CN117986386A

CN117986386A - Recombinant human interleukin-35 and preparation method and application thereof

Info

Publication number: CN117986386A
Application number: CN202410167256.5A
Authority: CN
Inventors: 王建刚; 张凯跃; 胡祥维; 韩倩; 乔磊; 郑彬彬; 吴明远
Original assignee: Kanglitai Biomedical Qingdao Co ltd
Current assignee: Kanglitai Biomedical Qingdao Co ltd
Priority date: 2024-02-06
Filing date: 2024-02-06
Publication date: 2024-05-07

Abstract

The application relates to the technical field of biological medicine, in particular to a recombinant human interleukin-35, a preparation method and application thereof. The recombinant human interleukin-35 has the biological activity of human interleukin-35 and is a heterodimer of a first fusion polypeptide and a second fusion polypeptide; from the N-terminal to the C-terminal, the first fusion polypeptide comprises a first active peptide shown as SEQ ID NO.11 and a first auxiliary peptide, the second fusion polypeptide comprises a second active peptide shown as SEQ ID NO. 15 and a second auxiliary peptide, and the first auxiliary peptide and the second auxiliary peptide form KIH structures. The application provides a recombinant IL-35 which has a KIH structure, and based on the KIH structure, EBI3 and p35 can be covalently combined to form a heterodimer, so that the recombinant IL-35 has good biological activity, and provides a new idea for subsequent expression and activity research of the cytokines.

Description

Recombinant human interleukin-35 and preparation method and application thereof

Technical Field

The application relates to the technical field of biological medicine, in particular to a recombinant human interleukin-35, a preparation method and application thereof.

Background

IL-12 family members are important cytokines, heterodimers consisting of an alpha chain (p 19, p28, p 35) and a beta chain (p 40 and EBI 3). Interleukin 35 (interleukin-35, IL-35) is a member of the IL-12 cytokine family, which has a similar structure to other members of the IL-12 family (IL-12, IL-23, IL-27, IL-39), and is a heterodimeric molecule composed of Epstein-Barr virus-induced gene 3 (EBI 3) and IL-12p35, secreted primarily by regulatory T cells (Treg) and regulatory B cells (Bregs).

IL-35, an anti-inflammatory and immunosuppressive cytokine, can play an important role in the development and progression of a variety of inflammatory and autoimmune diseases by regulating the differentiation and function of T cells, B cells, dendritic cells and macrophages. The results of the study also show that IL-35 is closely related to a variety of human diseases, including infectious diseases, sepsis, autoimmune diseases, inflammation and tumors. Therefore, IL-35 is expected to become a new biomarker reflecting disease progression and prognosis, or a new target for disease treatment, and has wide clinical treatment prospects.

IL-12p35 contains 7 cysteines, EBI3 contains 4 cysteines, and IL-12p35 and EBI3 subunits are capable of forming heterodimers by non-covalent binding to each other, while the subunits themselves bind to form p35: p35 or EBI3: EBI3 homodimers are multimeric structures. The receptor for IL-35 is a dimer composed of IL-12Rβ2 and gp130, sharing a receptor chain with IL-12 and IL-27. IL-35 signals through heterodimeric or homodimeric receptors consisting of IL-12Rβ2 and gp130 and produces different biological effects depending on the receptor composition. While IL-35 homodimers or multimers may still contribute to in vivo immunosuppression during information transfer, this weaker non-covalent binding and undefined combination of subunits results in a number of difficulties in the expression of human IL-35, and studies speculate that homodimeric hl-35 is not efficiently secreted from transfected cells due to the intracellular interactions of EBI3 and p 35.

There are current studies attempting to purify recombinant biologically active IL-35 from E.coli, but active IL-35 cannot be obtained. CN116478926a describes the following scheme: a double plasmid expression system with human interleukin-35 gene (the natural nucleotide sequence of p35 subunit is synthesized on pXC17.4 plasmid, the natural nucleotide sequence of EBI3 subunit is synthesized on pcDNA3.1 plasmid) is transiently transfected into the genome of CHO-K1 cell, and the high-efficiency expression human interleukin-35 cell strain is finally obtained by screening after multiple screening tests and biological activity detection, so that the expression quantity of IL-35 is improved, but because of the expressed molecules, the conditions of homodimer, multimer and the like still exist, the high-purity IL-35 protein with biological activity is difficult to purify and obtain. Attempts have also been made to design IL-35 as a heterodimeric protein covalently linked to EBI3 and p35, probably due to the influence of the spatial position of the binding of EBI3 and p35, and the activity of the obtained rhIL-35 is not stable. There have also been attempts to construct fusion proteins of an Fc fragment and IL-35, but the biological activity of the resulting recombinant IL-35 is to be improved, and conventional techniques for constructing fusion proteins of an Fc fragment and IL-35 are described, for example, in the patent application CN106075401A Hesuo, the "study of eukaryotic expression and biological function of recombinant human interleukin-35", in which:

CN106075401A describes the synthesis of amino acid sequences of EBI3, P35, linker and fusion proteins fused to IL-35, the synthesized IL-35 fusion protein being obtained by linking the Fc of human IgG1 at the C-terminus of IL-35. The Fc fusion protein has the advantages that part of antigen peptide of pathogen can be fused with IgG-Fc fragment to induce organism to generate antigen specific immune response, and enhance the disease resistance of IL-35. In the invention, single plasmid is transfected into an Expi293E cell, cell suspension is collected on the 6 th day of transfection, the obtained cell suspension is subjected to high-speed centrifugation, cell fragments are removed, IL-35 in supernatant is concentrated after ultrafiltration by a 0.45 mu m filter screen, the concentrated liquid is eluted until no impurity peak exists by a liquid exchange device, and IL-35 fusion protein is obtained, and the IL-35 fusion protein is successfully expressed by comparing with positive control protein.

"Eukaryotic expression of recombinant human interleukin-35 and study of biological function" describe the construction of IL-35 (EBI3+linker+P35)

Eukaryotic expression vector pSTEP-IL 35-Fc was transfected into HEK293T cells, the supernatant was harvested by culturing in a 10L reactor for 7 days, recombinant human IL-35-Fc protein was obtained by separation and purification using protein affinity chromatography columns, protein expression was identified by SDS-PAGE and Western Blot, and rhIL-35 bioactivity was detected.

Disclosure of Invention

Based on this, one or more embodiments of the present application provide a recombinant human interleukin-35, and a preparation method and application thereof. The method comprises the following technical scheme:

One or more embodiments of the present application provide a recombinant human interleukin-35 having human interleukin-35 biological activity as a heterodimer of a first fusion polypeptide and a second fusion polypeptide;

From the N end to the C end,

The first fusion polypeptide comprises a first active peptide shown as SEQ ID NO.11 and a first auxiliary peptide,

The second fusion polypeptide comprises a second active peptide shown as SEQ ID NO15 and a second auxiliary peptide,

The first helper peptide and the second helper peptide form a KIH structure.

In some embodiments of the application, the recombinant human interleukin-35 satisfies one or more of the following conditions:

(1) The first active peptide and the first auxiliary peptide are directly linked;

(2) The second active peptide and the second auxiliary peptide are directly linked; and

(3) The first helper peptide and the second helper peptide are each independently selected from mutant peptides of an Fc fragment of an antibody.

In some embodiments of the application, the first helper peptide and the second helper peptide are each independently selected from mutant peptides of an IgG antibody Fc fragment.

In some embodiments of the application, the N-terminus of the first fusion polypeptide and the second fusion polypeptide is further linked to a signal peptide.

In some embodiments of the application, the signal peptide is further linked at the N-terminus to a first protease cleavage site fragment.

In some embodiments of the application, the C-terminus of the first fusion polypeptide and the second fusion polypeptide is each independently linked to a second protease cleavage site fragment.

One or more embodiments of the application also provide a nucleic acid molecule encoding the recombinant human interleukin-35.

One or more embodiments of the application also provide a recombinant vector comprising the nucleic acid molecule.

One or more embodiments of the present application also provide a recombinant cell that expresses the recombinant human interleukin-35.

One or more embodiments of the present application also provide a method for constructing a recombinant cell, which includes the step of introducing the nucleic acid molecule into a host cell to construct a recombinant cell.

One or more embodiments of the present application also provide a method for preparing the recombinant human interleukin-35, which includes the steps of:

culturing said recombinant cell; and

Isolating said recombinant human interleukin-35 from the resulting culture.

One or more embodiments of the present application also provide an application of the recombinant human interleukin-35, the nucleic acid molecule, the recombinant vector or the recombinant cell in preparing a medicament for treating cancer, autoimmune diseases or inflammatory diseases.

One or more embodiments of the present application also provide a medicament comprising the recombinant human interleukin-35, the nucleic acid molecule, the recombinant vector or the recombinant cell, and a pharmaceutically acceptable carrier.

