Recombinant il-27 protein and methods of making same

Abstract

A p28 subunit of a recombinant IL-27 protein is disclosed, characterized in that it comprises an amino acid sequence selected from any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25. An EBI3 subunit of a recombinant IL-27 protein is also disclosed, characterized in that it comprises an amino acid sequence selected from any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22. Recombinant IL-27 proteins comprising these two subunits and methods for their preparation and screening are also disclosed.

Description

Recombinant IL-27 protein and preparation method thereof (1) Technical field

[0001] The present invention relates to the field of recombinant protein expression. Specifically, the present invention relates to a recombinant IL-27 protein and its modified subunits, as well as a method for preparing the recombinant IL-27 protein. (2) Background technology

[0002] IL-27 is a heterodimeric cytokine composed of p28 and EBI3, primarily expressed in macrophages, inflammatory monocytes, microglia, and dendritic cells, but also in plasma cells, endothelial cells, and epithelial cells. The IL-27 receptor, IL-27R, consists of the cytokine-binding protein α subunit (IL-27Rα / Wsx-1) and the signaling protein β subunit (gp130). IL-27Rα is expressed at the highest level in T cells, with higher levels also found in B cells and macrophages.

[0003] IL-27 is a pleiotropic cytokine. As an immune activator, IL-27 can prevent the development of cancer and infectious diseases; as an immunomodulator, IL-27 can inhibit the occurrence and development of autoimmune diseases. IL-27 is an attractive candidate target for the treatment of tumor immunotherapy, infectious diseases, and autoimmune diseases.

[0004] Recombinant human IL-27 protein is currently available from several suppliers, including R&D Systems (recombinant human IL-27 protein 2526-IL-010: R&D Systems (rndsystems.com)) and Sino Biological (recombinant human IL-27 protein, CHO stable cells, tagged, 10076-H08S | Sino Biological). However, these products utilize a (GGS)n linker to link the p28 and EBI3 subunits together for single protein expression. This results in significant protein aggregation, and after multiple purification steps, the product purity is only >90%, which does not meet clinical needs. To improve half-life, some have also linked it to the N-terminus of an Fc fragment, expressing and purifying it as an Fc fusion protein. However, this molecule exhibits severe aggregation, resulting in extremely low monomer yields and prohibiting large-scale production.

[0005] Regardless of the form of IL-27 molecules, they will produce severe aggregates, limiting their production and clinical application.

[0006] Therefore, there is a need in the art for a method for stably producing IL-27 that overcomes the generation of IL-27 molecule aggregates. (3) Summary of the invention

[0007] One purpose of the present invention is to solve the problem of IL-27 molecules generating aggregates during the production process.

[0008] In response to the serious aggregation problem of IL-27 protein, the inventors introduced disulfide bonds into the IL-27 protein to reduce aggregation, thereby increasing the protein's yield and clinical application potential.

[0009] In one aspect of the present invention, a method for preparing recombinant IL-27 protein is provided:

[0010] The amino acid sequence of p28 of the recombinant IL-27 protein MGQTAGDLGWRLSLLLLPLLLVQAGVWGFPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMRLDLRDLQRHLRFQVLAAGFNLPEEEEEEEEEEEEERKGLLPGALGSALQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPTLSPQP (SEQ ID NO: 26) was substituted with cysteine.

[0011] Cysteine ​​substitution was performed on the amino acid sequence MTPQLLLALVLWASCPPCSGRKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK (SEQ ID NO: 27) of the recombinant IL-27 protein.

[0012] Artificially synthesizing two modified subunits, assembling them and obtaining recombinant IL-27 protein; or, obtaining the coding sequence of the subunits, transfecting them into an expression vector, and expressing them in cells to obtain recombinant IL-27 protein;

[0013] The activity of the recombinant IL-27 protein was tested and compared with wild-type IL-27, thereby obtaining a recombinant IL-27 protein having higher activity than wild-type IL-27.

[0014] In one embodiment of this aspect, the activity of the recombinant IL-27 protein is preferably tested by molecular weight comparison with wild-type IL-27, preferably by chromatography, more preferably LC-MS, an immunological test, preferably ELISA, or an in vitro cell assay, preferably an inhibition cytopathic assay.

