CN104628870A - Human IL12Rbeta1-CHR protein and Fc fusion protein thereof - Google Patents

Abstract

The invention discloses a human IL12Rbeta1-CHR protein and an Fc fusion protein of the human IL12Rbeta1-CHR protein, a gene encoding the protein and use of the protein in treating autoimmune diseases. The protein disclosed by the invention has high affinity on hIL-12/IL-23 and can antagonize differentiation and development of IL-12/IL-23 induced Th1/Th17 cells in vitro and in vivo and moreover, the protein can inhibit secretion of inflammatory factors such as inflammatory cytokines IFN-gamma, IL-17A, IL-22 and the like and can be used for treating chronic diseases associated with Th1/Th17 dysfunction such as autoimmune diseases or chronic infection and the like. Compared with the IL12Rbeta1-CHR/Fc protein, the IL12Rbeta1-CHR/Fc protein provided by the invention has the advantages of good stability, high activity, long half-life in vivo and the like. The invention includes a molecular design, expression and purification, treatment of diseases and the like of the protein.

Description

A kind of human IL12Rβ1-CHR protein and its Fc fusion protein

technical field

The invention belongs to the field of biotechnology, and in particular relates to a recombinant human IL12Rβ1-CHR protein capable of binding IL-12/IL-23 and antagonizing its effect and its Fc fusion protein and the use of such protein in treating autoimmune diseases.

Background technique

Autoimmune disease is the third most common disease after cardiovascular disease and cancer, and it is one of the main chronic diseases that seriously threaten public health worldwide, and its overall incidence accounts for 3%-5% of the world's population. %. The pathogenesis of autoimmune diseases is not yet fully understood, and it is related to abnormal immune cells and immune regulatory dysfunction caused by viral infection, genetics, environment, drugs and other reasons. The abnormality of CD4 ⁺ regulatory T cells (T helper cells, Th) is one of the most important pathogenesis of autoimmune diseases. In the past two decades, the classification of Th1 and Th2 cells has constituted the basis of people's understanding of CD4 ⁺ T cells. The basis of immunological functions and their role in the regulation of innate and adaptive immunity. Th1 cells play an important role in anti-infection and anti-tumor responses by secreting IFN-γ, IL-12 and other cytokines. At the same time, abnormally elevated Th1 activity is also one of the important mechanisms for the pathogenesis of organ-specific autoimmune diseases. Th2 cells promote the body's humoral immune response by secreting IL-4, IL-5, IL-13 and other cytokines, and on the other hand, they also participate in the pathogenesis of asthma and systemic autoimmune diseases. T regulatory cells (Treg) play an important role in maintaining the immune balance of the body. Through the above-mentioned functional classification of T cells, people's understanding of the immune regulatory network has been enhanced, and at the same time, it has provided a basis for intervening in diseases. However, with the deepening of research, it was found that the above T cell functional classification cannot fully explain the pathogenesis of some autoimmune diseases, infections and allergic reactions and other diseases.

Recent studies have found that a group of cell subpopulations different from Th1, Th2, and Treg, which do not express IL-4 or IFN-γ, but secrete IL-17 at a high level, are named Th17 cells. The discovery of Th17 cells is more related to the study of autoimmune diseases. It is a special cell subgroup in the process of T cell differentiation, and it exerts immunological functions by secreting the characteristic cytokine IL-17. IL-17 is a pro-inflammatory cytokine that can recruit and activate neutrophils, induce the expression of other inflammatory factors, mediate the infiltration of inflammatory cells in the lesion site and eventually cause tissue damage. Studies have shown that there are inappropriate activation and functional imbalance of Th17 cells in many patients with autoimmune diseases, manifested as abnormal up-regulation of IL-17 and transcription factor RORγt, so if the differentiation and expansion of Th17 cells in patients can be effectively blocked And the expression of related cytokines can prevent, delay or even prevent the occurrence and development of autoimmune diseases. A large number of clinical experimental studies have found that inhibiting the function of Th17 cells can significantly improve the symptoms of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus and psoriasis. Liver tissue has a differentiation advantage for Th17 cells. In terms of liver autoimmunity and other liver inflammatory diseases, the induction of Th17 cells by the liver microenvironment is very important, and in patients with hepatitis B virus infection and liver cancer, Th17 cells increase significantly. These phenomena are It has aroused extensive attention of researchers on Th17 cells.