Compared with the prior art, the application has the following beneficial effects:

The application provides a recombinant IL-35 which has a KIH structure, and based on the KIH structure, EBI3 and p35 can be covalently combined to form heterodimers, so that the recombinant IL-35 has good biological activity (including better combination with gp130 and promotion of cell proliferation of PBMCs), and provides a new idea for subsequent expression and activity research of cytokines of this kind.

Drawings

In order to more clearly illustrate the technical solution in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is evident that the figures in the following description are only some embodiments of the application, from which other figures can be obtained without inventive effort for a person skilled in the art.

FIG. 1 shows the results of a concentration test of IL-35 in the supernatant of 96h cells after transfection of the plasmid by Dot Blot detection in example 1;

FIG. 2 is a SDS-PAGE map of KIH-IL-35 in example 2 after purification;

FIG. 3 is a graph of Western Blot identification results in example 3;

FIG. 4 is a graph showing the results of the identification of KIH-IL-35 activity in example 4;

FIG. 5 is a graph showing the results of the activity of KIH-IL-35 in example 5 against PBMCs.

Detailed Description

The present application will be described in further detail with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by persons skilled in the art without departing from the spirit of the application, and equivalents thereof are also intended to fall within the scope of the application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the application, it being understood that the application may be practiced without one or more of these details.

Unless defined otherwise, 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 application belongs. The terminology used herein in the description of the application is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the application.

Terminology

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical schemes of all "logical or" connections), also include any and all combinations of A, B, C, D, i.e., the combinations of any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical schemes of all "logical and" connections).

The terms "plurality", "plural", "multiple", and the like in the present application refer to, unless otherwise specified, an index of 2 or more in number. For example, "one or more" means one kind or two or more kinds.

As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.

The "suitable" in the "suitable combination manner", "suitable manner", "any suitable manner" and the like herein refers to the fact that the technical scheme of the present application can be implemented, the technical problem of the present application is solved, and the technical effect expected by the present application is achieved.

Herein, "preferred", "better", "preferred" are merely to describe better embodiments or examples, and it should be understood that they do not limit the scope of the application.

In the present application, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the application.

In the present application, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

In the present application, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.

In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present application, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. Where a numerical range merely refers to integers within the numerical range, including both end integers of the numerical range, and each integer between the two ends, unless otherwise indicated, each integer is recited herein as directly, such as where t is an integer selected from 1 to 10, and where t is any integer selected from the group of integers consisting of 1,2,3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The temperature parameter in the present application is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations within a range such as + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.

In the present application,% (w/w) and wt% each represent weight percent,% (v/v) represents volume percent, and% (w/v) represents mass volume percent.

Over the last decade, an increasing number of studies have shown that IL-35 plays an important role in controlling immune-related diseases, including autoimmune diseases, infectious diseases, and cancer. However, a comprehensive understanding of IL-35 still faces some impediments. For example, the production of purified unlabeled rIL-35 remains a significant challenge limiting its functional analysis. Another important issue that has not been addressed is how to maintain and regulate the stability of heterodimers in vivo. With increased biological knowledge of IL-35, it is possible to find other functions of IL-35 than to inhibit T-cell proliferation, but it is currently uncertain whether IL-35 has other functions. As a pharmaceutical study, it is crucial to obtain correctly expressed proteins. However, there is currently no stable relevant IL-35 product available for research or commercial scale production.

The application relies on two subunits of IL-35 on classical Knobs-into-holes to ensure that the two subunits are expressed and assembled respectively to be mature, reduce the generation of mismatched byproducts, try to produce correctly assembled recombinant cytokines, and further verify the biological characteristics of the recombinant cytokines, thus providing a new thought for the expression and activity research of the cytokines.

Knobs-into-Holes (KIH) is essentially a sterically-based technique for promoting heterodimerization of heavy chains that solves the problem of heavy chain mismatch in heterologous antibodies. The KIH structure was originally the packaging model for amino acid side chains in the alpha helix proposed by Crick in 1953, and Genentech, john b.b.ridgway and his colleagues, based on this development and demonstrated that KIH technology for heavy chain assembly of such bispecific antibody molecules was effective in facilitating correct assembly of antibodies. In this technique, the first technical platform for bispecific antibody molecule heavy chain assembly was achieved by replacing a small amino acid with a large amino acid (e.g., T366W) and inserting it into a "Hole" (the large amino acid being replaced with a small amino acid). Mutations in the CH3 domains (such as T366Wk (knob) and T366Sh, L368Ah and Y407Vh (pore)) were found to produce small amounts of homodimeric complexes, but more products were heterodimeric complexes, overall more advantageous for the formation of constitutive heterodimeric complexes. At the same time, the method is suitable for expressing glycosylated proteins in eukaryotic expression systems or bacterial expression systems.

First aspect of embodiments of the application

The embodiment of the application provides a recombinant human interleukin-35, which has the biological activity of human interleukin-35 and is a heterodimer of a first fusion polypeptide and a second fusion polypeptide;

From the N end to the C end,

The first helper peptide and the second helper peptide form a KIH structure.

KIH, i.e., a pestle-and-socket structure, the polypeptide chains spatially form a convex "Knobs" type structure and a concave "Holes" type structure, respectively, and the "Knobs" type structure and the "Holes" type structure are snapped to form KIH structures.

In some examples, the first active peptide and the first helper peptide are directly linked. In some examples, the second active peptide and the second auxiliary peptide are directly linked.

"Directly linked" is relative to "indirectly linked" and means that the two polypeptides fused are not linked via an intermediate such as a flexible chain, but rather that the amino group at the N-terminus of one polypeptide is directly dehydrated and condensed with the carboxyl group at the C-terminus of the other polypeptide to form a peptide bond through which the two polypeptides are linked.

Based on the knowledge of the "KIH structure", it is understood that the present application is not limited to the first and second helper peptides, and that the first and second helper peptides may each be independently selected from, but not limited to, mutant peptides of the Fc fragment of the antibody, under conditions that allow the first and second helper peptides to form the KIH structure and ensure that the resulting recombinant human interleukin-35 has the biological activity of human interleukin-35. In the process of forming the "KIH structure", the first auxiliary peptide may have a "Knobs" type structure, and the second auxiliary peptide may have a "Holes" type structure, or vice versa, i.e., the first auxiliary peptide has a "Holes" type structure, and the second auxiliary peptide has a "Knobs" type structure. It will be appreciated that the "antibody Fc fragment" of the mutant peptide of the antibody Fc fragment, which specifically includes the hinge region, the CH2 region and the CH3 region in the present application, may be a natural antibody Fc fragment, or may be an antibody Fc fragment artificially modified based on a natural antibody Fc fragment, for example, an antibody Fc fragment shown in any one of SEQ ID No.21 to SEQ ID No. 29. The following specific examples illustrate the technical scheme of the present application by taking the construction of mutant peptides thereof as examples of the Fc fragment of the antibody shown in SEQ ID NO.29, and it is understood that the present application is not limited thereto.

Alternatively, the mutation site of the mutant peptide of the antibody Fc fragment is located in the CH3 region relative to the antibody Fc fragment. In some embodiments, the mutation site of the mutant peptide of the antibody Fc fragment comprises a T mutation of its CH3 region to W to form a "Knobs" type structure to correspond to one of the first and second helper peptides, and the mutation site of the mutant peptide of the antibody Fc fragment comprises a T mutation of its CH3 region to S and an L mutation to A, Y to V to form a "Holes" type structure to correspond to the other of the first and second helper peptides.

The type of the mutant peptide of the Fc fragment of the antibody is not particularly limited in the present application, and may be selected from, but not limited to, mutant peptides of the Fc fragment of an IgG antibody. It is to be understood that the present application is not particularly limited to mutant peptides of the Fc fragment of an IgG antibody, for example: the mutant peptide of the Fc fragment of the IgG1 antibody, the mutant peptide of the Fc fragment of the natural IgG2 antibody, the mutant peptide of the Fc fragment of the natural IgG3 antibody, or the mutation of the Fc fragment of the natural IgG4 antibody can be used. The sequence of the CH3 region of the Fc fragment varies from antibody to antibody, as does the location of the mutation. For example, the Fc fragment shown in SEQ ID No.29 is mutated to form a "Knobs" type structure by T366W to correspond to one of the first and second helper peptides (optionally the first helper peptide) and mutated to form a "Holes" type structure by T366S, L368A and Y407V to correspond to the other of the first and second helper peptides (optionally the second helper peptide). Mutant peptides of the Fc fragment of the antibodies of the present application include, but are not limited to, the helper peptide combinations shown in SEQ ID NO.13 and SEQ ID NO.17, as well as SEQ ID NO.30 and SEQ ID NO. 31. The following examples illustrate the technical scheme of the present application by taking a group of auxiliary peptides shown in SEQ ID No.13 and SEQ ID No.17 as examples, and it is understood that the present application is not limited thereto.