[0015] In another aspect of the present invention, a p28 subunit of a recombinant IL-27 protein is provided, characterized in that it comprises an amino acid sequence selected from any one of SEQ ID NO: 1 (p28_S91C), SEQ ID NO: 3 (p28_D89C), SEQ ID NO: 5 (p28_L83C), SEQ ID NO: 7 (p28_Q86C), SEQ ID NO: 9 (p28_F94C), SEQ ID NO: 11 (p28_Q95C), SEQ ID NO: 13 (p28_L80C), SEQ ID NO: 15 (p28_P82C), SEQ ID NO: 17 (p28_G84C), SEQ ID NO: 19 (p28_E85C), SEQ ID NO: 21 (p28_L87C), SEQ ID NO: 23 (p28_L81C), and SEQ ID NO: 25 (p28_Y79C).

[0016] In the third aspect of the present invention, a recombinant IL-27 protein EBI3 subunit is provided, characterized in that it comprises an amino acid sequence selected from any one of SEQ ID NO: 2 (EBI3_S98C), SEQ ID NO: 4 (EBI3_M99C), SEQ ID NO: 6 (EBI3_Q95C), SEQ ID NO: 8 (EBI3_P41C), SEQ ID NO: 10 (EBI3_L96C), SEQ ID NO: 12 (EBI3_D93C), SEQ ID NO: 14 (EBI3_G69C), SEQ ID NO: 16 (EBI3_M70C), SEQ ID NO: 18 (EBI3_A100C), SEQ ID NO: 20 (EBI3_P101C), and SEQ ID NO: 22 (EBI3_P120C).

[0017] In a fourth aspect of the present invention, a recombinant IL-27 protein is provided, characterized in that it comprises a p28 subunit and an EBI3 subunit, wherein the p28 subunit comprises an amino acid sequence selected from any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25, and the EBI13 subunit comprises an amino acid sequence selected from any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22.

[0018] In a preferred embodiment of this aspect, the p28 subunit and the EBI3 subunit are selected from the following combinations:

[0019] SEQ ID NO: 1 and SEQ ID NO: 2 (p28_S91C / EBI3_S98C), SEQ ID NO: 3 and SEQ ID NO: 2 (p28_D89C / EBI3_S98C), SEQ ID NO: 3 and SEQ ID NO: 4 (p28_D89C / EBI3_M99C), SEQ ID NO: 5 and SEQ ID NO: 4 (p28_L83C / EBI3_M99C), SEQ ID NO: 7 and SEQ ID NO: 4 (p28_Q86C / EBI3_M99C), SEQ ID NO: 17 and SEQ ID NO: 4 (p28_G84C / EBI3_M99C), SEQ ID NO: 19 and SEQ ID NO: 4 (p28_E85C / EBI3_M99C), SEQ ID NO: 21 and SEQ ID NO: 4 (p28_L87C / EBI3_M99C), SEQ ID NO: 22 and SEQ ID NO: 4 (p28_L87C / EBI3_M99C), SEQ ID NO: 23 and SEQ ID NO: 4 (p28_L87C / EBI3_M99C), SEQ ID NO: 24 and SEQ ID NO: 4 (p28_L87C / EBI3_M99C), SEQ ID NO: 25 and SEQ ID NO: 4 (p28_L87C / EBI3_M99C), NO: 1 and SEQ ID NO: 6 (p28_L87C / EBI3_M99C), SEQ ID NO: 9 and SEQ ID NO: 8 (p28_F94C / EBI3_P41C), SEQ ID NO: 9 and SEQ ID NO: 10 (p28_F94C / EBI3_L96C), SEQ ID NO: 11 and SEQ ID NO: 12 (p28_Q95C / EBI3_D93C), SEQ ID NO: 5 and SEQ ID NO: 14 (p28_L83C / EBI3_G69C), SEQ ID NO: 5 and SEQ ID NO: 16 (p28_L83C / EBI3_M70C), SEQ ID NO: 13 and SEQ ID NO: 16 (p28_L80C / EBI3_M70C), SEQ ID NO: 15 and SEQ ID NO: 16 (p28_L80C / EBI3_M70C), SEQ ID NO: 16 and SEQ ID NO: 17 (p28_L87C / EBI3_M99C), SEQ ID NO: 17 and SEQ ID NO: 18 (p28_L87C / EBI3_P41C), SEQ ID NO: 19 and SEQ ID NO: 20 (p28_F94C / EBI3_L96C), NO: 16 (p28_P82C / EBI3_M70C), SEQ ID NO: 5 and SEQ ID NO: 18 (p28_L83C / EBI3_A100C), SEQ ID NO: 23 and SEQ ID NO: 18 (p28_L81C / EBI3_A100C), SEQ ID NO: 13 and SEQ ID NO: 20 (p28_L80C / EBI3_P101C), SEQ ID NO: 13 and SEQ ID NO: 22 (p28_L80C / EBI3_P120C), SEQ ID NO: 25 and SEQ ID NO: 22 (p28_Y79C / EBI3_P120C).