IL-23, belonging to the IL-12 family, shares the IL-12p40 subunit with IL-12, and also has a unique p19 subunit. The p40 and p19 subunits can interact with the IL-12Rβ1 of the IL-23 receptor complex respectively Binds to IL-23R subunit and plays a role. IL-23 is an important factor in the process of differentiation, development and function maintenance of Th17 cells. It plays a pro-inflammatory role by stimulating T cells to secrete IL-17. The absence of IL-23 will significantly reduce the production of Th17 cells, and at the same time reduce the autoimmunity of model mice The incidence of disease, reducing the severity of its onset. Numerous evidences have proved that the IL-23/Th17/IL-17 axis plays an important role in the development of autoimmune diseases, suggesting that we use IL-23/Th17 as a new drug target to obtain better disease Treatment prospects. During the long-term evolution process, the binding affinity between receptor and ligand is generally very high, and since soluble receptor is the extracellular region of cell receptor, its closed site is exactly the region where ligand binds to cell receptor. The FDA-approved drug Enbrel (TNFαR-Fc fusion protein) for the treatment of moderate to severe rheumatoid arthritis has performed well in clinical practice, proving that this receptor protein can also achieve the same or even better efficacy than antibodies.

Protein drugs are a new hotspot in the treatment of autoimmune diseases. These drugs are highly targeted and effective, but due to their short half-life, they have to be administered repeatedly in animal experiments or clinical studies, and their application is limited. How to prolong the plasma half-life and enhance the stability of recombinant proteins is a hotspot in the field of biopharmaceuticals, among which constructing Fc fusion proteins to achieve this goal is a highly feasible strategy. Fc fusion protein refers to a new type of recombinant protein produced by fusing a certain biologically active functional protein molecule (soluble ligand, receptor or other biologically active substances that need to prolong the half-life) and Fc fragments by genetic engineering and other technologies. This type of fusion protein not only retains the biological activity of functional protein molecules, but also has many special properties: the Fc fragment can bind to FcRn in a pH-dependent manner, which can prolong the half-life of the fusion protein and improve its stability, making it more suitable for the human body. Function; the interchain disulfide bonds of the Fc fragment are conducive to the formation of multimers of the fusion molecule, thereby enhancing the ligand binding ability and improving biological activity; the introduction of the Fc fragment is conducive to improving the expression level of the fusion molecule in mammalian cells.

To sum up, the present invention aims at IL-12/IL-23p40 subunit, and synthesizes human IL12Rβ1-CHR protein and its Fc fusion protein through genetic engineering means, and affinity experiments have confirmed that such protein can bind to human IL-12/IL-23, Furthermore, by establishing mouse and human Th1/Th17 cell differentiation and development platforms and related autoimmune disease animal models, the in vivo and in vitro activities of such protein molecules are studied. The human IL12Rβ1-CHR protein and its Fc fusion protein of the present invention can specifically block IL-12/Th1 and IL-23/Th17 signaling pathways, reduce abnormally increased Th1/Th17 cell levels in vivo and in vitro, and improve disease symptoms. Moreover, the IL12Rβ1-CHR/Fc fusion protein of the present invention has the advantages of good stability, high activity and long half-life in vivo compared with IL12Rβ1-CHR protein.

Contents of the invention

The present invention selects the p40 subunit as the target molecule, constructs and expresses the human IL12Rβ1 extracellular region receptor cytokine binding homology region (CHR) and its Fc fusion protein, in order to exert an effect similar to that of an antibody, and provide a therapeutic solution for autoimmune diseases and Treatment of chronic infectious diseases offers new options.

The first problem to be solved by the present invention is to provide the gene sequence encoding hIL12Rβ1-CHR, which is connected into prokaryotic expression vectors such as PGEX-4T-2, pet 22b, pet 32a, etc., and transformed into BL21(DE3), ROSETTA and other strains for expression.

The second problem solved by the present invention is that the protein expressed by the PGEX-hIL12Rβ1-CHR vector can be purified by a GST affinity column to obtain a GST fusion protein, and then the specific site can be cut with thrombin, and the flow-through can be collected by the GST affinity column Obtain unlabeled hIL12Rβ1-CHR protein, and detect the protein by SDS-PAGE and Western blot.