In some examples, the first fusion polypeptide and the second fusion polypeptide are further linked at their N-terminus to a signal peptide.

The term "signal peptide" refers to fragments that are secreted from biologically active molecular drugs and fusion proteins and are cleaved post-translationally in the host cell. The characteristics of signal peptides are well known in the art, and signal peptides typically have 16 to 30 amino acids, but they may comprise a greater or lesser number of amino acid residues. Conventional signal peptides consist of three regions, a basic N-terminal region, a central hydrophobic region and a more polar C-terminal region. The central hydrophobic region comprises 4 to 12 hydrophobic residues, which immobilize the signal peptide through the membrane lipid bilayer during translocation of the immature polypeptide. After initiation, the signal peptide is cleaved in the lumen of the ER by a cellular enzyme commonly referred to as signal peptidase. Specifically, the signal peptide may be a secretory signal peptide of tissue plasminogen activation (tPa), a signal peptide of herpes simplex virus glycoprotein D (HSV gDs), or a growth hormone. Alternatively, a secretion signal peptide used in higher eukaryotic cells including mammals and the like can be used. In addition, as the secretion signal peptide, a signal peptide contained in wild-type IL-7 may be used, or it may be used after substitution with a codon with a high expression frequency in a host cell. The signal peptide of the present application may be selected from, but not limited to, the signal peptide shown in SEQ ID NO.19, the signal peptides shown in SEQ ID NO.1 to 20, the signal peptides shown in SEQ ID NO.3 to 1 to 22, and optionally the signal peptide shown in SEQ ID NO. 19. Further, the N-terminus of the signal peptide is also linked to a first protease cleavage site fragment, including but not limited to KL. In one example, the first fusion polypeptide and the second fusion polypeptide are further linked at their C-terminus to a second protease cleavage site fragment, including but not limited to EF.

Second aspect of embodiments of the application

The embodiment of the application provides a nucleic acid molecule which codes for the recombinant human interleukin-35.

Alternatively, the nucleic acid molecules of the present application include the nucleic acid molecules shown in SEQ ID NO.8 at positions 70 to 1383 and the nucleic acid molecules shown in SEQ ID NO.10 at positions 70 to 1347. Further, the nucleic acid molecule may further comprise one or more of a nucleic acid molecule encoding a signal peptide, a nucleic acid molecule encoding said first protease cleavage site, a nucleic acid molecule encoding a second protease cleavage site.

The "nucleic acid molecule encoding a signal peptide" of the present application is a polynucleotide encoding an amino acid sequence that initiates movement of a protein across the Endoplasmic Reticulum (ER) membrane.

Third aspect of embodiments of the application

Embodiments of the present application provide a recombinant vector comprising the nucleic acid molecule.

An "expression system" or "expression vector (or vector)" refers to a nucleic acid sequence containing the desired coding and control sequences in operable linkage such that a host transformed with these sequences is capable of producing the encoded protein. To effect transformation, the expression system may be contained on a vector; however, the relevant nucleic acid molecules may subsequently also integrate into the host chromosome.

In some examples, the recombinant vector comprises a first recombinant vector and a second recombinant vector,

One recombinant vector of the first recombinant vector and the second recombinant vector comprises nucleic acid molecules shown in 70 th to 1383 th positions of SEQ ID NO.8, and one recombinant vector comprises nucleic acid molecules shown in 70 th to 1347 th positions of SEQ ID NO. 10.

In some of these examples, the first recombinant vector and the second recombinant vector are each independently selected from the pxc17.4 plasmid or pcdna3.1 (+).

In some examples, the first recombinant vector is pcdna3.1 (+) and comprises the nucleic acid molecule shown in SEQ ID No.8 at positions 70-1383, and the second recombinant vector is a pxc17.4 plasmid and comprises the nucleic acid molecule shown in SEQ ID No.10 at positions 70-1347.

Fourth aspect of embodiments of the application

The embodiment of the application provides a recombinant cell which expresses the recombinant human interleukin-35.

In some examples, the cell comprises a eukaryotic cell. In some examples, the cell is an animal cell. In some examples, the animal cell is a mammalian cell. In some examples, the source of mammalian cells includes mice, including but not limited to hamsters. In some examples, the mammalian cells include, but are not limited to, ovarian cells, such as CHO-K1 cells.

Fifth aspect of embodiments of the application

The embodiment of the application provides a construction method of the recombinant cell, which comprises the step of introducing the nucleic acid molecule into a host cell to construct the recombinant cell.

Optionally, the construction method includes: constructing the recombinant vector; and transfecting the host cell with the recombinant vector to construct the recombinant cell.

"Introduced", "transferred", "transfected", "stably transfected" or "transiently transfected" refers to the uptake of the expression vector by the host cell, whether or not any coding sequence is actually expressed. A variety of transfection methods are known to those skilled in the art. For example, transfection is accomplished by electroporation, by using phage or viral expression vectors to insert host cells, by mechanical insertion of nucleic acids, or even by culturing host cells in the presence of unpackaged (unpackaged) nucleic acid fragments in the presence of an expression vector and high concentrations of calcium phosphate. The success of transfection is generally confirmed when any indication of the vector of interest is manipulated to occur in the host cell.

Sixth aspect of embodiments of the application

The embodiment of the application provides a preparation method of recombinant human interleukin-35, which comprises the following steps:

culturing said recombinant cell; and

Isolating said recombinant human interleukin-35 from the resulting culture.

It will be appreciated that the culturing is carried out under conditions suitable for the recombinant cells, and that different types of recombinant cells, and corresponding culturing conditions (optimum medium, temperature, pH, etc.), may differ.

Seventh aspect of embodiments of the application

The embodiment of the application provides an application of the recombinant human interleukin-35, the nucleic acid molecule, the recombinant vector or the recombinant cell in preparing medicines for treating cancers, immune diseases or inflammatory diseases.

"Cancer" refers generally to the presence of cells that have characteristics typical of oncogenic cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological characteristics. It is generally observed that cancer cells aggregate into tumors, but such cells may be present alone in an animal subject, or may be non-tumorigenic cancer cells, such as leukemia cells. Thus, the term "cancer" may encompass reference to a solid tumor, a soft tissue tumor, or a metastatic lesion. As used herein, the term "cancer" includes pre-cancerous as well as malignant cancers, including but not limited to, hepatocellular carcinoma, human lung adenocarcinoma, hodgkin's lymphoma, acute myeloid leukemia, esophageal cancer, pancreatic ductal adenocarcinoma, cervical cancer, and colorectal cancer.

Immune diseases (immune diseases), diseases caused by the imbalance of immune regulation affecting the immune response of the body. Generalized immune diseases also include structural or functional abnormalities of the immune system caused by congenital or acquired causes, including but not limited to psoriasis, lupus erythematosus, immune diabetes, autoimmune uveitis, rheumatoid arthritis.

Inflammatory diseases, including generally autoimmune inflammatory diseases, and inflammatory diseases caused by infection with other pathogenic microorganisms, including, but not limited to, ulcerative colitis, lupus nephritis, and neuroinflammation.

Eighth aspect of embodiments of the application

The embodiment of the application provides a medicine which comprises the recombinant human interleukin-35 and a pharmaceutically acceptable carrier.

The medicament of the present application may be a medicament suitable for injectable use including sterile aqueous solutions (water-soluble) or dispersions, as well as sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, cremophor ELTM (BASF, parsippany, N.J.), or Phosphate Buffered Saline (PBS). In all cases, the drug should be sterile and should be a fluid that is easy to inject. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. For example, proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants (e.g. sodium lauryl sulphate). The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). In many cases, isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol) and/or sodium chloride will typically be included in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents which delay absorption (e.g., aluminum monostearate and gelatin).

The sterile injectable solution may be prepared by the following manner: the active ingredient is incorporated in the desired amount in an appropriate solvent optionally with one or a combination of the ingredients listed above, followed by filter sterilization. Typically, dispersions are prepared by incorporating the active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In some cases, the medicaments of the present application are prepared with carriers that protect the active ingredient from rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Such formulations may be prepared using standard techniques. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

Ninth aspect of embodiments of the application

Embodiments of the present application provide a method of alleviating cancer, an immune disorder or an inflammatory disorder, the method comprising administering to the subject the recombinant human interleukin-35 described in the first aspect, the nucleic acid molecule described in the second aspect, the vector described in the third aspect, the recombinant cell described in the fourth aspect or the drug described in the eighth aspect.