[0020] The sequences described herein are defined in the sequence listing in the computer readable form of the specification, the contents of which are incorporated herein by reference.

[0021] In one embodiment of the present invention, a signal peptide may be further included at the N-terminus of the amino acid sequence of the p28 subunit and the EBI3 subunit of the recombinant IL-27 protein. In a preferred embodiment, the signal peptide has the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 24).

[0022] In another aspect, the present invention also includes an isolated nucleic acid molecule encoding the recombinant IL-27 protein or its subunit of the present invention, wherein the nucleic acid is preferably DNA.

[0023] In another aspect of the present invention, an expression vector comprising and expressing the nucleic acid molecule of claim 6 is also included. The expression vector can be a transient or constitutive transfection vector, preferably a transient transfection vector. The vector can be selected from a plasmid or a viral vector. The vector is preferably a mammalian vector.

[0024] In another aspect of the present invention, an isolated host cell comprising the expression vector of the present invention is also included. Preferably, the cell is a mammalian cell, preferably a CHO cell. In another aspect of the present invention, the cell of the present invention may be a human cell. (4) Description of the accompanying drawings

[0025] Figure 1 depicts an SDS-PAGE gel analysis of recombinant IL-27 proteins 1-9.

[0026] FIG2 depicts the HPLC results of recombinant IL-27 proteins 1-9.

[0027] Figures 3 and 4 show the test results of the binding activity of recombinant IL-27 proteins 1-9 to human IL27RA-Fc.

[0028] Figures 5 and 6 show the changes in absorbance values ​​of HepG2 cells with concentration after incubation of cells with recombinant IL-27 proteins 1-9 and virus attack.

[0029] FIG7 depicts an SDS-PAGE gel analysis of recombinant IL-27 proteins 10-21.

[0030] FIG8 depicts the HPLC results of recombinant IL-27 proteins 10-21.

[0031] FIG9 shows the test results of the binding activity of recombinant IL-27 proteins 10-21 to human IL27RA-Fc.

[0032] FIG10 shows the change in absorbance of HepG2 cells with concentration after incubation of cells with recombinant IL-27 proteins 10-21 and virus attack. (5) Specific implementation methods

[0033] The examples discussed below are intended only to illustrate the present invention and should not be considered as limiting the present invention in any way. The examples are not intended to represent that the following experiments are all experiments or unique experiments performed. Efforts have been made to ensure accuracy regarding the quantities used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0034] definition

[0035] As used herein, unless the context clearly dictates otherwise, singular forms beginning with "a," "an," and "the" include plural references. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0036] As used herein, the terms "about," "approximately," or "approximately," when preceding a numerical value, refer to a range defined by approximately 1%, 2%, 3%, 4%, 5%, 10% or more around the specified value.

[0037] In the present disclosure, one or more features in one embodiment may be combined with any one or more features in another embodiment without departing from the spirit and concept of the present invention.

[0038] In this disclosure, unless otherwise specified, all ranges, including ranges defined as between two specified endpoints, include the specified endpoints. For example, a range between 1 and 10 means a range between 1 and 10 (inclusive).

[0039] In this document, when a duration, period or interval is expressed in days, and when a time point is expressed as a day or a certain day, it means that the duration or moment is measured or divided by days (days), and the numerical value is not required to represent a multiple of 24 hours.

[0040] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety into the disclosure herein. The citations are intended solely to illustrate the level of skill of those skilled in the art and do not constitute prior art prior to the present invention.

[0041] As used herein, "human recombinant IL-27," "human recombinant IL-27 protein," or "rhIL-27" refers to a genetically engineered mutant IL-27 protein that retains IL-27 biological activity. The p28 sequence subunit of the wild-type human IL-27 protein has the following amino acid sequence: MGQTAGDLGWRLSLLLLPLLLVQAGVWGFPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMRLDLRDLQRHLRFQVLAAGFNLPEEEEEEEEEEEEERKGLLPGALGSALQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPTLSPQP (SEQ ID NO: 26), where the underlined portion represents the native signal peptide sequence.