The third problem solved by the present invention is to provide the gene sequence of IL12Rβ1-CHR/Fc containing IL-12Rβ1 signal peptide, which is connected into eukaryotic expression vectors such as pcDNA3.1, pngfpN, PEE144 and the like.

The fourth problem solved by the present invention is to use cheap linear PEI for transfection, and reduce the transient transfection cost of collecting eukaryotic expression proteins in a large amount in a short period of time.

The fifth problem solved by the present invention is to transfect the eukaryotic expression vector into CHO cells by liposome transfection or electrotransfection technology, and G418 pressurized screening to obtain CHO monoclonal stably expressing IL12Rβ1-CHR/Fc fusion protein cell line.

The sixth problem to be solved by the present invention is to culture stable transfected cell lines in suspension, collect secreted proteins in large quantities, separate and purify, analyze and identify, and freeze-dry and store.

The seventh problem solved by the present invention is that the IL12Rβ1-CHR/Fc fusion protein can be purified by Protein A, Protein G affinity chromatography columns and other chromatography columns to obtain high-purity protein molecules, which can be detected and purified by SDS-PAGE and Western blot fusion protein.

The eighth problem to be solved by the present invention is that the affinity experiment proves that the expressed hIL12Rβ1-CHR protein and its Fc fusion protein can specifically bind to hIL-12/IL-23, and the IL12Rβ1-CHR/Fc fusion protein is more effective than the hIL12Rβ1-CHR protein. Stronger affinity.

The ninth problem solved by the present invention is to inject hIL12Rβ1-CHR and hIL12Rβ1-CHR/Fc fusion protein into the tail vein of Wistar rats. Orbital blood was collected at 32, 36, 48, 72, and 96 hours respectively, and the concentration of the drug was detected by ELISA, and the half-life was calculated by PKsolver software.

The tenth problem solved by the present invention is to induce the differentiation and development of Th1/Th17 cells in vitro. While stimulating T cells to differentiate into Th1/Th17 cells, different concentrations of IL12Rβ1-CHR and IL12Rβ1-CHR/Fc proteins are added, and through flow cytometry To detect the differentiation of Th1/Th17 cells, ELISA to detect the secretion and expression of inflammatory factors in the culture supernatant, and Q-PCR to detect the gene expression levels of Th1/Th17 cell-related factors, so as to study the influence of receptor proteins on the differentiation and development of Th1/Th17 cells .

The eleventh problem that the present invention solves is to establish multiple sclerosis animal model (EAE), set normal group, model group, IL12Rβ1-CHR group, IL12Rβ1-CHR/Fc group and cyclosporine positive drug group, research and To compare the therapeutic effects of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein on related diseases and their advantages compared with positive drugs.

The twelfth problem solved by the present invention is to establish a rheumatoid arthritis animal model (CIA), set normal group, model group, IL12Rβ1-CHR group, IL12Rβ1-CHR/Fc group and cyclosporine positive drug group, study And compare the therapeutic effects of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc protein on related diseases and their advantages compared with positive drugs.

The thirteenth problem solved by the present invention is to establish systemic lupus erythematosus (SLE) induced by chronic graft-versus-host disease (cGVHD), set up normal group, model group and IL12Rβ1-CHR/Fc group, study and compare IL12Rβ1-CHR The therapeutic effect of IL12Rβ1-CHR/Fc protein and IL12Rβ1-CHR/Fc protein on related diseases.

The fourteenth problem solved by the present invention is to establish a DSS-induced ulcerative colitis mouse model, set up normal group, model group, IL12Rβ1-CHR group and IL12Rβ1-CHR/Fc group, study and compare IL12Rβ1-CHR protein and IL12Rβ1 -Therapeutic effect of CHR/Fc protein on related diseases.

The fifteenth problem solved by the present invention is to establish an animal model of acute liver injury induced by ConA, set up normal group, model group, IL12Rβ1-CHR group and IL12Rβ1-CHR/Fc group, study and compare IL12Rβ1-CHR protein and IL12Rβ1-CHR Therapeutic effect of Fc protein on related diseases.

The sixteenth problem solved by the present invention is to establish a psoriasis-like inflammation model, set up normal group, model group, IL12Rβ1-CHR group and IL12Rβ1-CHR/Fc group, study and compare IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc The therapeutic effect of protein on related diseases.

