High-efficiency human interleukin-35 cell strain, preparation method and application thereof

Abstract

The present application provides a kind of high expression human interleukin-35 cell strain, its preparation method and application, belong to the biopharmaceutical technical field, can solve the problem that present technology cannot realize the stable expression of human interleukin-35 natural protein molecule. Among them, the preparation method of high expression human interleukin-35 cell strain is by the double plasmid expression system with human interleukin-35 gene sequence is transiently transfected into the genome of CHO-K1 cell, after multiple screening test and biological activity detection, finally screening is obtained, specific steps include: (1) plasmid system screening;(2) CHO cell strain screening;(3) gene sequence acquisition and optimization;(4) recombinant expression plasmid construction and cell transfection;(5) high expression human interleukin-35 cell strain screening etc. The present application can be applied to interleukin-35 natural molecule preparation.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and in particular relates to a cell line that expresses human interleukin-35 efficiently, its preparation method and its application. Background Technology

[0002] Human interleukin-35 is a heterodimer composed of α-chain p35 and β-chain EBV-induced gene 3 (EBI3) linked by disulfide bonds. It belongs to the IL-12 family and is currently the only cytokine in this family with immunosuppressive activity. Its biological functions mainly include: (1) inhibiting the proliferation of T lymphocytes or B lymphocytes; (2) promoting the proliferation of Treg cells; (3) inhibiting the polarization of Th17 cells and the production of IL-17; and (4) inducing naive T lymphocytes or B lymphocytes to transform into IL-35-secreting Tregs (IL-35-induced regulatory T cells, iTR35) or Bregs (IL-35+Breg). Based on the biological functions of IL-35, it may play an important role in the regulation of immunosuppression and the formation of the immune tolerance microenvironment. Therefore, achieving efficient and stable expression of the natural human interleukin-35 protein molecule is crucial for its future applications.

[0003] However, existing methods cannot achieve efficient and stable expression of interleukin-35. For example, Chinese patent application CN106075401A discloses an IL-35-Fc fusion protein and the use of interleukin-35 in the preparation of drugs for treating autoimmune skin diseases. The synthesized IL-35-Fc fusion protein is a novel protein obtained by linking the Fc of human IgG1 to the C-terminus of the IL-35 protein. Although this method successfully prepared the IL-35-Fc fusion protein, this protein is a non-human natural protein, which not only limits its application scope, but also cannot obtain a stably expressed natural interleukin-35 protein molecule based on the method disclosed in the document.

[0004] In summary, how to obtain a highly efficient and stable expression of the natural human interleukin-35 protein molecule is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] This invention addresses the problem of existing technologies failing to achieve stable expression of the natural human interleukin-35 protein molecule by proposing a highly efficient human interleukin-35 cell line, its preparation method, and its applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a cell line that efficiently expresses human interleukin-35 includes the following steps: Through a plasmid system screening step, a dual plasmid system was selected as the preparation of the highly efficient human interleukin-35 expression cell line. By comparing the expression levels of interleukin-35 in CHO-K1-IL-35 recombinant cells and CHO-DG44 recombinant cells, CHO-K1 cells were selected as the CHO cell line for preparing a cell line that expresses human interleukin-35 efficiently. Among them, the CHO-K1-IL-35 recombinant cells are Chinese hamster ovary cells CHO-K1-IL-35, with accession number CCTCC No: C202205, taxonomically named Chinese hamster ovary cells, and were deposited at the China Center for Type Culture Collection on December 29, 2021, located at the China Center for Type Culture Collection of Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province. By transiently transfecting a dual-plasmid expression system carrying the human interleukin-35 gene sequence into the genome of CHO-K1 cells, a cell line that efficiently expresses human interleukin-35 was obtained through screening experiments and bioactivity assays.

[0007] In one embodiment, the method for preparing a cell line that efficiently expresses human interleukin-35 includes the following steps: Gene sequence acquisition and optimization: The natural amino acid and natural nucleotide sequences of the p35 and EBI3 subunits of the human interleukin-35 gene were obtained from the NCBI database. The natural nucleotide sequences of the p35 and EBI3 subunits were optimized to obtain the optimized p35 and EBI3 subunit sequences. Construction of recombinant expression plasmids and cell transfection: The natural nucleotide sequence of p35 subunit, the optimized sequence of p35 subunit, the natural nucleotide sequence of EBI3 subunit, and the optimized sequence of EBI3 subunit were combined with nucleotide sequences containing different signal peptides and synthesized into a dual plasmid system, which was then transfected into the genome of CHO-K1 cells to obtain four different CHO-K1 recombinant cells. Screening of cell lines expressing high-efficiency human interleukin-35: After the four different CHO-K1 recombinant cells were cultured for a period of time, the expression level of interleukin-35 in the cell supernatant was compared. After screening by small library, single cloning and bioactivity detection, the CHO-K1 recombinant cells with the highest expression level were finally screened as the cell lines expressing high-efficiency human interleukin-35.

[0008] In one embodiment, the p35 subunit optimized sequence is shown in SEQ ID NO.14, and the EBI3 subunit optimized sequence is shown in SEQ ID NO.10; The nucleotide sequences containing different signal peptides include natural signal peptides and murine signal peptides. The natural nucleotide sequence of the p35 subunit containing the natural signal peptide is shown in SEQ ID NO.13, the optimized nucleotide sequence of the p35 subunit containing the murine signal peptide is shown in SEQ ID NO.16, the natural nucleotide sequence of the EBI3 subunit containing the natural signal peptide is shown in SEQ ID NO.9, and the optimized nucleotide sequence of the p35 subunit containing the murine signal peptide is shown in SEQ ID NO.12.

[0009] In one embodiment, in the recombinant expression plasmid construction and cell transfection steps, the dual plasmid system is pcDNA3.1 plasmid and pXC17.4 plasmid, wherein the natural nucleotide sequence of the p35 subunit is synthesized onto the pXC17.4 plasmid, and the natural nucleotide sequence of the EBI3 subunit is synthesized onto the pcDNA3.1 plasmid; The four different CHO-K1 recombinant cells are: CHO-K1 recombinant cells with natural p35 subunit and EBI3 subunit natural nucleotide sequences of natural signal peptides, CHO-K1 recombinant cells with natural p35 subunit and EBI3 subunit natural nucleotide sequences of mouse signal peptides, CHO-K1 recombinant cells with optimized p35 subunit and EBI3 subunit sequences of natural signal peptides, and CHO-K1 recombinant cells with optimized p35 subunit and EBI3 subunit sequences of mouse signal peptides.

[0010] In one embodiment, the culture medium formula used for pressure screening in the high-efficiency expression of human interleukin-35 cell line screening step is: EmCD CHO medium + (0.5%-5.0%) H / T + (0.1%-2.0%) Anti-clumping Agent + (10μM-30μM) GS System 100ml Methionine sulfoximine.

[0011] The optimal culture medium formulation is: EmCD CHO medium (Eminence) + 1% H / T (Gibco) + 0.5% Anti-clumping Agent (Gibco) + 6mM L-glutamine (Gibco).