In the present application, "alleviating" has the same meaning as "preventing and/or treating" and may be used interchangeably. In the present application, "alleviation" includes aspects of prevention, treatment, adjuvant therapy, and the like. As used herein, "alleviating" refers to alleviating, slowing progression, attenuation, prevention, or maintenance of an existing disease or condition. "alleviating" also includes curing, preventing the development of, or alleviating to some extent one or more symptoms of a disease or disorder.

In the present application, the subject is a mammal. In some examples, the mammal is a human, a mouse.

The definitions of cancer, immune diseases, inflammatory diseases are defined with reference to the seventh aspect.

Embodiments of the present application will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present application, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

EXAMPLE 1 expression of IL-35 recombinant proteins

In this example, recombinant expression plasmid (KIH-IL-35) fused with KIH-structure IL-35 was constructed, transfected into CHO-K1 cells, cultured for 96 hours, and the supernatant was collected and purified to determine the optimal mode. The specific flow is as follows:

construction of KIH-IL-35 expression plasmid

The natural nucleotide and amino acid sequences of human p35 and EBI3 were retrieved from NCBI network database: the amino acid sequence of the EBI3 subunit is shown as a sequence SEQ ID NO.1, and the nucleotide sequence is shown as a sequence SEQ ID NO. 2; the amino acid sequence of the P35 subunit is shown as a sequence SEQ ID NO.3, and the nucleotide sequence is shown as a sequence SEQ ID NO. 4. In addition, the amino acid sequence and the nucleotide sequence of the signal peptide are shown as SEQ ID NO.5 and SEQ ID NO. 6.

The sequences of peptide chains P35-Fch (well), peptide chains EBI3-Fck (knob) were designed and optimized based on IgG ₄ Fc using T366W (knob) and T366S, L368A and Y407V (well) mutations, and plasmid synthesis was performed by entrusted with the large genes, wherein P35-Fch was constructed on the pXC17.4 plasmid and EBI3-Fck was constructed on pcDNA3.1 (+). The amino acid sequence of EBI3-Fck is shown as sequence SEQ ID NO.7, and the optimized nucleotide sequence is shown as sequence SEQ ID NO. 8; the amino acid sequence of P35-Fch is shown as SEQ ID NO.9, and the optimized nucleotide sequence is shown as SEQ ID NO. 10.

Then the pXC17.4-EBI3-Fck and pcDNA3.1 (+) -p35-Fch vectors of the replacement vector are constructed by using an enzyme digestion connection method. The pcDNA3.1 (+), pXC17.4 empty plasmids were linearized with EcoRI and HindIII and the linearized vector was recovered by gel, and the desired fragments were recovered by restriction enzyme digestion of pcDNA3.1 (+) -EBI3-Fck and pXC17.4-p35-Fch using the same endonuclease and ligated with the linearized vector using DNA ligase to obtain pXC17.4-EBI3-Fck and pcDNA3.1 (+) -p35-Fch.

2. Amplification of plasmids

(1) Transformation of plasmids: ① Taking 100 mu L of DH5 alpha competent bacteria, adding a proper amount of the plasmid DNA prepared in the step 1 on ice, gently mixing, ice-bathing for 20min, heat-shocking for 90s at 32 ℃, and ice-bathing for 3min; ② 200. Mu.L of LB medium is added, the culture is carried out for 1h by shaking at 37 ℃ and 150rpm, a proper amount of bacterial liquid is coated on an LB medium plate containing ampicillin, and the culture is carried out overnight at 37 ℃.

(2) Screening and identification of clones: ① Streaking the strain on LB medium (containing ampicillin) plate, and culturing overnight at 37 ℃; ② After bacterial colonies grow out the next day, selecting single round bacterial plaques, picking out the bacterial plaques by using a gun tip, adding the bacterial plaques into 1mL of liquid LB culture medium with ampicillin resistance, placing the liquid LB culture medium into a shaking table at 37 ℃ for culturing for 4 hours at 220rpm, adding 10 mu L of culture solution into 100mL of liquid LB culture medium with ampicillin resistance, and placing the liquid LB culture medium into the shaking table at 37 ℃ for culturing for 12-16 hours at 220 rpm;

(3) Endotoxin-free extraction of recombinant expression plasmid (using the nuuzan large extraction kit): 1) 100mL of the overnight cultured bacterial liquid is taken, the bacterial liquid is centrifuged at about 11,000rpm (12,000Xg) for 1-2min, and the supernatant is discarded to collect bacterial cells. 2) 7.5mL Buffer P1 containing RNase A is added into a centrifuge tube with bacterial sediment, and the mixture is blown or vortex-vibrated by a pipette for uniform mixing. 3) Adding 7.5mL Buffer P2 into the bacterial suspension prepared in the step 2), gently mixing the bacterial suspension for 6 to 8 times in an upside down way, and standing the bacterial suspension at room temperature for 4 to 5 minutes. 4) 7.5mL Buffer P4 was added to the bacterial suspension prepared in step 3), and the solution was gently turned upside down 6-8 times immediately to thoroughly neutralize Buffer P2. At this point white flocculent precipitate should appear, and centrifuge at greater than about 10,000rpm (11,000Xg) for 10-15min, carefully aspirate the supernatant to a new 50mL round bottom fresh tube, avoiding aspiration of the floating white precipitate. 5) Add 0.1 volume (10% of the volume of the supernatant, about 2.2 mL) of endotoxin scavenger to the supernatant from step 4), mix upside down, place in ice bath or insert into crushed ice (or freezer compartment of refrigerator) for 5min until the solution becomes clear and transparent (or still slightly cloudy) from cloudiness, mix occasionally in the middle. 6) Placing in 37deg.C water bath for 10-15min, turning the solution into turbid, and mixing. 7) Centrifuge at about 11,000rpm (12,000Xg) for 10min to phase split (temperature greater than 25 ℃ C.). The upper aqueous phase contains DNA and the lower blue oily phase contains endotoxin and other impurities. The upper aqueous phase containing DNA was transferred to a new tube and the oily layer was discarded. 8) Adding 0.5 times volume of isopropanol (about 10 mL) into the upper water phase, mixing, transferring into adsorption column (the adsorption column is placed into collecting tube), centrifuging at about 11,000rpm (12,000Xg) for 1min, and pouring out waste liquid in the collecting tube. Until all the mixed solution passes through the column. 9) 10mL of the rinse solution PW with absolute ethanol added thereto was added at about 11,000rpm (12,000Xg), and the waste solution was discarded. The adsorption column was replaced in the collection tube, 10mL of rinse PW was added, and the rinsing was repeated once. 10 Sucking out ethanol possibly remained between the inner ring compression ring and column wall with gun head, placing the adsorption column back into the empty collecting tube, centrifuging at highest speed for 3min to dry ethanol remained on the matrix membrane, opening the cover, and air drying at room temperature for 3-5min.11 Taking out the adsorption column, placing into a clean centrifuge tube, adding 1-2mL Buffer TB preheated at 55deg.C into the middle part of the adsorption film, standing at room temperature for 3min, centrifuging at about 11,000rpm (12,000Xg) for 3min, eluting plasmid, sterilizing by passing through the film, and preserving at-20deg.C.

3. Transient transfection resuscitates CHO-K1 cells for subculture, and when the growth state of the cells is recovered to be normal, the cells are prepared for transfection. Cells in the logarithmic growth phase and well grown were observed and selected and inoculated into shake flasks at 3X 10 ⁶ cells/well in 30 mL. FectoPRO transfection reagent (Polyplus Transfection) was used, following plasmid: preparation of transfection complexes was performed at a ratio of FectoPRO (μg/. Mu.L) of 1:2. The transfection complexes were added to the above shake flask cells after incubation for 10min at room temperature, and the supernatant was collected after 96 cultures.

The CHO-K1 cell culture medium is: emCD CHO medium (Eminence) +1%H/T (Gibco) +0.5% Anti-clumping Agent (Gibco) +6mM L-glutamine (Gibco); the transfection medium was Opti-MEM (Gibco). The culture conditions of the cells are 37 ℃, 5% CO ₂, suspension culture is carried out, the rotation speed is 110rpm, the initial culture cell density is 3 multiplied by 10 ⁵cell/mL cell/mL-5×10⁵ cells/mL, and the cells are passaged once in 2-4 days.