The seventeenth problem solved by the present invention is to establish a mouse orthotopic liver cancer model, set up normal group, model group, IL12Rβ1-CHR group and IL12Rβ1-CHR/Fc group, study and compare IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc The therapeutic effect of protein on related diseases.

The eighteenth problem solved by the present invention is that in animal experiments, it is found that IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc protein are safer than traditional immunosuppressants, and have no obvious adverse effects on experimental animal weight, hemoglobin level and other indicators Toxic and side effects, it has a good advantage in clinical treatment of related diseases.

Description of drawings

Fig. 1 is the electrophoresis diagram of 1% nucleic acid of the PCR product of IL12Rβ1-CHR gene.

M: 150bp DNA Ladder Marker; 1: PCR product (650bp)

Fig. 2 is a diagram of double enzyme digestion identification of PGEX-IL12Rβ1-CHR.

M: 250bp DNA Ladder Marker; 1: PGEX-IL12Rβ1-CHR/BamH I+Xho I (4900bp, 650bp)

Fig. 3 is an SDS-PAGE analysis chart of the expressed GST-IL12Rβ1-CHR fusion protein.

M: Standard molecular weight protein marker; 1: Escherichia coli BL21(DE3) empty bacteria; 2: Whole bacteria not induced by PGEX-IL12Rβ1-CHRIPTG; 3-5: Whole bacteria induced by PGEX-IL12Rβ1-CHRIPTG

Fig. 4 is the SDS-PAGE analysis diagram of the purified GST-IL12Rβ1-CHR fusion protein.

M: standard molecular weight protein marker; 1: purified GST-IL12Rβ1-CHR

Figure 5 is a Western Blot analysis diagram of GST-IL12Rβ1-CHR fusion protein.

M: pre-stained protein marker; 1: PGEX-4T-2 empty plasmid; 2, 3: purified GST-IL12Rβ1-CHR

Fig. 6 is an SDS-PAGE analysis chart of recombinant IL12Rβ1-CHR protein without tags after digestion and purification.

M: Standard molecular weight protein marker; 1: Recombinant IL12Rβ1-CHR protein without tag after digestion and purification

Fig. 7 is an electrophoresis diagram of 1% nucleic acid of the PCR product of IL12Rβ1-CHR/Fc recombinant gene.

M: 250bp DNA Ladder Marker; 1: IL12Rβ1-CHR/Fc sequence overlap PCR product (1400bp); 2: IL12Rβ1-CHR sequence PCR product containing signal peptide (700bp); 3: Fc sequence PCR product of recombinant gene (700bp)

Fig. 8 is the nucleic acid electrophoresis diagram of double enzyme digestion identification of pcDNA3.1(+)-IL12Rβ1-CHR/Fc.

M: 250bp DNA Ladder Marker; 1: pcDNA3.1(+)-IL12Rβ1-CHR/Fc/Hind III+Xho I (5400bp, 1400bp); 2: Undigested pcDNA3.1(+)-IL12Rβ1-CHR/ Fc

Fig. 9 is a nucleic acid electrophoresis detection diagram of a linearized plasmid.

M: DNA Marker; 1, 2: pcDNA3.1(+)-IL12Rβ1-CHR/Fc plasmid digested with PvuI

Fig. 10 is a graph of PCR nucleic acid electrophoresis identification of the genome of a stable cell line.

M: DNA Marker; 1, 2: Genomic PCR products of two stable cell lines screened

Figure 11 is the SDS-PAGE electrophoresis of IL12Rβ1-CHR/Fc fusion protein.

1: Monomer form of IL12Rβ1-CHR/Fc fusion protein (reduced); 2: Dimeric form of IL12Rβ1-CHR/Fc fusion protein (non-reduced)

Figure 12 is a Western Blot analysis diagram of IL12Rβ1-CHR/Fc fusion protein.

1: Monomer form of IL12Rβ1-CHR/Fc fusion protein (reduced); 2: Dimeric form of IL12Rβ1-CHR/Fc fusion protein (non-reduced)

Figure 13 is the results of direct ELISA detection of the affinity of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein to IL-12/IL-23.