[0012] In one embodiment, the plasmid system screening step includes: The natural nucleotide sequences of the p35 and EBI3 subunits of the human interleukin-35 gene were obtained from the NCBI database. After codon optimization, they were synthesized into single and dual plasmid systems to construct expression plasmids, which were then transfected into the genome of CHO-DG44 cells to obtain CHO-DG44 recombinant cells I and II. After transient expression, the expression level of interleukin-35 in the cell supernatant of the two cells was compared to finally screen out the dual plasmid system as the plasmid system for preparing the high-efficiency human interleukin-35 expression cell line. The single plasmid system is pGN-M plasmid, and the dual plasmid system is pcDNA3.1 plasmid and pGN-M plasmid. The nucleotide sequence of the p35 subunit is synthesized into the pGN-M plasmid, and the nucleotide sequence of the EBI3 subunit is synthesized into the pcDNA3.1 plasmid.

[0013] In one implementation, the CHO cell line screening step includes: The natural nucleotide sequences of the p35 subunit and EBI3 subunit of the human interleukin-35 gene were obtained from the NCBI database. The sequences and the optimized sequences were synthesized into a dual plasmid system to construct expression plasmids, which were then transfected into the genome of CHO-K1 cells to obtain CHO-K1-IL-35 recombinant cells. After transient expression of CHO-K1-IL-35 in recombinant cells and CHO-DG44 in recombinant cells II, the expression levels of interleukin-35 in the cell supernatants of the two cells were compared, and CHO-K1 cells were finally selected as the CHO cell line for preparing a cell line that expresses human interleukin-35 efficiently.

[0014] The present invention also provides a highly efficient human interleukin-35 expression cell line, which is prepared by the method for preparing a highly efficient human interleukin-35 expression cell line described in any of the above embodiments.

[0015] In one embodiment, the expression level of the highly efficient human interleukin-35 cell line is 160-200 mg / L.

[0016] This invention also provides the application of a highly efficient human interleukin-35 cell line in the preparation of immunomodulators, antitumor drugs, and anti-inflammatory drugs.

[0017] Compared with the prior art, the advantages and positive effects of the present invention are as follows: 1. The present invention proposes a method for preparing a cell line that efficiently expresses human interleukin-35. This method combines the optimization and screening of single / dual plasmid systems, nucleotide sequences, and signal peptides. By synthesizing the human interleukin-35 gene sequence into a dual plasmid system, and then transfecting it into the genome of CHO-K1 cells by transient transfection, a cell line that can stably and efficiently express the natural protein molecule of human interleukin-35 is finally obtained after multiple screening experiments and bioactivity tests. 2. The method for preparing a high-efficiency human interleukin-35 cell line proposed in this invention has high interleukin-35 expression level and high biological activity, with the highest expression level reaching 160-200 mg / L, which can lay the foundation for subsequent large-scale production and practical application. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the Dot-blot measurement results of the single plasmid system and the dual plasmid system provided in the embodiments of the present invention; Figure 2 This is a schematic diagram of the Dot-blot assay results of the CHO-K1 cell dual plasmid system and the CHO-DG44 cell dual plasmid system provided in the embodiments of the present invention. Figure 3 This is a schematic diagram of the Dot-blot assay results of natural nucleotide series combined with different signal peptides provided in the embodiments of the present invention. Figure 4 This is a schematic diagram of the DOT detection results of cell supernatants from small-bank screening in 96-well plates of NN and NM groups provided in this embodiment of the invention; Figure 5 This is a 4x microscope image of a single-clone cell provided in an embodiment of the present invention. Figure 6 This is a schematic diagram of the Western-Blot identification results of monoclonal cells provided in an embodiment of the present invention; Figure 7 This is a schematic diagram of the batch results of the monoclonal cell line provided in the embodiments of the present invention; Figure 8 This is a schematic diagram showing the IL-35 quantification results of the supernatant of the superior monoclonal cell lines obtained from the 7 batches provided in this embodiment of the invention. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] This invention provides a method for preparing a cell line that efficiently expresses human interleukin-35, comprising the following steps: Through a plasmid system screening step, a dual plasmid system was selected as the preparation of the highly efficient human interleukin-35 expression cell line. By comparing the expression levels of interleukin-35 in CHO-K1-IL-35 recombinant cells and CHO-DG44 recombinant cells, CHO-K1 cells were selected as the CHO cell line for preparing a cell line that expresses human interleukin-35 efficiently. By transiently transfecting a dual-plasmid expression system carrying the human interleukin-35 gene sequence into the genome of CHO-K1 cells, a cell line that efficiently expresses human interleukin-35 was obtained through screening experiments and bioactivity assays.

[0021] In one specific implementation scheme, the method for preparing a cell line that efficiently expresses human interleukin-35 includes the following steps: S1. Gene Sequence Acquisition and Optimization: The natural amino acid and natural nucleotide sequences of the p35 and EBI3 subunits of the human interleukin-35 gene were obtained from the NCBI database. The natural nucleotide sequences of the p35 and EBI3 subunits were optimized to obtain the optimized p35 and EBI3 subunit sequences. In step S1 above, the sequence optimization is specifically performed on the GenScript codon optimization website (https: / / www.genscript.com / tools / gensmart-codon-optimization) to obtain an optimized sequence.

[0022] S2. Construction of recombinant expression plasmids and cell transfection: The natural nucleotide sequence of p35 subunit, the optimized sequence of p35 subunit, the natural nucleotide sequence of EBI3 subunit, and the optimized sequence of EBI3 subunit were combined with nucleotide sequences containing different signal peptides and synthesized into a dual plasmid system, which was then transfected into the genome of CHO-K1 cells to obtain four different CHO-K1 recombinant cells. S3. Screening of cell lines expressing high-efficiency human interleukin-35: After the four different CHO-K1 recombinant cells were cultured for a period of time, the expression level of interleukin-35 in the cell supernatant was compared. After screening by small library, single clonal screening and bioactivity detection, the CHO-K1 recombinant cells with the highest expression level were finally screened as cell lines expressing high-efficiency human interleukin-35.

[0023] In the above embodiments, the present invention proposes a method for preparing a cell line that efficiently expresses human interleukin-35. This method combines nucleotide sequence optimization and signal peptide screening. By synthesizing the human interleukin-35 gene sequence into a dual plasmid system, and then transfecting it into the genome of CHO-K1 cells via transient transfection, a cell line that can stably and efficiently express the natural human interleukin-35 protein molecule is finally obtained after multiple screening experiments and bioactivity tests. Using this cell line, the efficient and stable expression of the natural interleukin-35 protein molecule can be achieved, laying the foundation for subsequent large-scale production and application.

[0024] In one specific implementation, the p35 subunit optimized sequence is shown in SEQ ID NO.14, and the EBI3 subunit optimized sequence is shown in SEQ ID NO.10; The nucleotide sequences containing different signal peptides include natural signal peptides and murine signal peptides. The natural nucleotide sequence of the p35 subunit containing the natural signal peptide is shown in SEQ ID NO.13, the optimized nucleotide sequence of the p35 subunit containing the murine signal peptide is shown in SEQ ID NO.16, the natural nucleotide sequence of the EBI3 subunit containing the natural signal peptide is shown in SEQ ID NO.9, and the optimized nucleotide sequence of the p35 subunit containing the murine signal peptide is shown in SEQ ID NO.12.

[0025] In one specific implementation, in the recombinant expression plasmid construction and cell transfection steps, the dual plasmid system is pcDNA3.1 plasmid and pXC17.4 plasmid, wherein the natural nucleotide sequence of the p35 subunit is synthesized onto the pXC17.4 plasmid, and the natural nucleotide sequence of the EBI3 subunit is synthesized onto the pcDNA3.1 plasmid; The four different CHO-K1 recombinant cells are: CHO-K1 recombinant cells with natural p35 subunit and EBI3 subunit natural nucleotide sequences of natural signal peptides, CHO-K1 recombinant cells with natural p35 subunit and EBI3 subunit natural nucleotide sequences of mouse signal peptides, CHO-K1 recombinant cells with optimized p35 subunit and EBI3 subunit sequences of natural signal peptides, and CHO-K1 recombinant cells with optimized p35 subunit and EBI3 subunit sequences of mouse signal peptides.