4. Dot Blot detection of IL-35 in cell supernatants

① Loading: shearing a transfer filter membrane with proper size according to the number of samples, dripping 20 mu L of cell supernatant onto the transfer membrane twice, and airing in a fume hood; ② closure: 20mL of 5% milk-PBST is added for immersion, and the mixture is placed in a shaking table for sealing for 90min at room temperature; after the end of the closure, the milk was decanted and washed three times with 10mL PBST shaker for 5min each. ③ closure: sealing with 5% skimmed milk prepared in advance, and sealing with shaking table for 1 hr; ④ Incubating primary antibodies: after blocking was completed, the primary antibody was incubated overnight at 4 ℃. The antibody was either a Human/Mouse IL-12/IL-35p35 antibody or an anti-IL-27/IL-35 EBI3 antibody, diluted with PBST at a 1:1000 concentration. Washing the NC membrane after the prepared 1 XPBST buffer solution is washed overnight for 3 times for 10min each time; ⑤ Incubating a secondary antibody: secondary antibody Goat anti Mouse HRP (Genscript) was diluted 1:1000 with PBST. Incubating for 45min at room temperature, pouring out antibody, and washing with PBST three times for 5min each time; ⑥ exposure: according to the solution A: solution B = 1:1, and exposing the substrate. The film was removed and dried slightly and placed on a thin plastic film. Adding a freshly prepared exposure substrate, and reacting for 2min in a dark place to develop color.

Results: FIG. 1 shows the concentration of IL-35 in cell supernatants at 96h after transfection of plasmids by Dot Blot. As shown in FIG. 1, the expression level of the pcDNA3.1-EBI3-Fck and pXC17.4-p35-Fch was lower (right panel in FIG. 1) compared with the expression level of the combination of the replacement vector by Dot blot detection, so that the original vector was selected for subsequent experiments.

EXAMPLE 2 purification of KIH-IL-35 recombinant protein

The expression supernatant collected in example 1 was collected, centrifuged at 5000rpm for 10min, and the supernatant was filtered using a 0.22 μm filter membrane to obtain a supernatant free of impurities such as cell debris. The Protein A affinity column was equilibrated by washing 5-10CV with buffer A (1 XPBS) in advance, loading in the column pressure range, eluting again with buffer A, eluting with 0.1M Gly-HCl at pH3.0 to collect the elution peak, immediately neutralizing the eluate with NaOH or tris, and concentrating the eluate by ultrafiltration exchange with a 3K ultrafiltration tube. Finally, SDS-PAGE was used for verification.

Polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE method) according to the fifth method of the 2020 edition of four pharmacopoeias 0541 electrophoresis:

1. sample treatment

Mixing the sample with the buffer solution of the reduction type or the non-reduction type according to the volume of 3:1, and fully mixing on a vortex oscillator for electrophoresis. The sample to be tested of the reduced SDS-PAGE and the physicochemical reference substance are heated in a water bath or a metal bath heater at 100 ℃ for 5min before being loaded, and cooled to room temperature.

Non-reducing buffer (4×): 3.03g of tris (hydroxymethyl) aminomethane, 20mg of bromophenol blue and 8.0g of sodium dodecyl sulfate are weighed, 40mL of glycerin is weighed, water is added for dissolution and dilution to about 80mL, hydrochloric acid is used for adjusting the pH value to 6.8, and water is added for dilution to 100mL.

Reduction buffer (4×): 3.03g of tris (hydroxymethyl) aminomethane, 20mg of bromophenol blue and 8.0g of sodium dodecyl sulfate are weighed, 40mL of glycerin is weighed, water is added for dissolution and dilution to about 80mL, 20mL of beta-mercaptoethanol is added, the pH value is regulated to 6.8 by hydrochloric acid, and the water is diluted to 100mL.

2. Sample application and electrophoresis

2.1 After polymerization of the concentrated gel, carefully pulling out the sample comb, fixing the self-made gel on an electrophoresis device, placing the electrophoresis tank on ice, and adding diluted 1X electrode buffer solution into each of the inner and outer tanks. The inner groove of the electrophoresis tank is filled, and the buffer solution of the outer groove cannot overflow the inner groove and is not lower than 2/3 of the inner groove.

2.2, Adding the centrifuged sample and Marker into the gel well according to a preset sequence, wherein the total amount of the protein to be loaded is more than 10 mu g, and keeping caution in the protein loading operation, so as to avoid the overflow of the test sample to the adjacent lanes and prevent cross contamination and the loss of the test sample.

And 2.3, after sample loading is finished, connecting the electrophoresis device with a power supply, ensuring that the anode and the cathode of the power supply are correct, starting the power supply of the electrophoresis tank, performing constant voltage electrophoresis, and adjusting the initial voltage to be 80V and 120-140V when entering the separation gel. Bromophenol blue is used as electrophoresis indicator band, and the gel running time is enough to ensure that the sample is effectively separated and does not run out of gel. After electrophoresis, the gel is removed, the glass plate is gently pried off, the gel is taken down and put into a staining box, the gel is stained for 20min by using staining solution, and imaging is carried out after the decolorization of the decolorization solution for 2 h.

Results: FIG. 2 is a SDS-PAGE map of KIH-IL-35 after purification. As shown in the results of FIG. 2, the 120-150kD band was evident under non-reducing buffer treatment, indicating that KIH structure was effective in promoting IL-35 dimer formation; under the condition of treatment with the reduced buffer solution, a uniform band is formed at about 70kD, which is consistent with the expected size.

EXAMPLE 3 Western-Blot identification of KIH-IL-35

① Treating a sample: mixing the sample with 4 Xreduction buffer (same as in example 2), heating at 100deg.C for 5min, or mixing the sample with 4 Xnon-reduction buffer (same as in example 2); ② electrophoresis: the method comprises the following two steps: firstly, regulating the constant voltage of an electrophoresis apparatus to 80V, running electrophoresis for about 20min, regulating the voltage to 120V when a visual measurement sample runs to the separation gel, and ending electrophoresis after judging that the target protein is sufficiently separated; ③ film turning: and taking out the glass plate, and slowly prying the glass plate by using the rubber plate. Gently taking out the gel and soaking in a transfer membrane buffer solution; cutting off PVDF film with proper size, soaking in methanol for 1-2min for activation; after the protective paper on two sides of the PVDF film is completely soaked, putting the PVDF film into a film transfer buffer solution for soaking and rinsing, and fully soaking two film transfer sponge pads and filter paper; sequentially placing the black plate, the fiber pad, the filter paper, the gel-PVDF film, the filter paper, the fiber pad and the white plate, and lightly rolling away redundant bubbles by a glass rod every layer to prevent the bubbles from affecting the film transferring effect; placing the clamped plate into a film transfer instrument, regulating constant current to 200mA, and regulating film transfer time according to the size of protein; ④ closure: rinsing PVDF membrane with PBS for 10min after membrane transfer, and sealing with 5% skimmed milk powder at room temperature for 2h; ⑤ Incubation resistance: one antibody was either a Human/Mouse IL-12/IL-35p35 antibody or an anti-IL-27/IL-35 EBI3 antibody diluted with PBST at a 1:2500 concentration and incubated overnight at 4 ℃; ⑤ Secondary antibody incubation: taking out PVDF membrane after overnight incubation, rinsing 3 times with PBST at room temperature, and rinsing 10min each time; diluting the secondary antibody (Goat anti Mouse HRP (Genscript)) by PBST, soaking the PVDF membrane in the secondary antibody incubation liquid, and incubating for 2h at room temperature by a shaking table; ⑥ washing the membrane: after the secondary antibody incubation is completed, the PVDF membrane is fully washed by PBST for 3 times, and each time is 10min; washing with TBS for 3 times for 10min each time; ⑦ Chemiluminescence and imaging: mixing solution A and solution B with ECL color development kit (BIO-RED), adding into centrifuge tube, wrapping with tinfoil paper, shaking, and mixing. And carefully placing the rinsed PVDF film in a clean large dish with the right side facing upwards during development, uniformly dripping a proper amount of ECL developing solution, placing the cleaned PVDF film in a chassis of an imaging Quant LAS 4000mini imager, opening matched software, adjusting focal length and exposure time, and imaging to obtain a Western immunoblotting chemical development chart.

Results: FIG. 3 shows the Western Blot identification results. As the results in FIG. 3 show, the use of Human/Mouse IL-12/IL-35p35 antibodies, anti-IL-27/IL-35 EBI3 antibodies, respectively, detected KIH-IL-35, non-reduction and reduction results were consistent with expectations. The 120-150kD has obvious band under the treatment of non-reducing buffer solution, which shows that KIH structure can effectively promote the formation of IL-35 dimer; a uniform band is arranged at about 70kD under the treatment condition of the reduction buffer solution, which corresponds to the SDS-PAGE gel electrophoresis result.