[0042] The wild-type IL-27 EBI3 sequence has the following amino acid sequence:

[0043] MTPQLLLALVLWASCPPCSGRKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK (SEQ ID NO: 27), wherein the underlined portion represents the native signal peptide sequence. In the present invention, a cysteine ​​mutation is introduced at a suitable position at the interaction interface of the two IL-27 subunits, p28 and EBI3, respectively. The disulfide bond formed between the two subunits stabilizes the interaction between p28 and EBI3, thereby reducing aggregation. It should be understood that, excluding the specific cysteine ​​mutation modification, the remaining sequence can be at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to the naturally derived mature interleukin-27 and retain its biological activity. Interleukin-27 proteins that can be produced according to the methods of the present invention include various functional forms of recombinant proteins modified by cysteine ​​mutations. Commercially available products include R&D Corporation (recombinant human IL-27 protein 2526-IL-010: R&D Systems (rndsystems.com)) and Sino Biological (recombinant human IL-27 protein, CHO stable cells, tag, 10076-H08S | Sino Biological). The amino acid sequences of the p28 subunit and EB13 subunit of the recombinant IL-27 protein used in the Examples are shown in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25, and in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22, respectively. Conservative variants, homologous substitutions, or functional fragments thereof may also be used.

[0044] The mammalian cells described herein can be derived from a variety of mammalian sources, examples of which include, but are not limited to, primates (e.g., humans and non-human primates such as chimpanzees, baboons, or monkeys), dogs, cats, pigs, sheep, rabbits, mice, and rats. In some cases, the mammal can be a human.

[0045] Preferred mammalian cells are CHO cells and HEK293 cells. Other mammalian cells can also be used, such as NSO, etc.

[0046] The term "expression vector" refers to a nucleic acid molecule when used herein, which can guide the expression of a gene operatively connected thereto after entering a host cell. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, in which other DNA fragments can be joined. Another type of vector is a viral vector, in which other DNA fragments can be joined to a viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thus replicated together with the host genome. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (for example, electroporation, lipofection). Enzymatic reaction and purification techniques can be carried out according to the manufacturer's instructions or this area generally realized or as described herein. The aforementioned techniques and procedures can generally be performed according to the conventional methods described in the various general and more specific references as well-known in the art and as cited and discussed throughout the specification of the present invention.

[0047] The pharmaceutically acceptable compositions of the present invention comprise various forms or combinations of the recombinant IL-27 proteins described herein, nucleic acid molecules encoding them, recombinant expression vectors, host cells, and pharmaceutically acceptable excipients, such as carriers or diluents.

[0048] The advantages of the present invention are that, based on professional knowledge and experience in structural biology and protein engineering, 21 pairs of cysteine ​​mutation combinations are designed at the interaction interface of the two subunits of the human IL-27 protein, p28 and EBI3, to form disulfide bonds, thereby stabilizing the interaction between the two subunits, thereby reducing the phenomenon of protein aggregation caused by weak non-covalent interactions between the two subunits, and significantly reducing the aggregation of IL-27 protein during the production process, thereby making IL-27 protein production more economical and improving product quality.

[0049] Example 1 Expression and purification of recombinant human IL-27 protein

[0050] Recombinant human IL-27 proteins 1-9 were expressed and purified using the same method as follows.

[0051] 1. Materials and Equipment

[0052] The equipment and materials used in this study are shown in Tables 1 and 2 below.

[0053] Table 1 Equipment

[0054] Table 2: Reagents and Equipment

[0055] 2. Methods:

[0056] 1) Transient transfection plasmid construction method:

[0057] Each protein in the present invention contains two subunits, p28 and EBI3. The p28 gene (e.g., the corresponding p28 sequence in proteins 1-21) and the EBI3 gene (e.g., the corresponding EBI3 sequence in proteins 1-21) were synthesized and expressed as follows:

[0058] The recombinant human expression vectors pWX4.1 / p28 and pWX4.1 / EBI3 were constructed into the expression vector pWX4.1 (provided by Shanghai WuXi Biotech Co., Ltd., based on the commercial pTT5 vector backbone) and subsequently transformed into the E. coli BL21 / DH5α host strain. The recombinant human plasmids containing the indicated sequences were streaked onto solid LB plates containing ampicillin sodium and incubated inverted at 37°C overnight. A single colony was selected from the plate and expanded into 200 mL of LB medium (Sigma, Catalog # L7658) containing ampicillin sodium. Plasmid extraction was then performed according to the NucleoBond Xtra Midi EF Kit (MACHEREY-NAGEL, Catalog # 740420.50). Sufficient amounts of the recombinant human plasmids pWX4.1 / p28 and pWX4.1 / EBI3 were obtained. The pWX4.1 / p28 and pWX4.1 / EBI3 plasmids were diluted, and their concentrations were detected by Nano-Drop (Thermo scientific, model #: ND2000). Gene sequencing was performed to verify the expression of the plasmids for subsequent transfection.

[0059] 2) Transient transfection expression: Using the WuXi Biologics Protein Science fermentation platform process, CHO-K1 host cells were diluted to the specified density using the manufacturer's platform process medium. The pWX4.1 / p28 and pWX4.1 / EBI3 plasmids were mixed at a 1:1 mass ratio. The premixed plasmids and the transfection reagent, polyethyleneimine (PEI), were then added to the CHO-K1 cells to complete transfection (using the platform-specified amount). A 2 L volume was used for each host cell. The cells were then cultured in a shaker at 36.5°C, 120 RPM, 6% CO₂, and 85% humidity. The day of transfection was designated Day 0. On Day 1, the shaker settings were adjusted to 31°C, 120 RPM, 6% CO₂, and 85% humidity. Culture was continued under these shaker parameters until harvest on Day 7 after transfection. To ensure nutrient supply during cell culture, feed medium and glucose were added to the cells at a certain ratio on Day 0 and Day 4 after transfection according to the platform process. Cells were harvested on Day 7 for downstream purification.

[0060] 3) Affinity chromatography (AC): The cell fluid expressed on day 7 was centrifuged at 10,000 × g for 30 minutes using a floor-standing centrifuge. The supernatant was collected and filtered through a 0.22 μm filter membrane to obtain a clarified supernatant containing the target protein. First, 20 mL Ni-Excel was used to capture the target protein. Specifically, an AKTA chromatography system was used. The sample was first loaded at a flow rate of 20 ml / min. 50 mM PB, 200 mM NaCl, 10 mM imidazole, pH 8.0 was used as an EQ balance solution to balance five column volumes to flush the baseline. 50 mM PB, 200 mM NaCl, 10 mM imidazole, pH 8.0, 0.1% TX114 (V / V) was used as Wash2 (washing buffer) to wash for 10 column volumes, and the washing time was not less than 60 minutes to remove endotoxins. EQ balance solution was then used to wash for 7 column volumes to balance. 50 mM PB, 200 mM NaCl, 400 mM imidazole, pH 8.0 was used as an eluent to elute the target protein at a flow rate of 2.0 ml / min for 3 column volumes. 0.5 M Regenerate the column with NaOH at a flow rate of 5 ml / min for 15 minutes. Finally, use EQ equilibration solution for 10 column volumes to flush the AKTA baseline. Collect the eluate as the target protein obtained from the first affinity chromatography step and analyze it by SDS-PAGE.

[0061] 4) Molecular Sieve Chromatography (SEC): To remove aggregates and some impurities in the affinity chromatography elution sample, this study used molecular sieve chromatography for purification. PBS, pH 7.4, was selected as the equilibration buffer (Buffer A); the affinity-bound IL-27 protein solution was first loaded onto a 320 mL HiLoad 26 / 600 Superdex 200 pg column at a flow rate of 2.5 mL / min. After loading, the target protein peak gradually appeared. The target protein was collected to obtain pure IL-27. SDS-PAGE showed good protein purity, but some impurities were still present.

[0062] 5) SP-HP Fine Purification: To further purify the IL-27 protein, this study used an ion exchange chromatography column, SP-HP, for purification. 20 mM NaAC, pH 5.5, was used as the equilibration buffer (Buffer A), and 20 mM NaAC, pH 5.5, 1 M NaCl was used as the elution buffer (Buffer B). The SEC-purified IL-27 protein solution was first diluted tenfold with Buffer A to a conductivity below 8 ms and then loaded onto a 5 mL SP-HP column at a flow rate of 5 mL / min. Following loading, a gradient elution using Buffer B was performed, increasing the Buffer B ratio from 40% to 80% over 25 column volumes at a flow rate of 2.5 mL / min. The eluted protein was collected to obtain pure IL-27. SDS-PAGE gel analysis (Figures 1 and 7) demonstrates a significant increase in IL-27 purity after purification, and HPLC results indicate 100% protein purity (Figure 2).