[0026] In one specific implementation, the culture medium formula used for pressure screening in the high-efficiency expression of human interleukin-35 cell line screening step is: EmCD CHO medium + (0.5%-5.0%) H / T + (0.1%-2.0%) Anti-clumping Agent + (10μM-30μM) GS System 100ml Methionine sulfoximine.

[0027] In one specific implementation, the plasmid system screening step includes: The natural amino acid sequences of the p35 and EBI3 subunits of the human interleukin-35 gene were obtained from the NCBI database and their codons were optimized. They were then synthesized into a dual-plasmid system to construct expression plasmids, resulting in CHO-DG44 recombinant cells I. After adding a linker between the two subunits for codon optimization, the cells were transfected into the genome of CHO-DG44 cells to obtain CHO-DG44 recombinant cells II. After transient expression, the expression levels of interleukin-35 in the cell supernatants of the two cells were compared to finally select the dual-plasmid system as the plasmid system for preparing the high-efficiency human interleukin-35 expression cell line. The single plasmid system is pGN-M plasmid, and the dual plasmid system is pcDNA3.1 plasmid and pGN-M plasmid. The optimized nucleotide sequence of the p35 subunit is synthesized into the pGN-M plasmid, and the optimized nucleotide sequence of the EBI3 subunit is synthesized into the pcDNA3.1 plasmid.

[0028] In the above embodiments, in order to obtain a human interleukin-35 cell line with high efficiency and stable expression, the present invention further screened the plasmid system and CHO cell line used, and finally screened out the plasmid system and CHO cell line suitable for the construction of human interleukin-35 cell line.

[0029] In one specific implementation plan, the CHO cell line screening step includes: (1) Obtain the natural nucleotide sequences of the p35 subunit and EBI3 subunit of the human interleukin-35 gene from the NCBI database, synthesize the codon-optimized nucleotide sequences into the dual plasmid system to construct expression plasmids, and then transfect them into the genome of CHO-K1 cells to obtain CHO-K1-IL-35 recombinant cells; (2) After transient expression of CHO-K1-IL-35 recombinant cells and CHO-DG44 recombinant cells II, the expression level of interleukin-35 in the cell supernatant of the two cells was compared, and CHO-K1 cells were finally selected as the CHO cell line for preparing a cell line that expresses human interleukin-35 efficiently.

[0030] The present invention also provides a highly efficient human interleukin-35 expression cell line, which is prepared by the method for preparing a highly efficient human interleukin-35 expression cell line described in any of the above embodiments.

[0031] In one specific implementation, the expression level of the highly efficient human interleukin-35 cell line is 160-200 mg / L.

[0032] This invention also provides the application of a highly efficient human interleukin-35 cell line in the preparation of immunomodulators, antitumor drugs, and anti-inflammatory drugs.

[0033] This invention provides an immunomodulator, which is mainly prepared by the high-efficiency human interleukin-35 expression cell line described in any of the above embodiments. The immune diseases include, but are not limited to, psoriasis, lupus erythematosus, immune diabetes, and autoimmune uveitis.

[0034] This invention provides an anti-tumor drug, which is mainly prepared by the cell line that highly expresses human interleukin-35 as described in any of the above embodiments. The tumors include, but are not limited to, hepatocellular carcinoma, human lung adenocarcinoma, Hodgkin's lymphoma, acute myeloid leukemia, esophageal cancer, pancreatic ductal adenocarcinoma, cervical cancer, and colorectal cancer.

[0035] This invention provides an anti-inflammatory drug, which is mainly prepared by the high-efficiency human interleukin-35 expression cell line described in any of the above embodiments, wherein the inflammation includes, but is not limited to, ulcerative colitis, lupus nephritis and neuroinflammation.

[0036] To more clearly and in detail introduce the highly efficient human interleukin-35 expression cell line, its preparation method and its application provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0037] Example 1 This embodiment provides the plasmid system screening and CHO cell line screening steps in the preparation method of a high-efficiency human interleukin-35 expression cell line, specifically as follows: (1) Construction of IL-35 expression plasmid based on CHO-DG44 cells After retrieving the natural nucleotide sequences of the p35 and EBI3 subunits of the human interleukin-35 gene from the NCBI database, the two subunits were linked and optimized using a linker before being synthesized into the pGN-M plasmid (i.e., a single plasmid system). The optimized p35 subunit was synthesized into the pGN-M plasmid, and the optimized EBI3 subunit was synthesized into the pcDNA3.1 plasmid (the pGN-M plasmid and pcDNA3.1 plasmid constitute a dual plasmid system). The amino acid sequence and optimized nucleotide sequence of IL-35 are shown in SEQ ID NO. 7-8; the amino acid sequence and optimized nucleotide sequence of the EBI3 subunit of interleukin-35 are shown in SEQ ID NO. 1-2; and the amino acid sequence and optimized nucleotide sequence of the p35 subunit of interleukin-35 are shown in SEQ ID NO. 3-4. The amino acid sequence of the linker used in the single plasmid system is shown in SEQ ID NO. 5, and its nucleotide sequence is shown in SEQ ID NO. 6. (2) Construction of IL-35 expression plasmid based on CHO-K1 cells The natural nucleotide sequences of the p35 and EBI3 subunits of the human interleukin 35 gene were obtained from the NCBI database and synthesized into a dual plasmid system. The EBI3 subunit was synthesized into the pcDNA3.1 plasmid, and the p35 subunit was synthesized into the pXC17.4 plasmid. (3) Plasmid amplification (3-1) Plasmid transformation: ① Take 100 μl of competent bacteria, add an appropriate amount of plasmid DNA on ice, mix gently, incubate on ice for 20 min, heat shock at 32℃ for 90 s, and incubate on ice for 3 min; ② Add 200 μl of LB medium, incubate at 37℃ and shake at 150 rpm for 1 h, take an appropriate amount of bacterial solution and spread it on LB medium plates containing antibiotics, and incubate at 37℃ overnight; (3-2) Screening and identification of clones: The bacterial strain was streaked on LB medium (containing ampicillin) plates and incubated overnight at 37°C; after the colonies grew the next day, single and round colonies were selected, picked up with the tip of a pipette and added to 1 ml of liquid LB medium containing ampicillin resistance. The culture was then placed in a shaker at 37°C and incubated at 220 rpm / min for 4 h. 10 μl of the culture solution was added to 10 ml of liquid LB medium containing ampicillin resistance and incubated in a shaker at 37°C and 220 rpm / min for 14 h. (3-3) Endotoxin-free extraction of recombinant expression plasmid: Take 10 mL of bacterial culture, centrifuge at 5000×g for 10 min at room temperature to collect bacteria; discard the culture medium, add 500 μl of Solution I / RNase A mixture, and vortex to completely suspend the cells; transfer the solution to a new 2 mL centrifuge tube, add 500 μl of Solution II, gently invert to mix, and incubate at room temperature for 3 min; add 250 μl of pre-chilled N3 Buffer, and gently invert the centrifuge tube several times until a white flocculent precipitate forms; centrifuge at the maximum speed (13000×g) for 10 min at room temperature, and transfer the supernatant to a new 1.5 mL centrifuge tube; add 0.1 times the volume of ETR to the supernatant. Mix the solution by inverting it 10 times and incubate on ice for 10 minutes; incubate at 42°C for 5 minutes until the solution becomes cloudy again; centrifuge at 12000×g for 3 minutes at 25°C, transfer the upper aqueous phase (containing DNA) to a new 1.5mL centrifuge tube, add 0.5 times the volume of anhydrous ethanol, mix by inverting it 6-7 times, and incubate at room temperature for 1-2 minutes; transfer the mixture to a 2mL collection tube of a HiBind DNA binding column, centrifuge at 10000×g for 1 minute at room temperature, discard the filtrate, and repeat until all the mixture has been transferred through the column; reassemble the column into the collection tube, add 500μl of HBC Buffer, centrifuge at maximum speed for 5 minutes, reassemble the column into the collection tube, add 700μl of DNA Wash Buffer, centrifuge at maximum speed for 1 minute, discard the filtrate, repeat twice, centrifuge for 2 minutes each time. Add 60 μl of Elution Buffer, let stand for 1 min, centrifuge at maximum speed for 1 min to elute DNA, collect in a 1.5 cm sterile centrifuge tube, and store at -20 °C; (4) Instantaneous transfection Resuscitate CHO-DG44 and CHO-K1 cells for passage and adaptation culture. Once the cell growth status returns to normal, prepare for transfection. Observe and select cells in the logarithmic growth phase and those growing well. Transfect at a rate of 3 × 10⁻⁶ cells / year. 6 2 ml of the transfection solution was seeded into each well of a 6-well plate. The transfection reagent used was FectoPRO (purchased from Polyplus Transfection). The transfection complex was prepared by mixing plasmid and FectoPRO (µg / µl) at a ratio of 1:2. For the dual plasmid group, the two plasmids were added at a ratio of 1:1. After incubating the transfection complex at room temperature for 10 min, the cells were added to the 6-well plate. After culturing for 48 h, the supernatant was collected and the cells were screened to obtain CHO-DG44-P35+EBI3 recombinant cells, CHO-DG44-IL-35 recombinant cells, and CHO-K1-IL-35 recombinant cells.

