Expression and purification method of human recombinant interleukin-7

Abstract

The invention provides a fusion protein which is characterized in that the fusion protein comprises the following elements from the 5'end to the 3 'end: 5'-MBP-Furin restriction enzyme cutting site-rhIL-7-3 ', and a method for in-vitro expression and purification of recombinant human interleukin 7 in mammalian cells, and the method comprises the following steps: a, providing a first expression vector, the first expression vector comprises nucleic acid for coding the fusion protein; b, providing a second expression vector, wherein the second expression vector comprises nucleic acid for coding furin; c, co-transfecting the first expression vector and the second expression vector to mammalian cells, and performing fermentation culture to enable the cells to express recombinant human IL-7 protein; d, centrifuging to obtain fermentation supernate from the step c; and e, purifying by using POROS XS and CHT chromatographic methods to obtain the rhIL-7 fragment.

Description

Method for expressing and purifying human recombinant interleukin-7 (1) Technical field

[0001] The present invention relates to the production of recombinant human interleukin 7 (rhIL-7) in the field of immunotherapy, and in particular to a method for constructing a vector, efficiently expressing rhIL-7 in cells, and purifying rhIL-7 from cell fermentation broth. (2) Background technology

[0002] IL-7 belongs to the type I short-chain cytokine of the erythropoietin family. The gene open reading frame contains 534 base pairs, including 6 exons, encoding 177 amino acids. The mature IL-7 protein has 152 amino acids, a molecular weight of approximately 25kDa, and contains 3 N-glycosylation sites. The molecular weight of mature IL-7 without glycosylation modification is 17.4kDa. The α-helix is ​​the main conformational content in the molecule and is the core structure of the molecule. The three pairs of disulfide bonds formed by the six cysteines in the molecule are essential for its biological activity.

[0003] IL-7, as a member of the immunostimulatory cytokine, plays an important role in the adaptive immune system by promoting immune responses. This cytokine activates immune function by stimulating the survival and differentiation of T cells and B cells, the survival of lymphocytes, and the activity of natural killer (NK) cells. IL-7 also regulates the development of lymph nodes through lymphoid tissue induction (LTi) cells and promotes the survival and division of initial T cells or memory T cells. In addition, IL-7 enhances human immune response by promoting the secretion of IL-2 and interferon-γ. The receptor for IL-7 is a heterodimer and consists of IL-7Rα (CD127) and a shared γ chain (CD132). The γ chain is expressed on all hematopoietic cell types, while IL-7Rα is mainly expressed by lymphocytes (including B and T lymphoid precursors, initial T cells, and memory T cells). Compared to effector / initial T cells that express higher levels, low expression of IL-7Rα is observed on regulatory T cells. Therefore, CD127 is used as a surface marker to distinguish these two populations. IL-7Rα is also expressed on innate lymphoid cells (NK) and gut-associated lymphoid tissue (GALT)-derived T cells. The IL-7Rα (CD127) chain is shared with TSLP (tumor stromal lymphopoietin), and the CD132 (γ chain) is shared with IL-2, IL-4, IL-9, IL-15, and interleukin 21. Two major signaling pathways are induced through the CD127 / CD132 Janus kinase / STAT pathway (i.e., Jak-Stat-3 and 5) and the phosphoinositide-3 kinase pathway (i.e., PI3K-Akt).

[0004] IL-7 administration is well tolerated in patients and leads to expansion of CD8 and CD4 cells and a relative decrease in CD4+ T regulatory cells. Numerous animal studies have demonstrated that IL-7 can restore immune function through homeostatic proliferation of peripheral CD4+ and CD8+ T cells, promoting immune reconstitution after stem cell transplantation and chemotherapy. Furthermore, IL-7 can enhance antigen-specific T cell responses during vaccination, viral infection, and adoptive cell therapy. The mechanisms by which IL-7 exerts its effects include downregulating the expression of programmed cell death 1 (PD-1) and suppressor of cytokine signaling 3 (SOCS3) and upregulating the expression of the apoptosis-inhibiting gene BCL-2, thereby exerting its effects in acute and chronic viral infections by antagonizing the effects of related inhibitory networks.