[0063] 6) Separation chromatography (SEC): To further remove host proteins from the sample, this study used SEC for purification. PBS, pH 7.4, was used as the equilibration buffer (Buffer A). The IL-27 protein solution was loaded onto a 120 mL HiLoad 16 / 600 Superdex 200 pg column at a flow rate of 1.0 mL / min. After loading, the target protein was collected to obtain pure IL-27.

[0064] 7) LC-MS Verification: The molecular weight of the purified IL-27 was confirmed by LC-MS. The theoretical molecular weight of the protein and the molecular weight measured by LC-MS analysis are shown in the table below. The measured molecular weight is consistent with the theoretical molecular weight of the protein, confirming that both are IL-27 proteins.

[0065] Table 3 Results of protein molecular weight determination

[0066] 8) Host protein detection: The purpose of this experiment is to detect residual host cell proteins (HCP) in IL-27 protein. The kit (Cygnus, F550-1) uses a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with CHO host cell residual protein (CHO-HCP) antibodies, the specimen, standard, and HRP-labeled detection antibody are added in sequence, incubated, and thoroughly washed. The substrate TMB is used for color development. TMB is converted to blue under the catalysis of peroxidase and to the final yellow under the action of acid. The depth of the color is positively correlated with the residual CHO host cell protein (CHO-HCP) in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using a microplate reader to calculate the sample concentration. Table 4 provides the protein concentration analysis results.

[0067] Table 4 Host protein analysis results

[0068] 9) ELISA: The purpose of this study was to detect the binding activity of human IL-27 protein samples with human IL-27RA-Fc protein (Sino Biological, Cat: 10489-H02H). The experimental method is as follows:

[0069] i) Coating: Coat with IL27-His protein control (catalog number #10076-H08S) and proteins 1 to 9, 2 μg / mL, 100 μL / well, at 4°C overnight;

[0070] ii) Blocking: Shake off all liquid in the plate and pat dry. Add 2% BSA blocking buffer (300 μL / well), seal, and incubate at room temperature for 1 hour.

[0071] iii) Wash the plate: Wash twice with 300 μL / well PBS-T phosphate-Tween solution, and pat dry for the final wash;

[0072] iv) Sample dilution: Dilute IL27RA-Fc protein (Cat#10489-H02H) to 20,000 ng / mL with sample diluent (0.1% BSA in PBST), then continue dilutions according to the table below. Mix thoroughly after each dilution step.

[0073] Table 5 Sample dilution

[0074] v) Wash the plate: Wash the plate three times with 300 μL / well PBST, patting dry for the final wash.

[0075] vi) Add secondary antibody: Dilute the secondary antibody goat anti-human IgG (Fc) / HRP to 0.1 μg / mL and mix

[0076] Evenly add 100 μL / well and incubate at room temperature for 1 h;

[0077] vii) Washing the plate: same as step v);

[0078] viii) Color development: Mix Solution A and Solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 15 min.

[0079] ix) Stop: Add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0080] As shown in the results in Table 6 below, the binding activity data indicate that proteins 1 to 9 have good binding activity to human IL27RA-Fc protein (as shown in Figures 3 and 4).

[0081] Table 6 Binding activity data

[0082] Figure 9 shows the results of the test of the binding activity of recombinant IL-27 proteins 10-21 to human IL27RA-Fc. In addition, as shown in the data in Table 6 above, proteins 10-18 and 20-21 showed good binding activity to human IL27RA-Fc protein.

[0083] Example 2 Activity detection of recombinantly expressed IL-27:

[0084] In this example, the cytopathic effect inhibition assay (Seita J, et al. (2008) Interleukin-27 directly induces differentiation in hematopoietic stem cells. Blood. 111(4): 1903-12) was used to detect the activity of IL-27. The experimental method is as follows:

[0085] 1. Cell culture: HepG2 cells were expanded to the required cell number (1.92x10 6 cells);

[0086] 2. Cell seeding: Collect HepG2 cells, adjust to the desired cell density, and seed into 96-well plates. After completion, place the 96-well plates in a 37°C, 5% CO2 constant temperature incubator for incubation.