[0038] High-expressing monoclonal cell lines were identified using Dot-blot assays. The Dot-blot results are as follows: Figure 1-2 As shown.

[0039] from Figure 1 As can be seen, the expression level of the single plasmid system based on CHO-DG44 cells (left figure) is significantly lower than that of the dual plasmid system (right figure). Therefore, the dual plasmid system was ultimately chosen as the expression system for interleukin-35. Figure 2 As can be seen, even with the same dual-plasmid expression system, the expression level of CHO-K1 cells (right figure) is significantly higher than that of CHO-DG44 cells (left figure). Therefore, this invention selects the dual-plasmid system as the expression system for interleukin-35 and selects CHO-K1 cells as the cell line.

[0040] Example 2 This embodiment provides a method for preparing a high-efficiency human interleukin-35 cell line. Based on the experimental results obtained in Example 1, a dual-plasmid system with higher expression levels was selected for plasmid construction when preparing the interleukin-35 cell line. CHO-K1 was selected as the cell line, specifically as follows: (1) Gene sequence acquisition and optimization The natural nucleotide sequences of the p35 subunit and EBI3 subunit of the human interleukin-35 gene were obtained from the NCBI database as shown in SEQ ID NO.4 and SEQ ID NO.2. Codon optimization was performed on the natural nucleotide sequences of the p35 subunit and EBI3 subunit with the mouse signal peptide replaced, resulting in optimized p35 and EBI3 subunit sequences. Specifically, the natural nucleotide sequences of the p35 subunit of interleukin-35 containing both the natural and mouse signal peptides are shown in SEQ ID NO.13 and 14; the optimized nucleotide sequences of the p35 subunit of interleukin-35 containing both the natural and mouse signal peptides are shown in SEQ ID NO.15 and 16; the natural nucleotide sequences of the EBI3 subunit of interleukin-35 containing both the natural and mouse signal peptides are shown in SEQ ID NO.9-10; and the optimized nucleotide sequences of the EBI3 subunit of interleukin-35 containing both the natural and mouse signal peptides are shown in SEQ ID NO.11-12.

[0041] (2) Construction of recombinant expression plasmid The natural nucleotide sequences of the p35 subunit and the EBI3 subunit were combined with nucleotide sequences containing different signal peptides to obtain the following groups: A. Natural sequence + natural signal peptide (NN), B. Natural sequence + mouse signal peptide (NM); Codon optimization of A and B yielded the following groups: C. Optimized A sequence (ON), D. Optimized B sequence (OM). The above nucleotide sequences were synthesized into a dual plasmid system, with the EBI3 subunit synthesized into the pcDNA3.1 plasmid and the p35 subunit synthesized into the pXC17.4 plasmid.