[0005] Currently, IL-7 expression is mostly based on the E. coli BL21 strain. This prokaryotic expression system lacks natural sugar chain modifications, potentially impacting its biological activity and limiting its clinical application. Currently, there are 15 registered clinical trials incorporating rhIL-7 worldwide. The rhIL-7 (CYT107) currently being used in these studies is produced by eukaryotic cells. However, eukaryotic cell-expressed IL-7 suffers from low yields and difficult purification.

[0006] Therefore, there is still a great need in the art for new and efficient methods for producing IL-7.

[0007] (3) Summary of the invention

[0008] Therefore, unlike traditional FC-tag fusion, the present invention aims to achieve a method for efficiently producing recombinant human IL-7 protein. This method utilizes the MBP tag to improve the protein's water solubility and stability, increasing protein expression. A second co-transfection vector then expresses the furin enzyme to cleave the furin cleavage site between MBP and IL-7, efficiently yielding the native structure of the IL-7 protein.

[0009] The present invention provides a fusion protein, characterized in that the fusion protein comprises the following elements from the 5' end to the 3' end: 5'-MBP-Furin enzyme cleavage site-rhIL-7-3'.

[0010] In a preferred embodiment of this aspect, the amino acid sequence of maltose binding protein (MBP) is SEQ ID NO: 3; or the amino acid sequence of the Furin cleavage site is SEQ ID NO: 4.

[0011] In yet another preferred embodiment of this aspect, the recombinant human IL-7 (rhIL-7) is selected from the amino acid sequence of SEQ ID NO: 4, conservative variants thereof, or homologous substitutions.

[0012] In another preferred embodiment of this aspect, the fusion protein comprises the amino acid sequence of SEQ ID NO: 1. More preferably, the fusion protein consists of the amino acid sequence of SEQ ID NO: 1.

[0013] In another embodiment of this aspect, the fusion protein further comprises a tag for affinity purification, selected from, for example, a His tag, preferably (His)6, and the affinity tag can be inserted between the Furin cleavage site and the coding sequence of IL-7, and optionally, the tag is covalently linked thereto.

[0014] In another embodiment of this aspect, the fusion protein further comprises a second restriction enzyme cleavage site, which is preferably a TEV restriction enzyme cleavage site. The second restriction enzyme cleavage site can be inserted downstream of the Furin restriction enzyme cleavage site and upstream of the rhIL-7 coding sequence, preferably downstream of the affinity tag. Optionally, the second restriction enzyme cleavage site is covalently linked.

[0015] In another aspect of the present invention, a method for expressing and purifying recombinant human interleukin-7 in vitro in mammalian cells is disclosed, comprising the steps of:

[0016] a. Providing a first expression vector comprising a nucleic acid encoding a fusion protein of maltose binding protein (MBP) and recombinant human IL-7, wherein the fusion protein comprises the following elements from the 5' end to the 3' end:

[0017] 5'-MBP-Furin tag-rhIL-7-3' (SEQ ID NO: 1);

[0018] b. providing a second expression vector comprising a nucleic acid encoding furin (Furin) (SEQ ID NO: 6);

[0019] c. co-transfecting the first expression vector and the second expression vector into mammalian cells, and fermenting and culturing the cells to express recombinant human IL-7 protein;

[0020] d. Centrifugation to obtain the fermentation supernatant from step c above;

[0021] e. Purification by chromatography, preferably by POROS XS and CHT chromatography, to obtain rhIL-7 fragment;

[0022] In one embodiment of the method, the mammalian cell is a CHO-K1 cell or a HEK293 cell.

[0023] In another embodiment of the method, the affinity tag is a His tag, and the restriction enzyme cleavage site is a TEV restriction enzyme cleavage site.

[0024] In one embodiment of the method, the nucleic acid of the fusion protein is cloned into the SalI / NotI site of the first expression vector.

[0025] In one embodiment of this aspect, the Furin protein is cloned into the SalI / NotI site of the second expression vector.

[0026] In one embodiment of the method, the fermentation culture is a stable transfection culture, and the VCD reaches >10 for 14 days in a shaking incubator at 120 rpm. 6 / mL culture temperature is cooled from 36.5°C to 33°C; preferably, the supernatant of the fermentation culture is centrifuged at 4°C and 10,000 revolutions (rpm), and the supernatant after centrifugation is collected.