EXAMPLE 4 detection of KIH-IL-35 recombinant protein Activity

1. Principle of

The receptor for IL-35 consists of IL-12 beta 2 and gp130, and the gp130 molecule is one of the receptors for interleukin 6. The biological activity of IL-6 is mediated by its receptor IL-6 and gp130 together to signal IL-6 into the cell. IL-6 can induce the growth of mouse myeloid leukemia cells (mouse M1 cells) to stop their growth, cell differentiation and finally apoptosis, and it is presumed that the biological action of IL-6 can be inhibited if it has a biological activity of binding to its receptor chain gp 130. According to IL-6 signal transduction, KIH-IL-35 is combined with free gp130, IL-6 can be combined with free IL-6R and can be further combined with gp130 receptor on cell membrane so as to normally transmit signals, so that the IL-6 can inhibit the leukemia M1 cells of mice; if KIH-IL-35 is unable to bind to free gp130, free gp130 will block the complex of IL-6 and IL-6R after binding during culture without affecting the murine leukemia M1 cells. Therefore, IL-35 was added to IL-6 stimulated M1 cells at various concentrations, and IL-35 inhibition of IL-6 was examined.

2. Method of

① Cell culture: m1 mice myeloid leukemia cells were cultured in RPMI1640 medium with 10% fetal bovine serum at 37℃under 5% CO ₂ to logarithmic growth phase for the experiments.

② Activity detection: mouse leukemia M1 cells were diluted to a 4X 10 ⁵ cell/mL cell suspension containing 200ng/mL IL-6, 100ng/mL IL-6R, 800ng/mL gp130, and plated at 50. Mu.L/Kong Jiazhi 96-well cells; KIH-IL-35 was diluted with RPMI-1640 medium (containing double antibody P/S) +10% FBS medium and added to the cell culture plate at 50. Mu.L/well, with 6000ng/mL first well, three-fold gradient dilution, total 8 gradients. The medium was used as a blank. 96-well plates were placed in a 5% CO ₂ incubator at 37 ℃; after 96h of cell culture, 10% of the total volume, i.e., 10. Mu.L of CCK-8, was added to each well, and incubated in an incubator at 37℃for 3 to 4h, the absorbance at OD450nm was measured, and the inhibition of cell proliferation was calculated according to the following formula: cell proliferation inhibition (%) = [ (blank-experimental)/blank ] ×100%. The data processing adopts a four-parameter method: data were processed with GRAPHPAD PRISM software, OD values on the ordinate and dilution concentration on the abscissa, and fitted with four parameters to obtain the activity curve, EC50 values and R ² values, with the test standard requiring R ² values ∈ 0.95.

The biological activity is y= (A-D)/[ 1+ (x/C) B ] +D

Note that:

y: OD value;

x: concentration of rhIL-18;

a: an absorbance upper limit value representing the upper asymptote of the S-curve;

B: as an absorbance increase rate parameter, the slope of the curve is equivalent;

c: concentration values at which the rate of increase of absorbance begins to change, i.e., the EC50 value of rhIL-18;

d: the lower absorbance limit is indicated and represents the lower asymptote of the S-curve.

Results: FIG. 4 is a graph showing the results of the identification of KIH-IL-35 activity. The results showed that with increasing rhIL-35 concentration, the inhibition rate on M1 mouse leukemia cells also increased, and the data were processed using a four-parameter method to give an EC50 value of 1913ng/mL.

EXAMPLE 5 investigation of the Activity of the KIH-IL-35 recombinant protein against PBMCs

Cells from PBMCs cultured with anti-CD 3 and anti-CD 28 mAbs at a concentration of 500ng/mL were treated and 5. Mu.L of each of CD3 and CD56 flow antibodies was added, using cells without antibody as a blank.

Activated PBMCs were diluted to a 1X 10 ⁶ cell/mL cell suspension at 50. Mu.L/Kong Jiazhi 96 well cell culture plate; KIH-IL-35 was diluted with Dayou N500 medium and added to the cell culture plates at 50. Mu.L/well, with 6000ng/mL first well, three-fold gradient dilution, and total of 8 gradients. Gradient dilution KIH-IL-35 with DaeuN 500 Medium was added to the cell plates at 50. Mu.L/well, with medium as a blank. 96-well plates were placed in a 5% CO ₂ incubator at 37 ℃; after 96h of cell culture, 10% of the total volume, i.e., 10. Mu.L of CCK-8 reagent, was added to each well, and the mixture was incubated in an incubator at 37℃for 3h to 4h, and the absorbance at OD450nm was measured.

Results: FIG. 5 is a graph showing the results of KIH-IL-35 activity studies against PBMCs. The flow through of PBMCs from 17 days of induction culture showed a CD3+CD56+ cell fraction of 75.86%. The results for activating PBMCs showed that with increasing KIH-IL-35 concentration, a gradual increase in the detected OD, i.e., an increase in KIH-IL-35 concentration, has a proliferation-promoting effect on the PBMCs cells after induction culture, and an EC50 value of 5552ng/mL.

Sequence 1 (SEQ ID NO. 1) is the amino acid sequence of the signal peptide 1-EBI 3:

MTPQLLLALVLWASCPPCSG|RKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGM AARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAE RQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYG ELSDWSLPATATMSLGK.

sequence 2 (SEQ ID NO. 2) is the nucleotide sequence of the signal peptide 1-EBI 3-stop codon:

ATGACTCCACAACTACTACTAGCACTAGTGCTGTGGGCCTCTTGTCCTCCATGCTCTGGC|AGAAAG GGCCCTCCAGCCGCTCTGACACTGCCCAGAGTGCAGTGCCGCGCTTCCCGGTATCCTATCGCCGTGGACTGCTCCTGGACACTGCCTCCTGCCCCTAACTCCACCTCTCCCGTGTCCTTTATCGCCACCTACCGGCTGGGCATGGCCGCCAGAGGCCATTCTTGGCCTTGCCTGCAGCAGACCCCTACATCTACCTCCTGTACCATCACCGATGTGCAGCTGTTCTCCATGGCTCCTTACGTGCTGAACGTGACCGCCGTGCACCCTTGGGGCAGCTCCAGCTCCTTCGTGCCTTTTATCACCGAGCACATCATCAAGCCTGATCCCCCCGAAGGAGTCAGACTGTCTCCCCTGGCCGAAAGACAGCTGCAAGTGCAGTGGGAGCCTCCAGGATCTTGGCCTTTCCCTGAGATCTTCAGCCTGAAGTACTGGATCAGATACAAGCGGCAGGGCGCTGCTCGGTTCCACAGAGTGGGCCCCATTGAGGCCACCAGCTTCATCCTGAGAGCCGTGCGGCCTAGAGCTAGATACTACGTCCAGGTGGCCGCCCAGGACCTGACCGACTACGGCGAGCTGTCCGACTGGTCCCTGCCTGCTACCGCTACCATGTCTCTCGGCAAA|TGA.

Sequence 3 (SEQ ID NO. 3) is the amino acid sequence of the signal peptide 2-P35:

MCPARSLLLVATLVLLDHLSLA|RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKT MNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDR VMSYLNAS.

sequence 4 (SEQ ID NO. 4) is the nucleotide sequence of the signal peptide 2-P35:

ATGTGCCCAGCACGAAGTCTACTACTAGTGGCTACCCTGGTGCTGCTCGATCATCTGTCCCTGGCT|AGAAACCTGCCTGTGGCTACCCCTGATCCTGGCATGTTCCCTTGCTTGCACCACAGCCAGAACCTGCTGAGAGCCGTGTCCAACATGCTGCAGAAGGCCAGACAGACCCTGGAGTTCTACCCCTGTACCTCTGAGG AAATCGACCACGAGGACATCACCAAGGACAAGACATCTACCGTGGAGGCCTGCCTGCCTCTGGAACTGACCAAGAACGAGTCCTGTCTGAACTCCAGAGAGACCAGCTTCATCACCAATGGCTCCTGCCTGGCTTCTCGGAAGACATCCTTCATGATGGCCCTGTGCCTGTCTTCCATCTACGAGGATCTGAAGATGTACCAGGTGGAATTTAAGACCATGAACGCCAAACTGCTGATGGACCCCAAGCGGCAGATCTTCCTGGACCAAAACATGCTGGCTGTGATCGACGAGCTGATGCAGGCTCTGAACTTCAACTCCGAGACCGTGCCACAGAAAAGCTCTCTGGAAGAGCCTGACTTCTATAAGACCAAGATCAAGCTCTGCATCCTGCTGCACGCCTTTCGGATCAGAGCCGTCACAATTGACCGCGTGATGTCCTACCTGAATGCCTCC.

sequence 5 (SEQ ID NO. 5) is the amino acid sequence of signal peptide 3: ATMGWSCIILFLVATATGVHS.

Sequence 6 (SEQ ID NO. 6) is the nucleotide sequence of signal peptide 3:

GCCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGC。

Sequence 7 (SEQ ID NO. 7) is the amino acid sequence of the cleavage site-Signal peptide 3-EBI 3-Fck-cleavage site ：KL|ATMGWSCIILFLVATATGVHS|RKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK|AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG|EF.