[0042] (3) Plasmid amplification (3-1) Plasmid transformation: ① Take 100 μl of competent bacteria, add an appropriate amount of plasmid DNA on ice, mix gently, incubate on ice for 20 min, heat shock at 32℃ for 90 s, and incubate on ice for 3 min; ② Add 200 μl of LB medium, incubate at 37℃ and shake at 150 rpm for 1 h, take an appropriate amount of bacterial solution and spread it on LB medium plates containing antibiotics, and incubate at 37℃ overnight; (3-2) Screening and identification of clones: The bacterial strain was streaked on LB medium (containing ampicillin) plates and incubated overnight at 37°C; after the colonies grew the next day, single and round colonies were selected, picked up with the tip of a pipette and added to 1 ml of liquid LB medium containing ampicillin resistance. The culture was then placed in a shaker at 37°C and incubated at 220 rpm / min for 4 h. 10 μl of the culture solution was added to 10 ml of liquid LB medium containing ampicillin resistance and incubated in a shaker at 37°C and 220 rpm / min for 14 h. (3-3) Endotoxin-free extraction of recombinant expression plasmid: Take 10 mL of bacterial culture, centrifuge at 5000×g for 10 min at room temperature to collect bacteria; discard the culture medium, add 500 μl of Solution I / RNase A mixture, and vortex to completely suspend the cells; transfer the solution to a new 2 mL centrifuge tube, add 500 μl of Solution II, gently invert to mix, and incubate at room temperature for 3 min; add 250 μl of pre-chilled N3 Buffer, and gently invert the centrifuge tube several times until a white flocculent precipitate forms; centrifuge at the maximum speed (13000×g) for 10 min at room temperature, and transfer the supernatant to a new 1.5 mL centrifuge tube; add 0.1 times the volume of ETR to the supernatant. Mix the solution by inverting it 10 times and incubate on ice for 10 minutes; incubate at 42°C for 5 minutes until the solution becomes cloudy again; centrifuge at 12000×g for 3 minutes at 25°C, transfer the upper aqueous phase (containing DNA) to a new 1.5mL centrifuge tube, add 0.5 times the volume of anhydrous ethanol, mix by inverting it 6-7 times, and incubate at room temperature for 1-2 minutes; transfer the mixture to a 2mL collection tube in a HiBind DNA binding column, centrifuge at 10000×g for 1 minute at room temperature, discard the filtrate, and repeat until all the mixture has been transferred through the column; reassemble the column into the collection tube, add 500μl of HBC Buffer, centrifuge at maximum speed for 5 minutes, reassemble the column into the collection tube, add 700μl of DNAWash Buffer, centrifuge at maximum speed for 1 minute, discard the filtrate, repeat twice, centrifuge for 2 minutes each time. Add 60 μl of Elution Buffer, let stand for 1 min, centrifuge at maximum speed for 1 min to elute DNA, collect in a 1.5 cm sterile centrifuge tube, and store at -20 °C; (4) Instantaneous transfection Resuscitate CHO-K1 cells and passage them for adaptation culture. Once the cell growth status returns to normal, prepare for transfection. Observe and select cells in the logarithmic growth phase and those growing well. Transfect at a rate of 3 × 10⁻⁶ cells / year. 62 ml of the solution was seeded per well into a 6-well plate. The transfection reagent used was FectoPRO (purchased from Polyplus Transfection). The transfection complex was prepared at a ratio of 1:1 between the two plasmids and FectoPRO (µg / µl). After incubating the transfection complex at room temperature for 10 min, it was added to the cells in the 6-well plate. After culturing for 48 h, the supernatant was collected and the cells were screened to obtain different CHO-K1 cell lines. The CHO-K1 cell culture medium was: EmCD CHO medium (Eminence) + 1% H / T (Gibco) + 0.5% Anti-clumping Agent (Gibco) + 6 mM L-glutamine (Gibco). (5) Dot-Blot detection of IL-35 in cell supernatant ① Sample loading: Cut an appropriate size of transfer membrane according to the sample quantity, add 10 μl of cell supernatant to the transfer membrane, and air dry in a fume hood; ② Blocking: Immerse in 20 ml of 5% milk-PBST and place on a shaker at room temperature for 90 min; after blocking, discard the milk, and wash three times with 10 ml of PBST, immersing for 5 min each time; ③ Blocking: Block with pre-prepared 5% skim milk and shake for 1 h; ④ Incubation with primary antibody: After blocking, incubate with primary antibody at 4°C overnight; the primary antibody is Mouse anti-human IL-35 P35 (R&D), which is diluted with PBST at a concentration of 1:1000; wash the NC membrane after overnight incubation with prepared 1×PBST buffer, washing 3 times for 10 min each time; ⑤ Incubation with secondary antibody: the secondary antibody Goat anti MouseHRP (Genscript) is diluted with PBST at a concentration of 1:1000. Incubate at room temperature for 45 min, then discard the antibody. Wash three times with PBST, 5 min each time. ⑥ Exposure: Prepare the exposure substrate according to a 1:1 ratio of solution A to solution B. Remove the membrane and allow it to dry slightly before placing it on a thin plastic film. Add the freshly prepared exposure substrate and react in the dark for 2 min to develop color. Dot-blot detection results are as follows Figure 3 As shown, the results of different combinations of signal peptides and nucleotide sequences show that the expression level of natural sequence + mouse signal peptide group (NM) is the highest, followed by natural sequence + natural signal peptide group (NN).

[0043] Example 3 Based on the experimental results of Example 2, this embodiment performs monoclonal screening on two groups of cells obtained 48 hours after transient transfection with "natural sequence + mouse signal peptide group (NM)" and "natural sequence + natural signal peptide group (NN)". Specifically: Cells from the NM-8E wells with high expression levels and good cell condition, as described in Example 2, were selected for expansion culture. Once the cell viability reached 90%, single-clone selection was performed using the limiting dilution method. Cells were seeded at a density of 0.4 cells / well in a 96-well plate with 100 μl of culture medium per well. Cell clones in each well were observed and photographed under a microscope every other day (e.g., ...). Figure 5 After culturing for 14-21 days, the confluence of cells in the monoclonal cell wells was observed. Multiple monoclonal cell lines were selected for expanded culture. The monoclonal cells were seeded into 24-well plates, and the cell density and viability were continuously monitored. Cells with good growth were selected for batch experiments (i.e., batch control experiments), i.e., at a ratio of 5 × 10⁶ cells / well. 5 Cell lines were seeded into 24-well plates at a density of 1 cell / ml, and after 6 days of continuous culture, the supernatant was collected for DOT assay (see [link to DOT assay]). Figure 7 Based on the test results, the supernatant of 7 cell lines was selected, serially diluted, and compared with the serially diluted results of the positive sample to determine the cell line with the highest expression level.

[0044] Experimental results: A monoclonal cell line 3E9 with high expression was successfully obtained (see experimental results below). Figure 8 The expression level is 160-200 mg / L when cultured using the above method.

[0045] Furthermore, the IL-35 produced by the cell line of this invention has a significant inhibitory effect on the proliferation of some tumor cells, including human non-small cell lung cancer cells (A549), and has good binding activity to its receptor chain gp130.

[0046] Example 4 In this embodiment, the supernatant of stable monoclonal cells from Example 3 was collected, and the expression of IL-35 in the supernatant was identified using Western blotting. Specifically: Sample preparation: Mix the sample with 4× reducing Loading Buffer and heat at 100℃ for 5 minutes, or mix the sample with 4× non-reducing Loading Buffer; Electrophoresis: This is a two-step process: First, adjust the electrophoresis apparatus to a constant voltage of 80V and run the electrophoresis for about 20 minutes. When the sample reaches the separating gel, adjust the voltage to 120V. Once the target protein is sufficiently separated, the electrophoresis can be stopped; Transfer: Remove the glass plate and slowly pry it open with the gel plate. Gently remove the gel and immerse it in transfer buffer; cut a PVDF membrane of appropriate size and immerse it in methanol for 1-2 minutes for activation; after the protective paper on both sides of the PVDF membrane is completely soaked, immerse and rinse the PVDF membrane in transfer buffer, while fully soaking two transfer sponge pads and filter paper; place the membrane in the following order: black plate - fiber pad - filter paper - gel - PVDF membrane - filter paper - fiber pad - white plate, and gently roll out excess air bubbles with a glass rod after each layer to prevent air bubbles from affecting the transfer effect; clamp the plate and place it in the transfer apparatus, adjust the constant current to 200mA, and adjust the transfer time according to the protein size; blocking: after the transfer, rinse the PVDF membrane with PBS for 10 minutes, and block it with 5% skim milk powder on a shaker at room temperature for 2 hours; primary antibody incubation: dilute Mouse anti-human IL-35 with PBST according to the instructions for the P35 antibody. P35 (R&D) antibody, incubated overnight at 4°C; Secondary antibody incubation: After overnight incubation, remove the PVDF membrane and wash it three times with PBST at room temperature, 10 min each time; Dilute Goat anti-Mouse HRP (Genscript) with PBST, immerse the PVDF membrane in the secondary antibody incubation solution, and incubate on a shaker at room temperature for 2 h; Washing: After secondary antibody incubation, wash the PVDF membrane thoroughly three times with PBST, 10 min each time; then wash three times with TBS, 10 min each time; Chemiluminescence and imaging: Use the ECL colorimetric kit (BIO-RED), mix solutions A and B in equal proportions in a centrifuge tube, wrap with aluminum foil to protect from light, and shake well. For development, carefully place the washed PVDF membrane face up in a clean dish, evenly add an appropriate amount of ECL developing solution, place it on the ImageQuant LAS 4000 mini imaging instrument chassis, open the accompanying software, adjust the focus and exposure time, and obtain the protein immunoblot chemiluminescence image. The results are as follows. Figure 6 As shown.