[0027] In another embodiment of this method, the fermentation culture is transiently transfected and cultured on a shaker at 150 rpm for 6-7 days. The culture temperature is lowered from 36.5° C. to 33° C. 24 hours after the transfection. The fermentation culture supernatant is centrifuged at 4° C. and 10,000 rpm, and the supernatant is collected.

[0028] In one embodiment of the method, the chromatography is performed using POROS XS and CHT chromatography columns. The chromatography system uses an AKTA system. The equilibration buffer used for POROS XS contains 20 mM HEPES, 50 mM NaCl, pH 7.2; the elution buffer contains 20 mM HEPES, 1000 mM NaCl, pH 7.2, and the target protein is separated from impurities by gradient elution, with elution parameters of 0-100%, 10CV. The equilibration buffer used for CHT contains 5 mM PB, 100 mM NaCl, 0.1 mM CaCl2, pH 7.0; the elution buffer contains 150 mM PB, pH 7.0, and the target protein is separated from impurities by gradient elution, with elution parameters of 0-75%, 12CV.

[0029] In another embodiment of this method, including an affinity His tag and a TEV cleavage site, the affinity chromatography column is a Niexcel column, the wash buffer comprises 50 mM PB, 200 mM NaCl, 10 mM imidazole, pH 8.0, and the elution buffer comprises 50 mM PB, 200 mM NaCl, 400 mM imidazole, pH 8.0. Aggregates are removed using a Superdex 200 pg molecular sieve SEC column in PBS. Optionally, further removal can be performed by cation exchange SDS-PAGE to enhance purity.

[0030] The method of the present invention has the following advantages:

[0031] The method offers simple operating conditions and excellent reproducibility, yielding hrIL-7 in its native form with high purity. Mass spectrometry analysis indicates that the purified IL-7 is over 95% pure, with varying glycosylation patterns matching the theoretical molecular weight. Cell proliferation assays demonstrate that rhIL-7 expressed in mammalian cells exhibits potent cell proliferation-promoting effects, confirming its functional activity, even superior to that of its native form. (4) Description of the accompanying drawings

[0032] Figure 1 depicts the plasmid maps of the stable transfection vector pWX039 / IL7, the recombinant expression vector pWX4.1 / IL-7 for transient transfection (Figure 1a), and the stable transfection vector pWX040 / Furin and the transient transfection vector pWX155 / Furin (Figure 1b);

[0033] Figures 2a and 2b depict the AKTA chromatogram and SDS-PAGE analysis, respectively, of the POROS XS purification of CHO-K1-expressed IL-7. 1 represents the supernatant; 2 represents the flow-through; 3, 4, 5, and 6 represent the different elution fractions; M represents the standard protein; the lane to the left of M represents the non-reduced state; the lane to the right of M represents the reduced state.

[0034] Figures 3a and 3b show the AKTA chromatogram and SDSPAGE gel image of the AKT purified by POROS XS and then purified by CHT; the symbols represent: M: standard protein; NR: non-reduced state; R: reduced state; 1-8 represent different collection solutions of CHT column purification elution.

[0035] FIG4 is an LC-MS liquid chromatography tandem mass spectrometry analysis spectrum, showing the desugared non-reducing LC-MS spectrum and the post-translationally modified molecular weight.

[0036] Figures 5a and 5b depict the AKTA chromatogram and SDS-PAGE gel image of a His-affinity purified protein with a His-tag and TEV cleavage site. Symbols indicate: M: standard protein; NR: non-reduced state; R: reduced state; L: supernatant; FT: flow-through; TX114: Triton114 wash; and E: eluate.

[0037] Figures 6a and 6b show the chromatogram and SDSPAGE gel image of the eluate after Ni column affinity purification by molecular sieve SEC purification. The symbols represent: M: standard protein; NR: non-reduced state; R: reduced state; the remaining lanes represent different fractions collected by molecular sieve.

[0038] Figure 7a and Figure 7b are the AKTA chromatogram and SDSPAGE gel image after SPHP purification, respectively.