Fig. 14 is a blood concentration-time curve of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein in rats.

Figure 15 is the ELISA detection results of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein inhibiting the secretion and expression of mouse inflammatory factors IFN-γ, IL-17A and IL-22 in vitro.

Fig. 16 is a flow cytometric analysis diagram of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein inhibiting mouse Th1/Th17 differentiation in vitro.

Figure 17 is a graph showing the results of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein inhibiting gene expression of mouse Th1/Th17 cell transcription factors T-bet and RORγt in vitro.

Fig. 18 is a flow cytometric analysis diagram of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein inhibiting human Th1/Th17 differentiation in vitro.

Fig. 19 is the ELISA detection result of L12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein inhibiting the secretion and expression of human IFN-γ and IL-17A in vitro.

Fig. 20 is a graph showing the results of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein inhibiting gene expression of human Th1/Th17 cell transcription factors T-bet and RORγr in vitro.

Fig. 21 is a graph showing the clinical scores of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein in treating multiple sclerosis mouse EAE model.

Fig. 22 is HE pathological sections of mouse brain and spinal cord in EAE model group, IL12Rβ1-CHR administration group and IL12Rβ1-CHR/Fc administration group.

Figure 23 is the ELISA detection results of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein inhibiting the secretion of inflammatory factors IFN-γ and IL-17A in the serum of EAE mice.

Fig. 24 is a flow cytometric analysis diagram of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein inhibiting Th1/Th17 cell differentiation in splenocytes of EAE mice.

Fig. 25 is a graph showing the clinical scores of IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein in treating CIA model of rheumatoid arthritis rats.

Figure 26 shows the X-ray imaging results of rats in the normal group, CIA model group, IL12Rβ1-CHR administration group and IL12Rβ1-CHR/Fc administration group.

Fig. 27 is HE pathological slices of ankle joints of rats in normal group, CIA model group, IL12Rβ1-CHR administration group and IL12Rβ1-CHR/Fc administration group.

Fig. 28 shows the ELISA detection results of IL12Rβ1-CHR and IL12Rβ1-CHR/Fc fusion protein inhibiting the expression levels of cytokines such as TNF-α, IFN-γ and IL-17A in serum of CIA rats.

Fig. 29 is a comparison of the anti-ss-DNA antibody levels of mice in each group of SLE models at different time points.

Figure 30 is the immunofluorescence images of the kidneys of mice in each group of SLE models.

Figure 31 shows the results of HE and PAS staining of kidney pathological sections of mice in various groups of SLE models.

Fig. 32 is an ELISA detection graph of IL12Rβ1-CHR and IL12Rβ1-CHR/Fc fusion protein inhibiting the secretion and expression of IFN-γ and IL-17A in the serum of SLE mice.

Figure 33 is the DAI score of DSS-induced ulcerative colitis mouse model treated with IL12Rβ1-CHR and IL12Rβ1-CHR/Fc fusion protein.

Fig. 34 is the pathological sections of the colon tissues of mice in the ulcerative colitis model group, the IL12Rβ1-CHR administration group and the IL12Rβ1-CHR/Fc administration group.

Fig. 35 is an ELISA detection chart of IL12Rβ1-CHR and IL12Rβ1-CHR/Fc fusion protein inhibiting the expression of inflammatory factors IFN-γ and IL-17A in the colon tissue of mice with ulcerative colitis.

Fig. 36 is a graph showing the results of serum transaminase (ALT) levels of mice in each experimental group of the acute liver injury model.

Fig. 37 is HE pathological sections of liver tissues in the normal group, the acute liver injury model group and the IL12Rβ1-CHR/Fc administration group.

Fig. 38 is an ELISA detection graph of IL12Rβ1-CHR and IL12Rβ1-CHR/Fc fusion protein inhibiting the expression of inflammatory factors TNF-α and IL-6 in the serum of acute liver injury model mice.

Fig. 39 is a picture of HE staining of the skin of mice in each experimental group of the psoriasis-like model after being sacrificed on day 14.

Fig. 40 is an ELISA detection chart of IL12Rβ1-CHR and IL12Rβ1-CHR/Fc fusion protein inhibiting the expression of inflammatory factors IL-17A, IL-22 and IFN-γ in serum of psoriasis-like model mice.

