pCDNA3.1-6×His-p19; a double strep purification tag was added to the N-terminus of the IL-12B (p40) subunit gene to obtain the recombinant expression vector pCDNA3.1-strep-(GGGS)2GGSA-strep-p40. The recombinant expression vectors pCDNA3.1-6×His-p19 and pCDNA3.1-strep-(GGGS)2GGSA-strep-p40 were first transformed into DH5α competent cells to screen positive clones, and then pCDNA3.1-6×His-p19 and pCDNA3.1-strep-(GGGS)2GGSA-strep-p40 of the positive clones were extracted and co-transformed into HEK293F cells. After culture, the cell culture fluid was collected.

[0087] According to the example, the cell culture fluid was subjected to strep tag affinity chromatography and His tag affinity chromatography in sequence, and the yield of the obtained IL23 heterodimer was 0.55 mg / 100 mL, and the protein purity was 95%.

[0088] Comparative Example 4

[0089] A (GGGS)3 flexible connecting peptide and a 6×His purification tag were added to the N-terminus of the IL-23A (p19) subunit gene of mouse IL23 to obtain the recombinant expression vector pCDNA3.1-6×His-(GGGS)3-p19; a single strep purification tag was added to the N-terminus of the IL-12B (p40) subunit gene to obtain the recombinant expression vector pCDNA3.1-strep-p40. The recombinant expression vectors pCDNA3.1-6×His-(GGGS)3-p19 and pCDNA3.1-strep-p40 were first transformed into DH5α competent cells to screen positive clones, and then pCDNA3.1-6×His-(GGGS)3-p19 and pCDNA3.1-strep-p40 of the positive clones were extracted and co-transformed into HEK293F cells. After culture, the cell culture fluid was collected.

[0090] When the cell culture medium was subjected to strep tag affinity chromatography according to the embodiment, the target protein could not bind to the matrix, and ultimately the IL23 heterodimer could not be purified.

[0091] Effect example

[0092] The IL23 heterodimers purified in Example 1 and Comparative Examples 1-2 were tested for protein activity. IL-23 can promote Th17 cells to secrete cytokines such as IL-17A, IL-17F and IL-22, mediating the production of inflammation and the occurrence of autoimmune diseases. Therefore, in this embodiment, the biological activity of the protein is verified by the ability of mouse spleen cells to secrete IL-17A after IL-23 treatment. The stronger its activity, the more IL-17A stimulated to be secreted. By detecting the content of IL17A, the activity of the IL-23 protein can be reflected. Specifically, the method comprises the following steps: (1) taking mouse spleen cells, resuspending them in complete culture medium (RPMI1640 (purchased from Seville, catalog number G4531-500ML) + 10% fetal bovine serum (FBS, purchased from Thermo Fisher, catalog number A5669701) + 1% sodium pyruvate (purchased from Procell, catalog number PB180422) + 1% double antibody (penicillin-streptomycin (P / S), purchased from Source Bio, catalog number S110JV) + 50 μM 2-mercaptoethanol (2-ME, purchased from Sigma, catalog number Sigma) + 10 μg / mL phytohemagglutinin (PHA-M, purchased from Yisheng Bio, catalog number 40110ES08) and counting them, and adjusting the cell density to 1×10 with complete culture medium. 6 (1) The IL-17A concentration was determined by ELISA kit (purchased from ABclonal, catalog number RK00039) and the mouse IL-17A protein concentration was used as the horizontal axis and the sample solution OD was used as the horizontal axis. 450 The value is used to draw the curve on the ordinate, and the result is shown in Figure 4 .