[0039] Figure 8a and Figure 8b are the SDSPAGE gel image and SEC-HPLC results of the final product after three-step purification, respectively.

[0040] FIG9 shows the cell activity assay of the positive control and hrIL-7 produced in one embodiment.

[0041] FIG10 shows the cell activity assay of hrIL-7 (with a His tag) produced in a positive control and another embodiment. (5) Specific implementation methods

[0042] definition

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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).

[0047] 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.

[0048] 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.

[0049] As used herein, "human recombinant IL-7," "human recombinant IL-7 protein," or "rhIL-7" can be any genetically engineered modified IL-7 protein that retains IL-7 biological activity. It may 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-7 (P13232) and retain its biological activity. The interleukin-7 proteins targeted by the methods of the present invention include various functional forms of recombinant proteins. Commercially available products include, for example, those from Beijing Sino-Biotech Co., Ltd. The amino acid sequence of the recombinant IL-7 protein used in the Examples is shown in SEQ ID NO: 6, and conservative variants, homologous substitutions, or functional fragments thereof may also be used.

[0050] 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.

[0051] Preferred mammalian cells are CHO cells and HEK293 cells. Other mammalian cells can also be used, such as NSO, etc.

[0052] 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.

[0053] As used herein, the term "fusion protein" refers to a protein composed of at least two domains encoded by separate genes that have been linked together to be transcribed and translated as a single unit, thereby producing a polypeptide. Currently, almost all recombinant proteins in the art are prepared using a fusion domain (also known as a "tag"). Fusion proteins are also known as fusion-tagged proteins or chimeric proteins.

[0054] The dual-vector mammalian cell expression system provided by the present invention shows a higher IL-7 expression level, especially compared with natural IL-7, has the same glycosylation modification and disulfide bond pairing, and retains the complete IL-7 protein biochemical activity.

[0055] The first vector, A, contains MBP and Furin cleavage sites. Additionally, a purification tag (A1) and a cleavage site (A2) can be inserted into Vector A as needed. The second vector, B, expresses Furin, which cleaves the proteolytic site during fusion protein secretion to produce IL-7 protein.

[0056] The MBP tag (Maltose binging protein) is a preferred fusion partner. MBP is a natural periplasmic protein of Escherichia coli, part of a large class of proteins that facilitate the absorption of small molecules. It has been demonstrated in the art that the solubility and stability of several proteins expressed in prokaryotic and eukaryotic host cells can be enhanced by fusing them to MBP. The presence of MBP can further significantly increase the yield of the target protein compared to the unfused protein.

[0057] However, solubilizers (such as GST, MBP, etc.) in fusion proteins may interfere with the biological function of their fusion partner, the target protein, which may lead to a significant decrease in enzyme activity. Therefore, they need to be enzymatically cleaved during protein expression and purification. This step can be performed by using a specific protease to recognize the cleavage site between the two fusion protein components.

[0058] Regarding the selection of protease hydrolysis sites, the inventors prefer to select a sequence containing 5-30 amino acids and representing a naturally occurring recognition sequence. The present invention uses the Furin cleavage site. Furin is a ubiquitously expressed protease that is present in the trans-Golgi apparatus and processes protein precursors during or during protein precursor secretion. The purpose of the cleavage site is to allow cleavage between MBP and A1 / A2 / A3, usually during protein secretion into the cell medium or during its secretion into the extracellular medium. When a second vector is present, the fusion protein is cleaved at its hydrolysis site, separating the protein from the elements connected to the hydrolysis site, thereby isolating the target protein.

[0059] The target protein is then released into the extracellular medium, and proteolysis of the fusion protein occurs on the outer membrane of the host cell. Furin catalyzes the cleavage of the Arg-Xaa-Yaa-Arg (Xaa is any amino acid, Yaa is Arg or Lys) carboxyl-terminal peptide bond in proteins. It is the best-characterized member of the mammalian subtilisin invertase family to date. Furin's enzymatic activity and substrate specificity were evaluated through cell co-expression and in vitro studies.