Fig. 41 is a HE staining diagram of liver tumor tissues of mice in each experimental group of the primary liver cancer model after 14 days of sacrifice.

Figure 42 is a graph of Q-PCR detection of receptor protein inhibiting the expression levels of IL-17A and IFN-γmRNA in the liver tissue of mice with primary liver cancer.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention, but are not used to limit the scope of the present invention. - Base synonymous mutations of CHR/Fc fusion proteins, Fc substitutions, vector substitutions, changes in host bacteria and expression cells, changes in purification methods, construction of animal models of related autoimmune diseases and increase in indications all belong to this The scope of protection claimed by the invention.

Example 1 Construction of pGEX-IL12Rβ1-CHR prokaryotic expression vector

1. PCR amplification of IL12Rβ1-CHR gene

Total RNA was extracted from normal human peripheral blood leukocytes, the first strand of cDNA was synthesized by reverse transcription, and the IL12Rβ1-CHR sequence was amplified using it as a template. The primer sequences used are as follows:

Upstream primer: F: 5′CGGGATCC TGCAGAACCAGTGAGTGCT 3′(BamH I)

Downstream primer: R: 5′CCGCTCGAGTTAAAAATACAAAATTAGCCGGG 3′(Xho 1)

The PCR reaction conditions are as follows:

Step 1: Pre-denaturation at 98°C for 30s, 1 cycle;

Step 2: Denaturation at 98°C for 30s, annealing at 55°C for 15s, extension at 72°C for 90s, a total of 30 cycles;

Step 3: Extend at 72°C for 10 min, 1 cycle.

The PCR product was identified by 1% nucleic acid electrophoresis, and a target band of about 650bp could be seen (Fig. 1).

2. Construction of pGEX-IL12Rβ1-CHR prokaryotic expression vector

The IL12Rβ1-CHR gene fragment recovered from rubber tapping was digested with PGEX-4T-2 vector respectively, and then ligated with T4 ligase overnight at 16°C. The obtained linker was transformed into Escherichia coli E.coli DH5α competent cells, the transformation product was spread on the LB medium plate containing 100 μg/mL Amp, cultivated in a 37°C incubator for 16 hours, picked a single colony to screen positive clones, double enzyme digestion and Sequencing identified a correctly constructed expression vector containing the target gene (Fig. 2).

Example 2 Expression and separation and purification of pGEX-IL12Rβ1-CHR prokaryotic expression vector

1. Induced expression of pGEX-IL12Rβ1-CHR prokaryotic expression vector

Transform pGEX-IL12Rβ1-CHR into Escherichia coli E.coli BL21(DE3), spread the transformation product on the LB medium plate containing 100 μg/mL Amp, incubate in a 37°C incubator for 16 hours, and pick a single colony into 3 mL medium containing 100 μg/mL Amp In LB medium, culture at 220rpm in a shaker at 37°C until the OD ₆₀₀ of the bacterial solution is 0.6-0.8, add 1.0mM IPTG and continue to culture at 37°C for 6 hours to induce the expression of the target protein, and the target protein at 50KDa can be seen by SDS-PAGE electrophoresis of the whole bacteria belt (Figure 3).

2. Expanded culture of pGEX-IL12Rβ1-CHR

Expand the seed-preserving bacteria solution to 1L LB medium, culture in a 37°C shaker at 220rpm until the OD ₆₀₀ of the bacteria solution is 0.6-0.8, add 0.2mMIPTG and culture overnight at 16°C on a shaker. 8500rpm, centrifuge at 4°C for 10min to collect the bacteria, and weigh the wet bacteria. Add bacteriostasis buffer at 10mL/g wet bacterial weight, stir well until the bacterial cells are completely resuspended. Power 400w, ultrasound 3s, interval 3s, ice bath ultrasound 15min. The obtained ultrasonic mixture was centrifuged at 12000 rpm for 20 min in an ultra-fast refrigerated centrifuge, and the supernatant was collected. The fusion protein is present in the supernatant in its native, soluble form.