[0047] Depend on Figure 6 It can be seen that the recombinant human IL-35 expressed by CHO cells is a glycosylated protein with a molecular weight of approximately 69 kDa. As shown in the figure below, the first band is IL-35 in its unreduced state, and the third band is the P35 subunit of the reduced IL-35. SEQUENCE LISTING <110> Kanglitai Biopharmaceutical (Qingdao) Co., Ltd. <120> Highly efficient human interleukin-35 expression cell line, its preparation method and application <130> 20220111 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 229 <212> PRT <213> The amino acid sequence of the EBI3 subunit of IL-35 (containing the natural signal peptide). <400> 1 Met Thr Pro Gln Leu Leu Leu Ala Leu Val Leu Trp Ala Ser Cys Pro 1 5 10 15 Pro Cys Ser Gly Arg Lys Gly Pro Pro Ala Ala Leu Thr Leu Pro Arg 20 25 30 Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala Val Asp Cys Ser Trp 35 40 45 Thr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro Val Ser Phe Ile Ala 50 55 60 Thr Tyr Arg Leu Gly Met Ala Ala Arg Gly His Ser Trp Pro Cys Leu 65 70 75 80 Gln Gln Thr Pro Thr Ser Thr Ser Cys Thr Ile Thr Asp Val Gln Leu 85 90 95 Phe Ser Met Ala Pro Tyr Val Leu Asn Val Thr Ala Val His Pro Trp 100 105 110 Gly Ser Ser Ser Ser Phe Val Pro Phe Ile Thr Glu His Ile Ile Lys 115 120 125 Pro Asp Pro Pro Glu Gly Val Arg Leu Ser Pro Leu Ala Glu Arg Gln 130 135 140 Leu Gln Val Gln Trp Glu Pro Pro Gly Ser Trp Pro Phe Pro Glu Ile 145 150 155 160 Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg Gln Gly Ala Ala Arg 165 170 175 Phe His Arg Val Gly Pro Ile Glu Ala Thr Ser Phe Ile Leu Arg Ala 180 185 190 Val Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val Ala Ala Gln Asp Leu 195 200 205 Thr Asp Tyr Gly Glu Leu Ser Asp Trp Ser Leu Pro Ala Thr Ala Thr 210 215 220 Met Ser Leu Gly Lys 225 <210> 2 <211> 687 <212> DNA <213> Optimized nucleotide sequence of the EBI3 subunit of IL-35 (containing natural signal peptide) <400> 2 atgactccac aactactact agcactagtg ctgtgggcct cctgtccacc ctgttctggc 60 agaaagggcc ctcccgcagc cctgaccctg cccagggtcc agtgcagagc ctctcggtat 120 cctatcgctg tggactgctc ttggaccctg cctccagccc ctaactccac cagccccgtg 180 tccttcatcg ctacctaccg gctcggaatg gccgctagag gccttcctg gccttgcctg 240 cagcagaccc ctacctctac atcctgcaca atcaccgacg tgcagctgtt ttccatggct 300 ccttacgtgc tgaacgtgac cgccgtgcac ccttggggat cttctagctc cttcgtgcca 360 tttatcaccg agcacatcat caagcctgac ccccctgaag gcgtgcggct gtctcctctg gccgagagac agctgcaggt gcagtggga cctcctggct cctggccttt ccctgagatc 480 ttctctctga aatactggat cagatacaag cggcagggcg ctgctcgctt ccacagagtg ggccctatcg aggccacctc cttcattctg aggctgtcc ggcctagagc ccggtactac 600 gtgcaagtgg ccgcccagga tctgaccgat tacggcgagc tgtccgactg gagcctgccc 660 gccacagcta ccatgtccct gggcaag 687 <210> 3 <211> 219 <212> PRT <213> IL-35 or P35 is a slightly less expensive type of IL-35. <400> 3 Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val Leu Leu 1 5 10 15 Asp His Leu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro Asp Pro 20 25 30 Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val 35 40 45 Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys 50 55 60 Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser 65 70 75 80 Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys 85 90 95 Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala 100 105 110 Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr 115 120 125 Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys 130 135 140 Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu 145 150 155 160 Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr 165 170 175 Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys 180 185 190 Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr 195 200 205 Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser 210 215 <210> 4 <211> 657 <212> DNA <213> Optimized nucleotide sequence of IL-35's P35 subunit (containing natural signal peptide) <400> 4 atgtgcccag cacgaagtct actactagtg gctaccctgg tgctgctcga tcatctgtcc 60 ctggctagaa acctgcctgt ggctacccct gatcctggca tgttcccttg cttgcaccac 120 agccagaacc tgctgagagc cgtgtccaac atgctgcaga aggccagaca gaccctggag 180 ttctacccct gtacctctga ggaaatcgac cacaggaca tcaccaagga caagacatct 240 accgtggagg cctgcctgcc tctggaactg accaagaacg agtcctgtct gaactccaga 300 gagaccagct tcatcaccaa tggctcctgc ctggcttctc ggaagacatc cttcatgatg 360 gccctgtgcc tgtcttccat ctacgaggat ctgaagatgt accaggtgga atttaagacc 420 atgaacgcca aactgctgat ggaccccaag cggcagatct tcctggacca aaacatgctg 480 gctgtgatcg acgagctgat gcaggctctg aacttcaact ccgagaccgt gccacagaaa 540 agctctctgg aagagcctga cttctataag accaagatca agctctgcat cctgctgcac 600 gcctttcgga tcagagccgt cacaattgac cgcgtgatgt cctacctgaa tgcctcc 657 <210> 5 <211> 15 <212> PRT <213> The amino acid sequence of the linker <400> 5 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 <210> 6 <211> 45 <212> DNA <213> Linker nucleotide sequence <400> 6 ggtggtggtg gttctggtgg tggtggtggt tctggtggtg gttct 45 <210> 7 <211> 441 <212> PRT <213> The amino acid sequence of IL-35 (including the natural signal peptide). <400> 7 Met Thr Pro Gln Leu Leu Leu Ala Leu Val Leu Trp Ala Ser Cys Pro 1 5 10 15 Pro Cys Ser Gly Arg Lys Gly Pro Pro Ala Ala Leu Thr Leu Pro Arg 20 25 30 Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala Val Asp Cys Ser Trp 35 40 45 Thr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro Val Ser Phe Ile Ala 50 55 60 Thr Tyr Arg Leu Gly Met Ala Ala Arg Gly His Ser Trp Pro Cys Leu 65 70 75 80 Gln Gln Thr Pro Thr Ser Thr Ser Cys Thr Ile Thr Asp Val Gln Leu 85 90 95 Phe Ser Met Ala Pro Tyr Val Leu Asn Val Thr Ala Val His Pro Trp 100 105 110 Gly Ser Ser Ser Ser Phe Val Pro Phe Ile Thr Glu His Ile Ile Lys 115 120 125 Pro Asp Pro Pro Glu Gly Val Arg Leu Ser Pro Leu Ala Glu Arg Gln 130 135 140 Leu Gln Val Gln Trp Glu Pro Pro Gly Ser Trp Pro Phe