[0060] The fusion protein of the present invention has the following structure from 5' to 3':

[0061] 5'-MBP-Furin tag-rhIL-7-3', the amino acid sequence of which contains:

[0062] Sequence 1. MBP, Furin restriction site, and IL7 amino acid sequence on vector A

[0063] (The italicized text indicates the Furin cleavage site, the underlined text indicates the glycosylation site, and the bold underlined text indicates the paired cysteine)

[0064] in:

[0065] 1. MBP tag (SEQ ID NO: 3) is in bold.

[0066] 2. The Furin cleavage site (SEQ ID NO: 4) is in italics, not underlined, and not bold. The amino acid sequence of recombinant IL-7 (SEQ ID NO: 5) is in normal font, not gray shading, starting from the first amino acid after the slash.

[0067] 3. The fusion protein may optionally have an IgG signal peptide sequence at the 5' end, preferably, for example

[0068] MGWSCIILFLVATATGVHS (SEQ ID NO: 2).

[0069] The present invention also provides the amino acid sequence of Furin protease on the second vector (SEQ ID NO: 6):

[0070] [Corrected 06.11.2024 according to Rule 26] A His tag with an affinity tag and a TEV cleavage site can be inserted between the furin cleavage site and the IL7 sequence. The fusion protein sequence is as follows (SEQ ID NO: 7).

[0071] (Wherein the italics are the Furin cleavage site (SEQ ID NO: 4), the double underline is the MBP tag, the underlined wavy line is the IgG signal peptide sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 2), the His tag is HHHHHH (SEQ ID NO: 9), the bold underline is the TEV cleavage site (SEQ ID NO: 8), followed by the amino acid sequence of recombinant IL-7 (SEQ ID NO: 5), in normal font.

[0072] The MBP-IL7 fusion protein expressed by vector A is hydrolyzed outside mammalian cells by the Furin enzyme expressed by vector B to produce IL-7 protein, MBP tag, and Furin enzyme. In the present invention, POROS XS and CHT chromatography methods are used to remove these impurities.

[0073] The POROS XS chromatography method used in this article is a cation exchange chromatography method that uses charge differences to separate target molecules. Proteins with positive surface charges bind to the chromatographic medium, while negatively charged proteins do not. Proteins bound to the column are separated using a gradient elution method with increasing salt concentration.

[0074] The CHT hydroxyapatite used in this article is a composite filler that has both cation exchange chromatography and metal chelation functions, and is eluted and separated by adding phosphate.

[0075] As used herein, "affinity chromatography" is a chromatographic method that utilizes the binding properties of a stationary phase to separate molecules. Affinity chromatography involves attaching molecules with a certain binding capacity to the substance to be separated to a chromatographic column. A preferred affinity chromatography method for use in the present invention is an affinity chromatography ligand for the His tag, nickel ions, chelated to a macroporous affinity solid support, IMAC resin. Alternatively, commercially available affinity columns such as Ni-Excel (Cytiva, Catalog No. 17371201) for the tagged peptide can be used.

[0076] The molecular weight of the rhIL-7 of the present invention can be determined by LC-MS, etc., and the proliferation stimulating ability of IL-7 can be verified using, for example, the Mo7e cell proliferation assay (Avanzi GC, Brizzi MF, Giannotti J, Ciarletta A, Yang YC, Pegoraro L, Clark SC. M-07e human leukemic factor-dependent cell line provides a rapid and sensitive bioassay for the human cytokines GM-CSF and IL-3. J Cell Physiol. 1990 Dec; 145(3): 458-64. doi: 10.1002 / jcp.1041450310. PMID: 2273055).

[0077] Example 1 Expression and purification of recombinant human IL-7

[0078] 1. Materials and Methods

[0079] 1) The equipment and materials used in this study are shown in Tables 1 and 2

[0080] Table 1 Equipment

[0081] Table 2: Reagents and Equipment

[0082] 2) Methods:

[0083] i) Recombinant Vector Construction: The fully synthesized expression components of the present invention, the MBP-Furin-IL7 gene (coding sequence of the fusion protein shown in SEQ ID NO: 1) and the Furin gene (SEQ ID NO: 6), were constructed into the expression vectors pWX039 and pWXO40, respectively (provided by Shanghai WuXi Biotech Co., Ltd., using the commercial pTT5 vector backbone). These recombinant expression vectors pWX039IL-7 and pWX040Furin were then transformed into Top10 competent cells. The recombinant plasmids containing the specified sequences were streaked onto 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 (Sangon, Catalog #: A507002-0250) containing ampicillin sodium. Plasmid extraction was then performed according to the instructions for the NucleoBond Xtra Midi EF Kit (MACHEREY-NAGEL, Catalog #: 740420.50). Sufficient amounts of recombinant plasmids were obtained. The pWX039 / IL7 and pWX040 / Furin plasmids were diluted, concentrations were determined using a Nano-Drop (Thermo Scientific, Model #: ND2000), and gene sequencing was performed to facilitate subsequent transfection and expression. In another embodiment, the expression vectors used for transient transfection were pWX4.1 and pWX155 (provided by Shanghai WuXi Biologics Co., Ltd., and the backbone of these vectors is the commercial pTT5 vector backbone).

[0084] ii) Stable transfection expression: According to the fermentation platform process of WuXi Biologics Protein Science Department, CHO-K1 host cells were enriched by centrifugation and the supernatant was removed. The cells were resuspended to the specified density using the electroporation reagent provided by the manufacturer. The pWX039-IL7 and pWX040-Furin plasmids were mixed at a molar ratio of 10:1 and then added to the cell suspension. After mixing, the cells were placed in an electroporation cup for electroporation. After electroporation, an appropriate amount of culture medium was added and the cells were cultured in a shaker with the shaker set at 36.5°C, 120 RPM, and 6% CO2. The day of transfection was recorded as Day 0 (Day 0). On the first day after transfection (Day 1), pressurized culture medium containing antibiotics was added. Depending on the cell growth rate, the cells were passaged every 2-4 days until the cell viability recovered to >95% on Day 17.

[0085] Subsequently, the seed cells were added to the expression medium at an inoculation density of 4E5 / mL. The shake flask was placed in a shaker for culture, and the shaker setting parameters were 36.5°C, 120RPM, and 6% CO2. To ensure nutrient supply during the cell culture process, a certain proportion of feed medium and glucose were added to the cells every 2-3 days according to the platform process. At the same time, when the VCD reached >10E6 / mL, the shaker was cooled and the temperature was adjusted to 33°C, 120RPM, and 6% CO2. After harvesting on the 14th day, it was handed over to downstream purification.

[0086] iii) Transient transfection expression:

[0087] Using the WuXi Biologics Protein Science platform fermentation process, CHO-K1 host cells were diluted to the specified density using the manufacturer's platform process medium. The pWX4.1 / IL7 and pWX155 / Furin plasmids were then mixed at a mass ratio of 5:1. The premixed plasmids and the transfection reagent, polyethyleneimine (PEI), were then added to the CHO-K1 cells in the amounts specified for the platform. A 200 ml volume was used for expression. 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 continued under these shaking conditions until Day 7, when the cells were harvested. 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.

[0088] iv) POROS XS chromatography: The cell suspension expressed on day 7 was centrifuged at 10,000*g / 30 minutes using a floor-standing centrifuge. The supernatant was collected and filtered through a 0.22um filter to obtain a clarified supernatant containing the target protein. First, 8mL of POROS XS was used to capture the target protein. Using the AKTA chromatography system, the sample was first loaded at a flow rate of 3ml / min. 20mM HEPES, 50mM NaCl, pH 7.2 was used as the EQ balance solution to balance five column volumes to flush the baseline. The target protein was eluted using a gradient elution method with 20mM HEPES, 1000mM NaCl, pH 7.2 at a flow rate of 3ml / min for 10 column volumes. The column was then regenerated with 0.5M NaOH at a flow rate of 5ml / min for 15 minutes. Finally, EQ balance solution was used to balance the AKTA baseline for 10 column volumes, as shown in Figure 2a. The eluted proteins were collected in separate sections and analyzed by SDS-PAGE. Figure 2b shows the supernatant, flow-through, and eluate from CHO-K1 expressed IL-7, along with non-reducing and reducing gels. This single-step purification yielded a relatively pure IL-7 protein.