Sequence 8 (SEQ ID NO. 8) is the nucleotide sequence of the cleavage site-signal peptide 3-EBI 3-Fck-cleavage site:

AAGCTT|GCCACCATGGGCTGGTCCTGCATTATCCTGTTTCTGGTGGCCACCGCCACCGGCGTTCATTCC|AGGAAAGGACCTCCTGCTGCTCTGACCCTGCCTAGGGTGCAGTGTAGAGCTTCCAGGTATCCCATTGCTGTGGACTGTTCCTGGACACTGCCCCCTGCTCCTAATTCCACATCCCCCGTGTCCTTTATCGCCACATACAGGCTGGGCATGGCCGCCAGAGGACATTCCTGGCCTTGTCTGCAACAAACACCCACATCCACCTCCTGCACCATCACCGACGTTCAGCTGTTTTCCATGGCCCCCTATGTGCTGAACGTGACCGCTGTGCACCCTTGGGGATCTTCCTCCTCCTTTGTGCCTTTCATCACCGAGCACATCATCAAGCCCGACCCCCCCGAAGGAGTGAGGTTGTCCCCTTTGGCTGAGAGGCAACTGCAAGTGCAATGGGAACCCCCTGGCTCCTGGCCTTTTCCTGAAATTTTTTCCCTGAAGTACTGGATCAGGTACAAGAGGCAGGGCGCCGCCAGGTTTCATAGGGTGGGACCTATCGAGGCCACATCCTTTATCCTGAGGGCCGTGAGGCCCAGGGCTAGGTATTATGTGCAAGTGGCCGCTCAGGACCTGACCGACTATGGCGAACTGTCCGATTGGTCCCTGCCCGCTACAGCTACAATGTCCCTGGGAAAA|GCTGAATCCAAATACGGCCCTCCTTGCCCCCCTTGTCCTGCTCCTGAAGCTGCTGGAGGACCTTCCGTTTTTCTGTTCCCTCCTAAACCCAAAGACACCCTGATGATCTCCAGGACCCCCGAGGTTACATGTGTGGTGGTGGATGTGTCCCAGGAGGACCCCGAAGTGCAATTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCTAAGACAAAGCCCAGGGAGGAGCAGTTTAACTCCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCATCAAGACTGGCTGAATGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCTCCTCCATTGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGGGAACCTCAAGTGTATACCCTGCCCCCTTCCCAGGAGGAAATGACCAAAAATCAGGTGTCCCTGTGGTGCCTGGTGAAGGGCTTTTATCCCTCCGACATCGCCGTGGAGTGGGAGTCCAATGGCCAACCCGAAAATAACTACAAGACCACCCCCCCCGTGCTGGACTCCGATGGATCTTTTTTTCTGTACTCCAGGCTGACCGTGGACAAGTCCAGGTGGCAAGAAGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCATAATCACTACACCCAGAAATCCCTGTCCCTGTCCCTGGGC|GAATTC.

sequence 9 (SEQ ID NO. 9) is the amino acid sequence of the cleavage site-Signal peptide 3-P35-Fch-cleavage site ：KL|ATMGWSCIILFLVATATGVHS|RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS|AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG|EF.

Sequence 10 (SEQ ID NO. 10) is the nucleotide sequence of the cleavage site-Signal peptide 3-P35-Fch-cleavage site:

AAGCTT|GCCACCATGGGCTGGTCCTGCATCATTCTGTTCCTGGTGGCCACCGCCACCGGCGTGCATTCC|AGGAACTTGCCTGTGGCTACCCCCGATCCTGGAATGTTTCCCTGTCTGCACCATTCCCAAAACCTGCTGAGGGCCGTGTCCAACATGCTGCAGAAAGCCAGGCAGACCCTGGAGTTTTACCCCTGCACATCCGAGGAGATCGACCACGAGGATATCACCAAGGACAAGACCTCCACCGTGGAGGCCTGCCTGCCTCTGGAACTGACAAAAAATGAATCCTGCCTGAACTCCAGGGAAACCTCCTTCATCACCAACGGCTCCTGCCTGGCCTCTAGGAAAACATCCTTTATGATGGCCCTGTGCCTGTCCTCCATCTACGAGGACCTGAAGATGTACCAGGTGGAGTTCAAGACCATGAACGCCAAGCTGCTGATGGACCCCAAGAGGCAGATTTTCCTGGACCAGAACATGCTGGCCGTGATCGACGAGCTGATGCAGGCCCTGAACTTCAACTCCGAAACCGTGCCCCAAAAGTCCTCCCTGGAGGAACCTGACTTCTACAAGACCAAGATCAAGCTGTGCATCCTGCTGCACGCCTTCAGGATCAGGGCCGTGACTATTGATAGGGTGATGTCCTACCTGAACGCCTCC|GCCGAATCCAAGTACGGCCCTCCTTGTCCCCCTTGTCCTGCTCCTGAAGCTGCCGGAGGACCTTCCGTGTTTCTGTTCCCCCCTAAACCTAAAGACACCCTGATGATCTCCAGGACCCCCGAGGTTACATGCGTGGTGGTGGATGTGTCCCAAGAGGACCCCGAAGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAGACAAAGCCCAGGGAGGAGCAGTTCAACTCCACCTACAGGGTGGTGTCCGTGCTGACCGTTCTGCATCAGGATTGGCTGAATGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCTCCTCTATTGAGAAAACCATTTCCAAGGCCAAGGGCCAGCCCAGGGAACCCCAAGTTTATACACTGCCCCCTTCCCAAGAAGAGATGACCAAAAACCAGGTGTCCCTGTCCTGCGCCGTGAAGGGATTTTATCCCTCCGACATTGCCGTGGAGTGGGAGTCCAATGGACAGCCCGAAAATAACTACAAGACCACCCCCCCCGTGCTGGACTCCGATGGATCCTTTTTTCTGGTGTCCAGGCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGAAATGTGTTCTCCTGTTCCGTGATGCACGAGGCCCTGCACAATCACTATACCCAGAAGTCCCTGTCCCTGTCCCTGGGC|GAATTC.

sequence 11 (SEQ ID NO. 11) is the amino acid sequence of EBI 3:

RKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK.

sequence 12 (SEQ ID NO. 12) is the nucleotide sequence of the optimized EBI 3:

AGGAAAGGACCTCCTGCTGCTCTGACCCTGCCTAGGGTGCAGTGTAGAGCTTCCAGGTATCCCATTGCTGTGGACTGTTCCTGGACACTGCCCCCTGCTCCTAATTCCACATCCCCCGTGTCCTTTATCGCCACATACAGGCTGGGCATGGCCGCCAGAGGACATTCCTGGCCTTGTCTGCAACAAACACCCACATCCACCTCCTGCACCATCACCGACGTTCAGCTGTTTTCCATGGCCCCCTATGTGCTGAACGTGACCGCTGTGCACCCTTGGGGATCTTCCTCCTCCTTTGTGCCTTTCATCACCGAGCACATCATCAAGCCCGACCCCCCCGAAGGAGTGAGGTTGTCCCCTTTGGCTGAGAGGCAACTGCAAGTGCAATGGGAACCCCCTGGCTCCTGGCCTTTTCCTGAAATTTTTTCCCTGAAGTACTGGATCAGGTACAAGAGGCAGGGCGCCGCCAGGTTTCATAGGGTGGGACCTATCGAGGCCACATCCTTTATCCTGAGGGCCGTGAGGCCCAGGGCTAGGTATTATGTGCAAGTGGCCGCTCAGGACCTGACCGACTATGGCGAACTGTCCGATTGGTCCCTGCCCGCTACAGCTACAATGTCCCTGGGAAAA.

Amino acid sequence of sequence 13 (SEQ ID NO. 13) is Fck:

AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

nucleotide sequence 14 (SEQ ID NO. 14) is Fck:

GCTGAATCCAAATACGGCCCTCCTTGCCCCCCTTGTCCTGCTCCTGAAGCTGCTGGAGGACCTTCCGTTTTTCTGTTCCCTCCTAAACCCAAAGACACCCTGATGATCTCCAGGACCCCCGAGGTTACATGTGTGGTGGTGGATGTGTCCCAGGAGGACCCCGAAGTGCAATTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCTAAGACAAAGCCCAGGGAGGAGCAGTTTAACTCCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCATCAAGACTGGCTGAATGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCTCCTCCATTGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGGGAACCTCAAGTGTATACCCTGCCCCCTTCCCAGGAGGAAATGACCAAAAATCAGGTGTCCCTGTGGTGCCTGGTGAAGGGCTTTTATCCCTCCGACATCGCCGTGGAGTGGGAGTCCAATGGCCAACCCGAAAATAACTACAAGACCACCCCCCCCGTGCTGGACTCCGATGGATCTTTTTTTCTGTACTCCAGGCTGACCGTGGACAAGTCCAGGTGGCAAGAAGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCATAATCACTACACCCAGAAATCCCTGTCCCTGTCCCTGGGC.

sequence 15 (SEQ ID NO. 15) is the amino acid sequence of P35:

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS.