[0093] According to the curve calculation, the half effective concentration (EC) of the IL23 protein purified in Example 1 and Comparative Examples 1-2 for stimulating spleen cells to produce IL-17A was 50 ) values ​​were 0.0582ng / mL, 1.17588ng / mL, and 1.19708ng / mL, respectively. It can be seen that the IL23 heterodimer protein expressed by the method of the present invention can effectively stimulate mouse spleen cells to secrete IL-17A, and its biological activity is higher and has greater application value.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a mouse interleukin-23 heterodimer, characterized in that: The following steps are involved: S1. Constructing a dual-vector expression system, the dual-vector expression system is composed of a first expression vector and a second expression vector, the first expression vector comprises a p19 fusion gene, the p19 fusion gene comprises a first purification tag, a first flexible connecting peptide and a p19 gene from upstream to downstream, the second expression vector comprises a p40 fusion gene, the p40 fusion gene comprises a second purification tag, a second flexible connecting peptide and a p40 gene from upstream to downstream, and the first purification tag and the second purification tag are different; S2. Co-transform eukaryotic host cells with the dual vector expression system, collect the cell culture fluid after cultivation, and sequentially perform affinity purification through the affinity chromatography matrix of the first purification tag and the second purification tag to obtain mouse interleukin 23 heterodimer.

2. The method for preparing mouse interleukin-23 heterodimer according to claim 1, characterized in that: The first purification tag and the second purification tag are selected from one of 6×His, 8×His, Trx, Flag, strep, HA, GFP, cMyc, and mFC.

3. The method for preparing mouse interleukin-23 heterodimer according to claim 2, characterized in that: The first purification tag is a 6×His tag, and the second purification tag is a strep tag.

4. The method for preparing mouse interleukin-23 heterodimer according to claim 3, characterized in that: The affinity purification comprises the following steps: S21, centrifuge the cell culture medium, take the supernatant and mix it with the balanced Strep tag affinity chromatography matrix, and incubate at 15-20rpm and 4℃ for 3h; S22, flowing through the cell culture medium, and eluting the Strep tag affinity chromatography matrix with 10-15 column volumes of a first binding buffer, wherein the first binding buffer is PBS; S23, eluting the Strep tag affinity chromatography matrix using a first elution buffer to collect a first eluate, wherein the first elution buffer is PBS containing 5 mM D-biotin; S24, mixing the first eluate with the equilibrated His tag affinity chromatography matrix, and incubating at 15-20 rpm and 4° C. for 3 h; S25, flowing through the first eluent, and eluting the His tag affinity chromatography matrix with 10-15 column volumes of a second binding buffer, wherein the formula of the second binding buffer is: 250 mM NaCl, 20 mM Tris, 10 mM imidazole and 10% glycerol; S26. Elute the His tag affinity chromatography matrix using a second elution buffer to collect a second eluate, wherein the formula of the second elution buffer is: 250 mM NaCl, 20 mM Tris, 10 mM-500 mM imidazole and 10% glycerol.

5. The method for preparing mouse interleukin-23 heterodimer according to claim 1, characterized in that: The first flexible connecting peptide and the second flexible connecting peptide are selected from one or more of (GGGGS)n, (GGGS)n, (GGGS)nGGSA, wherein n=2, 3, 4, 5 or 6.

6. The method for preparing mouse interleukin-23 heterodimer according to claim 5, characterized in that: The first flexible connecting peptide is (GGGS)3, and the second flexible connecting peptide is (GGGS)2GGSA.

7. The method for preparing mouse interleukin-23 heterodimer according to claim 1, characterized in that: The nucleic acid sequence of the p19 fusion gene is shown as SEQ ID NO.2, and / or the nucleic acid sequence of the p40 fusion gene is shown as SEQ ID NO.

4.

8. The method for preparing mouse interleukin-23 heterodimer according to claim 1, characterized in that: The original vectors of the first expression vector and the second expression vector are pCDNA3.

1.

9. The method for preparing mouse interleukin-23 heterodimer according to claim 8, characterized in that: Constructing the first expression vector and the second expression vector comprises the following steps: synthesizing the p19 fusion gene and the p40 fusion gene by total gene synthesis, and inserting the p19 fusion gene and the p40 fusion gene into the downstream of the signal peptide of the vector pCDNA3.1 by homologous recombination, respectively, to obtain the first expression vector and the second expression vector.

10. The method for preparing mouse interleukin-23 heterodimer according to claim 1, characterized in that: The eukaryotic host cell is HEK293F cell.

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