[0089] v) CHT Purification: To further improve purity, the purer eluates from the first step of POROSXS purification were collected, combined, and then exchanged into CHT equilibration buffer. A total of 45 mg was loaded onto a 5 mL CHT column. CHT equilibration buffer consisted of 5 mM PB, 100 mM NaCl, 0.1 mM CaCl2, pH 7.0. Gradient elution was performed using 150 mM PB, pH 7.0, with a 0-75% gradient over 12 CV. The column was then regenerated with 0.5 M NaOH at a flow rate of 5 mL / min for 15 minutes. Finally, the AKTA baseline was flushed using EQ equilibration buffer for 10 column volumes, as shown in Figure 3a.

[0090] The SDS-PAGE gel analysis, shown in Figure 3b, shows a significant improvement in IL-7 purity after purification. The target bands in the SDS-PAGE images in Figures 3a and 3b appear mushy, due to the numerous glycosylation sites in IL-7. This study ultimately yielded 11 mg of IL-7 protein expressed in 0.2 L of CHO K1 cells.

[0091] LC-MS verification: The molecular weight of the purified IL-7 was confirmed by LC-MS. As shown in Figure 4 and Table 3, the measured molecular weight was consistent with the theoretical molecular weight of the protein, confirming that it was IL-7 protein.

[0092] Table 3

[0093] vi) Purification of recombinant proteins with affinity His tags.

[0094] The cell suspension 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 to obtain a clarified supernatant containing the target protein. The sample was first loaded onto a 5 ml Niexcel column using an equilibration buffer of 50 mM PB, 200 mM NaCl, 10 mM imidazole, pH 8.0. The column was equilibrated with this buffer for 5 CV, and then the sample was loaded at a flow rate of 2 ml / min. After loading, the column was washed with equilibration buffer for 15 CV, followed by elution with a buffer containing Triton 114 (50 mM PB, 200 mM NaCl, 10 mM imidazole, pH 8.0, 0.1% TX114) at a flow rate of 1 ml / min for 60 minutes. The column was then washed with equilibration buffer for 10 CV, and the target protein was eluted with an elution buffer of 50 mM PB, 200 mM NaCl, 400 mM imidazole, pH 8.0. The gel run is shown in Figures 5a and 5b. The target protein pool was concentrated and loaded onto a Superdex 75pg molecular sieve column in PBS at a flow rate of 1 ml / min. The AKTA chromatogram is shown in Figure 6a. After molecular sieve separation, samples 1E3-1E10 were pooled and loaded onto a 1 ml SPHP column to further enhance purity. Equilibration buffer A was 20 mM NaAC-HAC, 50 mM NaCl, pH 5.5, and elution buffer B was 20 mM NaAC-HAC, 1 M NaCl, pH 5.5. Elution was performed first with 23% B for 10 CV, then with 23-100% B for 20 CV, and finally with 100% B for 10 CV. The AKTA chromatogram is shown in Figure 7a. The SPHP eluted samples were run on separate SDSPAGE gels, pooled with samples 1A12-1B2, and the final product was obtained after buffer exchange. The SDSPAGE of the final product is shown in Figure 8a. The target protein is very pure, and the varying sizes are due to glycosylation. As shown in FIG8b and Table 4, the purity of the product by SEC-HPLC was 95.7%.

[0095] Table 4 SEC-HPLC results

[0096] Example 2 IL-7 activity detection

[0097] The activity of IL7 obtained in Example 1 was verified using the Mo7e cell proliferation assay. The test samples were: hIL17 obtained in step v) of Example 1, batch number: 20221207_1011_M171, concentration 0.92 mg / ml; His-tagged hIL17 obtained in step vi) of Example 1, batch number: 20221129-Hil-7, concentration 0.26 mg / ml, and a commercial IL7 protein (from Beijing Sino Biological Technology Co., Ltd.) 11821-HNAE, batch number: ME16SE1515, concentration 2.24 mg / ml) was used as a positive control.

[0098] 1. Materials and Methods

[0099] The instruments and reagents used in this experiment are shown in Table 1 and Table 2 of Example 1 above.

[0100] 2. Test steps:

[0101] 1) Cell culture: Cultivate cells until the flask is 80%-90% full.