Pro Glu Ile 145 150 155 160 Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg Gln Gly Ala Ala Arg 165 170 175 Phe His Arg Val Gly Pro Ile Glu Ala Thr Ser Phe Ile Leu Arg Ala 180 185 190 Val Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val Ala Ala Gln Asp Leu 195 200 205 Thr Asp Tyr Gly Glu Leu Ser Asp Trp Ser Leu Pro Ala Thr Ala Thr 210 215 220 Met Ser Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser 225 230 235 240 Gly Gly Gly Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met 245 250 255 Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn 260 265 270 Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser 275 280 285 Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val 290 295 300 Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn 305 310 315 320 Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg 325 330 335 Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp 340 345 350 Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu 355 360 365 Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val 370 375 380 Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro 385 390 395 400 Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys 405 410 415 Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp 420 425 430 Arg Val Met Ser Tyr Leu Asn Ala Ser 435 440 <210> 8 <211> 1323 <212> DNA <213> IL-35 optimized nucleotide sequence (containing natural signal peptide) <400> 8 atgactccac aactactact agcactagtg ctgtgggcct cttgtcctcc ttgttctggc 60 agaaaaggac ctccagctgc tctgaccctg cctcgggtgc agtgccgggc aagtcggtac 120 cccattgccg tggactgctc ctggaccctg cctcctgctc ctaactccac ctcccctgtg 180 tccttcatcg ccacctacag actgggcatg gccgctagag gccattcttg gccctgtctg 240 cagcagaccc ccacctccac atcttgcacc atcaccgacg tgcagctgtt ctccatggct 300 ccttacgtgc tgaacgtgac cgccgtgcac ccttggggct ctagctcttc tttcgtgccc 360 ttcatcaccg agcacatcat caagcctgat ccccctgagg gcgtgcggct gtccccactc 420 gccgagagac agctgcaagt gcagtgggag cccccaggct cctggccttt tcctgaaatc 480 ttcagcctga agtactggat cagatacaag agacagggcg ccgccagatt ccacagagtg 540 ggcccaatcg aggctacctc tttcatcctg agagccgtca ggcctagagc cagatactac 600 gtgcaagtgg ctgctcagga cctgaccgac tacggcgagc tgtccgactg gtccctgcct 660 gccaccgcta caatgtccct aggcaagggt ggtggtggtt ctggtggtgg tggtggttct 720 ggtggtggtt ctcggaacct gcccgtggcc acccctgatc ctggcatgtt cccctgtctg 780 caccattccc agaacctgct gcgcgccgtc tccaacatgc tgcagaaggc cagacagact 840 ctcgaatttt atccttgcac atctgaggaa atcgatcacg aggacatcac aaaagacaag 900 acctctaccg tggaagcctg cctgcctctg gaactgacca agaacgagtc ttgcctgaat 960 agccgggaga caagctttat caccaacgga tcttgcctgg ccagccgcaa aacatccttc 1020 atgatggccc tgtgcttgtc ttccatctac gaggatctga agatgtacca ggttgaattc 1080 aagaccatga acgctaagct gctgatggac cctaagcggc agatcttcct ggaccagaac 1140 atgctggctg tgatcgacga gctgatgcag gctctgaact tcaactccga gaccgtgcct 1200 cagaagtcca gcctggaaga gcctgatttc tacaagacca agatcaagct gtgcatcctg 1260 ctgcacgcct tcagaatcag agctgtgacc atcgacaggg tcatgtccta cctgaatgcc 1320 tcc 1323 <210> 9 <211> 696 <212> DNA <213> Natural nucleotide sequence of EBI3 subunit (including natural signal peptide) <400> 9 gccaccatga ccccgcagct tctcctggcc cttgtcctct gggccagctg cccgccctgc 60 agtggaagga aagggccccc agcagctctg acactgcccc gggtgcaatg ccgagcctct 120 cggtacccga tcgccgtgga ttgctcctgg accctgccgc ctgctccaaa ctccaccagc 180 cccgtgtcct tcattgccac gtacaggctc ggcatggctg cccggggcca cagctggccc 240 tgcctgcagc agacgccaac gtccaccagc tgcaccatca cggatgtcca gctgttctcc 300 atggctccct acgtgctcaa tgtcaccgcc gtccacccct ggggctccag cagcagcttc 360 gtgcctttca taacagagca catcatcaag cccgaccctc cagaaggcgt gcgcctaagc 420 cccctcgctg agcgccagct acaggtgcag tgggagcctc ccgggtcctg gcccttccca 480 gagatcttct cactgaagta ctggatccgt tacaagcgtc agggagctgc gcgcttccac 540 cgggtggggc ccattgaagc cacgtccttc atcctcaggg ctgtgcggcc ccgagccagg 600 tactacgtcc aagtggcggc tcaggacctc acagactacg gggaactgag tgactggagt 660 ctccccgcca ctgccacaat gagcctgggc aagtaa 696 <210> 10 <211> 693 <212> DNA <213> Natural nucleotide sequence of the EBI3 subunit (including murine signal peptide) <400> 10 gccaccatgg gctggagctg catcatcctg ttcctggtgg ccaccgccac cggcgtgcac 60 agcaggaaag ggcccccagc agctctgaca ctgccccggg tgcaatgccg agcctctcgg 120 tacccgatcg ccgtggattg ctcctggacc ctgccgcctg ctccaaactc caccagcccc 180 gtgtccttca ttgccacgta caggctcggc atggctgccc ggggccacag ctggccctgc 240 ctgcagcaga cgccaacgtc caccagctgc accatcacgg atgtccagct gttctccatg 300 gctccctacg tgctcaatgt caccgccgtc cacccctggg gctccagcag cagcttcgtg 360 cctttcataa cagagcacat catcaagccc gaccctccag aaggcgtgcg cctaagcccc 420 ctcgctgagc gccagctaca ggtgcagtgg gagcctcccg ggtcctggcc cttcccagag 480 atcttctcac tgaagtactg gatccgttac aagcgtcagg gagctgcgcg cttccaccgg 540 gtggggccca ttgaagccac gtccttcatc ctcagggctg tgcggccccg agccaggtac 600 tacgtccaag tggcggctca ggacctcaca gactacgggg aactgagtga ctggagtctc 660 cccgccactg ccacaatgag cctgggcaag taa 693 <210> 11 <211> 698 <212> DNA <213> Optimized nucleotide sequence of EBI3 subunit (including natural signal peptide) <400> 11 gccaccatga ctccacaact actactagca ctggtgctgt gggcctcttg tcctccttgt tctggcagaa agggccctcc agccgccctg accctgccta gagtccagtg cagagcctcc cggtatccta tcgccgtgga ctgctcctgg accctcccac ccgctcctaa ctccaccagc 180 cccgtgtcct tcattgccac ctacagactg ggcatggctg ccgaggcca ctcctggcct 240 tgcctgcagc agacccctc atctacatcc tgcaccatca ccgatgtgca gctgttctcc atggcccctt acgtgctgaa cgtgaccgcc gtgcatcctt ggggatcctc ttctagcttc 360 gtgccttca tcaccgagca catcatcaag ccagatcctc ccgagggcgt gcggctgtct 420 cctctggccg aaagacagct gcaggtgcag tgggaacccc ctggctcttg gcccttccct 480 gagatcttta gcctgaagta ctggatccgg tacaagcggc agggcgctgc tcggttccac 540 agagtgggcc ccatcgaggc cacctccttt atcctgagag ctgtcagacc tcggggcccgc 600 tactacgtgc aagtggccgc tcaggacctg acagactcg gcgagctgtc cgactggtcc 660 ctgcctgcta ccgctaccat gtctctggga aaatgtaa 698 <210> 12 <211> 696 <212> DNA <213> Optimized