[0087] 3. Sample addition: dilute the sample in a 5-fold gradient, for a total of 10 dilutions, with 2 replicates for each dilution, add the sample, and incubate overnight;

[0088] 4. Add virus: Take out the VSV virus, dilute it a certain multiple, add it to the 96-well plate, and incubate overnight;

[0089] 5. Detection: Add MTT, incubate for 4 hours, then add the triple solution, incubate overnight in a constant temperature incubator at 37°C and 5% carbon dioxide, and read the plate with a microplate reader.

[0090] Using HepG2 cells as experimental cells, the samples were incubated with different concentrations of the samples, then challenged with a virus to induce cytopathic effects. Finally, the cell OD values ​​were measured using MTT. The results are shown in Figures 5, 6, and 10, as well as Table 7. Comparison with the positive control demonstrates that the samples have a significant protective effect on the cells, indicating that proteins 1-9 and 10-21 have biological activity in inhibiting HepG2 cytopathic effects.

[0091] Table 7 Summary of activity results

[0092] The results in Table 7 clearly show that protein 1-21 has a better effect than the existing commercial IL-27 recombinant protein.

[0093] All publications, patents, patent applications, or other documents cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

[0094] Amino acid sequence

Claims

1. A p28 subunit of a recombinant IL-27 protein, characterized in that, Comprises an amino acid sequence selected from any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25.

2. The EBI3 subunit of a recombinant IL-27 protein, characterized in that, Comprises an amino acid sequence selected from any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22.

3. A recombinant IL-27 protein, characterized in that, Comprises a p28 subunit and an EBI3 subunit, wherein the p28 subunit comprises an amino acid sequence selected from any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and the EBI13 subunit comprises an amino acid sequence selected from any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22.

4. The recombinant IL-27 protein according to claim 3, wherein the p28 subunit and the EI3 subunit are selected from any one of the following combinations shown by the SEQ ID NO encoding: 1 and 2, 3 and 2, 3 and 4, 5 and 4, 7 and 4, 17 and 4, 19 and 4, 21 and 4, 1 and 6, 9 and 8, 9 and 10, 11 and 12, 5 and 14, 5 and 16, 13 and 16, 15 and 16, 5 and 18, 23 and 18, 13 and 20, 13 and 22, 25 and 22.

5. The recombinant IL-27 or its subunit according to any one of claims 1, 2 or 3, characterized in that, The sequence further includes a signal peptide sequence at the 5' end: MGWSCIILFLVATATGVHS (SEQ ID NO: 24).

6. An isolated nucleic acid molecule encoding the recombinant IL-27 protein or its subunit according to any one of claims 1-5.

7. An expression vector comprising and expressing the nucleic acid molecule according to claim 6.

8. An isolated host cell comprising the expression vector according to claim 7, preferably the cell is a CHO cell.

9. A method for preparing a recombinant IL-27 protein: Perform cysteine substitution on the amino acid sequence of p28 of the recombinant IL-27 protein M G Q T A G D L G W R L S L L L L P L L L V Q A G V W G F P R P P G R P Q L S L Q E L R R E F T V S L H L A R K L L S E V R G Q A H R F A E S H L P G V N L Y L L P L G E Q L P D V S L T F Q A W R R L S D P E R L C F I S T T L Q P F H A L L G G L G T Q G R W T N M E R M Q L W A M R L D L R D L Q R H L R F Q V L A A G F N L P E E E E E E E E E E E E R K G L L P G A L G S A L Q G P A Q V S W P Q L L S T Y R L L H S L E L V L S R A V R E L L L L S K A G H S V W P L G F P T L S P Q P (SEQ ID NO: 26). Cysteine substitutions were made on the amino acid sequence of EBI3 of recombinant IL-27 protein MTPQLLLALVLWASCPPCSGRKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK (SEQ ID NO: 27). Two modified subunits were artificially synthesized, assembled and recombinant IL-27 protein was obtained; or the coding sequences of the subunits were obtained, transfected into an expression vector and expressed in cells to obtain recombinant IL-27 protein. The activity of the recombinant IL-27 protein was tested, compared with wild-type IL-27, and a recombinant IL-27 protein with higher activity than wild-type IL-27 was obtained.