[0102] 2) Cell seeding: Collect Mo7e cells, count them, and seed them into 96-well plates at a density of 20k / well. Place them in a CO2 incubator and incubate them (incubator conditions: 37°C, 5% CO2);

[0103] 3) Sample addition: First dilute the sample from the initial concentration to 2000 ng / ml, then dilute it 5-fold in 9 steps, add the sample, and incubate for 120 hours.

[0104] 4) Detection: After the incubation, WST8 (2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylphenyl)-2H-tetrazole monosodium salt) was added to detect the OD450 value.

[0105] Analysis of the sample's effect on Mo7e cell proliferation, as shown in Figures 9 and 10, demonstrates that the IL-7 protein produced in Example 1 promoted Mo7e cell proliferation. Table 5 below lists the ED values ​​for stimulation. As shown in Tables 5 and 6, the ED50 value of IL-7 obtained in Example 1 was similar to that of the positive control, demonstrating excellent cell proliferation-promoting activity.

[0106] Table 5: Summary of sample activity results

[0107] Table 6: Summary of activity results of samples with His tag

[0108] Although the above disclosure has been described in some detail by way of illustration and example for the purpose of clarity of understanding, it is obvious to those skilled in the art that certain changes and modifications may be made thereto without departing from the spirit and scope of the disclosure described in the appended claims. Therefore, the above description should not be construed as limiting the scope of the disclosure.

Claims

1. A fusion protein, characterized in that The fusion protein includes the following elements from the 5' end to the 3' end: 5'-MBP-Furin restriction site-rhIL-7-3'. 2 . The fusion protein according to claim 1 , wherein the amino acid sequence of maltose binding protein (MBP) is SEQ ID NO: 3; or the amino acid sequence of the Furin cleavage site is SEQ ID NO:

4. 3 .

3. The fusion protein of claim 1, wherein the recombinant human IL-7 (rhIL-7) is selected from the amino acid sequence of SEQ ID NO: 4, conservative variants thereof, or homologous substitutions. The fusion protein according to claim 1 , comprising the amino acid sequence of SEQ ID NO:

1. The fusion protein according to claim 1 , further comprising an affinity tag, preferably a His tag.

6. The fusion protein according to claim 1, further comprising a second restriction enzyme cleavage site, wherein the second restriction enzyme cleavage site is located downstream of the Furin restriction enzyme cleavage site and upstream of the rhIL-7 coding sequence, and is preferably a TEV restriction enzyme cleavage site.

7. A method for expressing and purifying recombinant human interleukin-7 in vitro in mammalian cells, comprising the steps of: a. Providing a first expression vector, the first expression vector comprising a nucleic acid encoding the fusion protein of any one of claims 1-6; b. providing a second expression vector, the second expression vector comprising a nucleic acid encoding Furin; c. co-transfecting the first expression vector and the second expression vector into mammalian cells, and fermenting and culturing the cells to allow the cells to express recombinant human IL-7 protein; d. Centrifuge the fermentation supernatant obtained from step c above; e. The rhIL-7 fragments were purified by POROS XS and CHT chromatography; rhIL-7 with affinity tags and restriction site elements was obtained by affinity tag purification and restriction cleavage.

8. The method of claim 7, wherein the mammalian cell is a CHO-K1 cell or a HEK293 cell; or wherein the nucleic acid encoding the fusion protein is cloned into the SalI / NotI site of the first expression vector, and / or the Furin protease is cloned into the SalI / NotI site of the second expression vector.

9. The method of claim 7, wherein the fermentation culture is a transient transfection, cultured in a shaking incubator at 150 rpm for 6-7 days, and the culture temperature is lowered from 36.5° C. to 33° C. 24 h after the completion of the transfection; preferably, the supernatant of the fermentation culture is centrifuged at 4° C. and 10,000 rpm, and the supernatant after centrifugation is collected.

10. The method of claim 7, wherein the fermentation culture is a stable transfection, cultured in a 120 rpm shaker for 14 days, VCD reaches >10 E6 / mL, and the culture temperature is cooled from 36.5°C to 33°C; preferably, the supernatant of the fermentation culture is centrifuged at 4°C and 10,000 rpm, and the supernatant after centrifugation is collected.

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