nucleotide sequence of EBI3 subunit (including murine signal peptide) <400> 12 gccaccatgg gttggagttg catcatccta ttcctggtgg ccaccgccac cggcgttcat 60 tctcggaagg gcccccccgc cgctctgacc ctgcctagag tccagtgccg ggcttctcgc 120 taccctatcg ccgtggactg ctcttggacc ctcccacctg ctcctaactc caccagccct 180 gtgtccttta ttgccaccta cagactgggc atggccgcta gaggccactc ctggccttgc 240 ctgcagcaga cccctacatc taccagctgt acaatcaccg atgtgcagct gttctccatg 300 gccccttatg tgctgaacgt gaccgccgtg cacccttggg gctcttcttc ctccttcgtg 360 ccatttatca ccgagcacat catcaagccc gatcctcctg aaggcgtgcg gctgtctcct 420 ctggccgaga gacagctgca ggtgcagtgg gagcctccag gatcctggcc tttccccgaa 480 atcttctctc tgaagtactg gatccggtac aagcggcagg gcgccgccag attccacaga 540 gtgggcccta tcgaggccac ctccttcatc ctgagagccg tccggcctag agctcgctac 600 tacgtgcaag tggctgctca ggacctgacc gactacggcg agctgtccga ctggtccctg 660 cctgctaccg ctaccatgtc cctgggaaaa tgataa 696 <210> 13 <211> 624 <212> DNA <213> Natural nucleotide sequence of P35 subunit (including natural signal peptide) <400> 13 gccaccatgt gtccagcgcg cagcctcctc cttgtggcta ccctggtcct cctggaccac 60 ctcagtttgg ccagaaacct ccccgtggcc actccagacc caggaatgtt cccatgcctt 120 caccactccc aaaacctgct gagggccgtc agcaacatgc tccagaaggc cagacaaact 180 ctagaatttt acccttgcac ttctgaagag attgatcatg aagatatcac aaaagataaa 240 accagcacag tggaggcctg tttaccattg gaattaacca agaatgggag ttgcctggcc 300 tccagaaaga cctcttttat gatggccctg tgccttagta gtatttatga agacttgaag 360 atgtaccagg tggagttcaa gaccatgaat gcaaagcttc tgatggatcc taagaggcag 420 atctttctag atcaaaacat gctggcagtt attgatgagc tgatgcaggc cctgaatttc 480 aacagtgaga ctgtgccaca aaaatcctcc cttgaagaac cggattttta taaaactaaa 540 atcaagctct gcatacttct tcatgctttc agaattcggg cagtgactat tgatagagtg 600 atgagctatc tgaatgcttc ctaa 624 <210> 14 <211> 615 <212> DNA <213> Native nucleotide sequence of P35 subunit (including murine signal peptide) <400> 14 gccaccatgg gctggagctg catcatcctg ttcctggtgg ccaccgccac cggcgtgcac 60 agcagaaacc tccccgtggc cactccagac ccaggaatgt tcccatgcct tcaccactcc 120 caaaacctgc tgagggccgt cagcaacatg ctccagaagg ccagacaaac tctagaattt 180 tacccttgca cttctgaaga gattgatcat gaagatatca caaaagataa aaccagcaca 240 gtggaggcct gtttaccatt ggaattaacc aagaatggga gttgcctggc ctccagaaag 300 acctctttta tgatggccct gtgccttagt agtatttatg aagacttgaa gatgtaccag 360 gtggagttca agaccatgaa tgcaaagctt ctgatggatc ctaagaggca gatctttcta 420 gatcaaaaca tgctggcagt tattgatgag ctgatgcagg ccctgaattt caacagtgag 480 actgtgccac aaaaatcctc ccttgaagaa ccggattttt ataaaactaa aatcaagctc 540 tgcatacttc ttcatgcttt cagaattcgg gcagtgacta ttgatagagt gatgagctat 600 ctgaatgctt cctaa 615 <210> 15 <211> 669 <212> DNA <213> Optimized nucleotide sequence of P35 subunit (including natural signal peptide) <400> 15 gccaccatgt gcccagcacg aagtctacta ctcgtggcca ccctggtgct gctggatcac 60 ctgtctctgg cccggaacct gcctgtggct accccagatc ctggcatgtt cccctgtctg 120 caccactccc aaaatctgct gagagccgtg tccaacatgc tgcagaaggc cagacagacc 180 ctcgagtttt atccttgcac ctccgaggaa atcgaccacg aggacatcac caaggacaag 240 accagcacag tcgaggcctg tctgcctctg gaactgacca agaacgagtc ctgcctgaac 300 tccagagaga catctttcat taccaacggc tcttgcctgg cctccagaaa aacatccttc 360 atgatggccc tgtgcctgtc ctccatctac gaggatctga agatgtacca ggtggaattc 420 aagaccatga acgccaagct gctgatggac cccaagcgcc agatcttcct ggaccagaac 480 atgctggctg tgatcgacga gctgatgcag gctctgaact tcaactctga aaccgtgcct 540 cagaaaagca gcctggaaga gcctgacttc tacaagacca agatcaagct gtgcatcctg 600 ctgcatgctt ttcggatccg ggctgtgacc atcgacagag tgatgtccta cctgaatgct 660 tcttgataa 669 <210> 16 <211> 660 <212> DNA <213> Optimized nucleotide sequence of P35 subunit (including murine signal peptide) <400> 16 gccaccatgg gttggagttg catcatccta tttctggtgg ccaccgctac aggcgtgcac 60 tcccgcaacc tgcctgtggc tacacctgat cctggaatgt tcccatgtct gcaccactct 120 caaaatctcc tgagagccgt gtccaacatg ctgcagaaag ccagacagac cctggaattt 180 tatccctgca cctctgagga aatcgatcac gaggacatca ccaaggacaa gacctctacc 240 gtggaagcct gcttgcctct ggaactgacc aagaacgagt cctgtctgaa ctccagagag 300 acatctttca tcaccaacgg ctcctgcctg gcttctcgga aaaccagctt catgatggcc 360 ctgtgcctgt cctccatcta cgaggatctg aagatgtacc aggtcgagtt caagaccatg 420 aacgccaagc tgctgatgga ccccaagcgg cagatcttcc tggaccagaa catgctggcc 480 gtgatcgacg agctgatgca ggctctgaac ttcaactccg agaccgtgcc tcagaagtcc 540 agcctggaag agcctgactt ctacaagacc aagatcaagc tgtgcatcct gctgcatgcc 600 ttccggatta gagctgtgac catcgacaga gtgatgagct acctgaatgc cagctgataa 660

Claims

1. A method for preparing a cell line that efficiently expresses human interleukin-35, characterized in that, Includes the following steps: A dual-plasmid expression system carrying the human interleukin-35 gene sequence was transiently transfected into CHO-K1 cells to obtain a cell line that expresses human interleukin-35 efficiently. The dual plasmid system comprises a pXC17.4 plasmid containing the nucleotide sequence of the p35 subunit of a mouse signal peptide and a pcDNA3.1 plasmid containing the nucleotide sequence of the EBI3 subunit of a mouse signal peptide. The p35 subunit nucleotide sequence containing the mouse signal peptide is shown in SEQ ID NO.14, and the EBI3 subunit nucleotide sequence containing the mouse signal peptide is shown in SEQ ID NO.

10.

2. A cell line that efficiently expresses human interleukin-35, characterized in that, The high-efficiency human interleukin-35 expression cell line was prepared using the method described in claim 1.

3. The high-efficiency human interleukin-35 expression cell line according to claim 2, characterized in that, The expression level of the highly efficient human interleukin-35 cell line was 160-200 mg / L.

4. The application of the highly efficient human interleukin-35 expression cell line according to claim 2 or 3 in the preparation of immunomodulators and anti-inflammatory drugs.

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