SEQ ID NO.16 is the nucleotide sequence of optimized P35

AGGAACTTGCCTGTGGCTACCCCCGATCCTGGAATGTTTCCCTGTCTGCACCATTCCCAAAACCTGCTGAGGGCCGTGTCCAACATGCTGCAGAAAGCCAGGCAGACCCTGGAGTTTTACCCCTGCACATCCGAGGAGATCGACCACGAGGATATCACCAAGGACAAGACCTCCACCGTGGAGGCCTGCCTGCCTCTGGAACTGACAAAAAATGAATCCTGCCTGAACTCCAGGGAAACCTCCTTCATCACCAACGGCTCCTGCCTGGCCTCTAGGAAAACATCCTTTATGATGGCCCTGTGCCTGTCCTCCATCTACGAGGACCTGAAGATGTACCAGGTGGAGTTCAAGACCATGAACGCCAAGCTGCTGATGGACCCCAAGAGGCAGATTTTCCTGGACCAGAACATGCTGGCCGTGATCGACGAGCTGATGCAGGCCCTGAACTTCAACTCCGAAACCGTGCCCCAAAAGTCCTCCCTGGAGGAACCTGACTTCTACAAGACCAAGATCAAGCTGTGCATCCTGCTGCACGCCTTCAGGATCAGGGCCGTGACTATTGATAGGGTGATGTCCTACCTGAACGCCTCC.

Amino acid sequence of sequence 17 (SEQ ID NO. 17) Fch

AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

Nucleotide sequence 18 (SEQ ID NO. 18) is Fch:

GCCGAATCCAAGTACGGCCCTCCTTGTCCCCCTTGTCCTGCTCCTGAAGCTGCCGGAGGACCTTCCGTGTTTCTGTTCCCCCCTAAACCTAAAGACACCCTGATGATCTCCAGGACCCCCGAGGTTACATGCGTGGTGGTGGATGTGTCCCAAGAGGACCCCGAAGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAGACAAAGCCCAGGGAGGAGCAGTTCAACTCCACCTACAGGGTGGTGTCCGTGCTGACCGTTCTGCATCAGGATTGGCTGAATGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCTCCTCTATTGAGAAAACCATTTCCAAGGCCAAGGGCCAGCCCAGGGAACCCCAAGTTTATACACTGCCCCCTTCCCAAGAAGAGATGACCAAAAACCAGGTGTCCCTGTCCTGCGCCGTGAAGGGATTTTATCCCTCCGACATTGCCGTGGAGTGGGAGTCCAATGGACAGCCCGAAAATAACTACAAGACCACCCCCCCCGTGCTGGACTCCGATGGATCCTTTTTTCTGGTGTCCAGGCTGACCGTGGACAAGTCCAGGTGGCAAGAGGGAAATGTGTTCTCCTGTTCCGTGATGCACGAGGCCCTGCACAATCACTATACCCAGAAGTCCCTGTCCCTGTCCCTGGGC.

sequence 19 (SEQ ID NO. 19) is the nucleotide sequence 1 of the optimized signal peptide 3:

GCCACCATGGGCTGGTCCTGCATTATCCTGTTTCTGGTGGCCACCGCCACCGGCGTTCATTCC。

Sequence 20 (SEQ ID NO. 20) is the nucleotide sequence 2 of the optimized signal peptide 3:

GCCACCATGGGCTGGTCCTGCATCATTCTGTTCCTGGTGGCCACCGCCACCGGCGTGCATTCC。

Sequence 21 (SEQ ID NO. 21) is the amino acid sequence ：ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. of the Fc of IGG4 (P01861-1) (secreted; sIgG 4) wherein: the hinge region is 1 st to 12th, CH2 is 13 rd to 122 th, and CH3 is 123 rd to 229 th.

Sequence 22 (SEQ ID NO. 22) is the amino acid sequence of the Fc of IGG4 (P01861-2):

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLEL. Wherein: the hinge region is 1 st to 12 th, CH2 is 13 rd to 122 th, and CH3 is 123 rd to 229 th.

Sequence 23 (SEQ ID NO. 23) is the amino acid sequence of the Fc of IGG1 (P01857-1) (secreted; sIgG 1):

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. Wherein: the hinge region is from 1 st to 12 th, CH2 from 13 th to 125 th, and CH3 from 126 th to 232 th.

Sequence 24 (SEQ ID NO. 24) is the amino acid sequence of the Fc of IGG1 (P01857-2):

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPEL. Wherein: the hinge region is from 1 st to 12 th, CH2 from 13 th to 125 th, and CH3 from 126 th to 232 th.

Sequence 25 (SEQ ID NO. 25) is the amino acid sequence ：ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. of the Fc of IGG2 (P01859-1) (secreted; sIgG 2) wherein: the hinge region is 1 st to 12th, CH2 is 13 rd to 121 th, and CH3 is 122 th to 228 th.

Sequence 26 (SEQ ID NO. 26) is the amino acid sequence of the Fc of IGG2 (P01859-2):

ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPEL. Wherein: the hinge region is 1 st to 12 th, CH2 is 13 rd to 121 th, and CH3 is 122 th to 228 th.

Sequence 27 (SEQ ID NO. 27) is the amino acid sequence of the Fc of IGG3 (P01860-1) (secreted; sIgG 3):

ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG. Wherein: the hinge region is from 1 st to 62 nd, CH2 from 63 rd to 172 th, and CH3 from 173 rd to 278 th.

Sequence 28 (SEQ ID NO. 28) is the amino acid sequence of the Fc of IGG3 (P01860-2):

ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPE. Wherein: the hinge region is from 1 st to 62 nd, CH2 from 63 rd to 172 th, and CH3 from 173 rd to 278 th.

Sequence 29 (SEQ ID NO. 29) is the amino acid sequence of the Fc of IGG 4:

AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG. Wherein: the hinge region is from 1 st to 13 th, CH2 from 14 th to 123 th, and CH3 from 124 th to 229 th.

Sequence 30 (SEQ ID NO. 30) is the amino acid sequence of the Fc fragment (K chain) of the IGG1 antibody:

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Sequence 31 (SEQ ID NO. 31) is the amino acid sequence of the Fc fragment (H chain) of the IGG1 antibody:

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLEASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK.

The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.

The above examples merely illustrate a few embodiments of the present application, which are convenient for a specific and detailed understanding of the technical solutions of the present application, but should not be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims

1. Recombinant human interleukin-35 having human interleukin-35 bioactivity, being a heterodimer of a first fusion polypeptide and a second fusion polypeptide;

From the N end to the C end,

The first helper peptide and the second helper peptide form a KIH structure.

2. The recombinant human interleukin-35 of claim 1, which satisfies one or more of the following conditions:

(2) The second active peptide and the second auxiliary peptide are directly linked;

(3) The first helper peptide and the second helper peptide are each independently selected from mutant peptides of an Fc fragment of an antibody; alternatively, the first helper peptide and the second helper peptide are each independently selected from mutant peptides of an Fc fragment of an IgG antibody.

3. The recombinant human interleukin-35 of any one of claims 1 to 2, wherein the N-terminus of the first and second fusion polypeptides are each independently further linked to a signal peptide;

optionally, the N-terminal of the signal peptide is also connected with a first protease cleavage site fragment;

Optionally, the C-terminus of the first fusion polypeptide and the second fusion polypeptide are each independently linked to a second protease cleavage site fragment.

4. A nucleic acid molecule encoding the recombinant human interleukin-35 of any one of claims 1 to 3.

5. A recombinant vector comprising the nucleic acid molecule of claim 4.

6. A recombinant cell expressing the recombinant human interleukin-35 of any one of claims 1 to 3.

7. A method of constructing a recombinant cell according to claim 6, comprising the step of introducing the nucleic acid molecule according to claim 4 into a host cell to construct a recombinant cell.

8. A method of preparing the recombinant human interleukin-35 of any one of claims 1 to 3, comprising the steps of:

Culturing the recombinant cell of claim 6; and

Isolating said recombinant human interleukin-35 from the resulting culture.

9. Use of the recombinant human interleukin-35 of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the recombinant vector of claim 5 or the recombinant cell of claim 6 in the manufacture of a medicament for the treatment of cancer, an autoimmune disease or an inflammatory disease.

10. A medicament comprising the recombinant human interleukin-35 of any one of claims 1 to 9, the nucleic acid molecule of claim 4, the recombinant vector of claim 5 or the recombinant cell of claim 6, and a pharmaceutically acceptable carrier.