3. Separation and purification of GST-IL12Rβ1-CHR protein

First equilibrate the GST column with 10 times the column volume of bacteriostasis buffer, and then filter the ultrasonic supernatant obtained by centrifugation through a 0.45 μm filter and load the sample at a flow rate of 0.6-1.0 mL/min. The fusion protein with the GST tag will be adsorbed on the affinity column superior. Pay attention to maintain a low temperature environment when loading samples to prevent protein denaturation. After loading the sample, equilibrate the column with 10 times the column volume of bacteriostasis buffer to wash away the loosely bound impurities. Slowly elute with freshly prepared 10mM pH7.5 bacteriostasis buffer containing GSH, and collect the eluate in a 1.5mL EP tube. The collected protein solution was analyzed by SDS-PAGE and Western Blotting, and the purity was as high as 90% (Fig. 4, Fig. 5).

4. Isolation and purification of IL12Rβ1-CHR protein

Purify the obtained GST fusion protein, add thrombin according to the amount of 1U digested 2mg protein, digest at 4°C for 48 hours, pass the proteolytic digestion product through the GST column, and the collected flow-through is IL12Rβ1-CHR protein without GST tag, SDS -PAGE electrophoresis identified the target protein at 24KDa (Figure 6).

Example 3 Construction of pcDNA3.1(+)-IL12Rβ1-CHR/Fc Expression Vector

1. Over-lap PCR construction of IL12Rβ1-CHR/Fc fusion gene

Total RNA was extracted from normal human peripheral blood leukocytes, reverse-transcribed to synthesize the first strand of cDNA, and then used as a template to amplify IL12Rβ1-CHR sequence containing signal peptide and IgG1Fc with P1, P2, P3, and P4 upstream and downstream primers respectively Gene, the primer sequences used are as follows:

IL-12Rβ1 upstream primer: P1: 5′CCCAAGCTTATGGAGCCGCTGGTGA 3′(Hind III)

IL-12Rβ1 downstream primer: P2: 5′GGGGTTTTTCAGGGGGAAC 3′

Fc upstream primer: P3: 5'GTTCCCCCTGAAAACCCCCAAAGGGCCCTTCTGTG 3'

Fc downstream primer: P4: 5′-CCTCTCGAGTCACTTGCCGGGGGAC-3′(Xho I)

The PCR reaction conditions are as follows:

Step 1: Pre-denaturation at 98°C for 30s, 1 cycle;

Step 2: Denaturation at 98°C for 30s, annealing at 55°C for 15s, extension at 72°C for 90s, a total of 30 cycles;

Step 3: Extend at 72°C for 10 min, 1 cycle.

The PCR product was identified by 1% nucleic acid electrophoresis, and the IL12Rβ1-CHR sequence band containing the signal peptide of about 700bp and the IgG1Fc sequence band of about 700bp were seen ( FIG. 7 ).

The two fragments recovered from rubber tapping were spliced by Over-lap PCR using P1 and P4 as primers. In order to improve specificity, the Touchdown method was used. The PCR reaction conditions were as follows:

Step 1: Pre-denaturation at 98°C for 30s, 1 cycle;

Step 2: Denaturation at 98°C for 30s, annealing at 65°C for 15s, extension at 72°C for 90s, and then decreasing by 1°C for each cycle until 55°C, a total of 10 cycles;

Step 3: Denaturation at 98°C for 30s, annealing at 55°C for 15s, extension at 72°C for 90s, 20 cycles;

Step 4: Extend at 72°C for 5 minutes, 1 cycle.

The PCR product was identified by 1% nucleic acid electrophoresis, and the IL12Rβ1-CHR/Fc target gene band of about 1400bp was seen ( FIG. 7 ).

2. Construction of pcDNA3.1(+)-IL-12Rβ1-CHR/Fc expression vector

The IL-12Rβ1-CHR/Fc gene fragment recovered from rubber tapping was digested with the pcDNA3.1(+) vector separately, and ligated overnight at 16°C by T4 ligase. Take the full amount of linker to transform E. coli DH5α competent, spread the transformation product on the LB medium plate containing 100μg/mL Amp, culture in a 37°C incubator for 16h, pick a single colony to screen positive clones, sequence identification and double enzyme The correct eukaryotic expression vector containing IL12Rβ1-CHR/Fc target gene was obtained through cutting identification ( FIG. 8 ).

Example 4 Transient transfection expression of IL-12Rβ1-CHR/Fc fusion protein

1. Preparation of recombinant plasmids for transfection

The plasmid extraction process for transfection refers to the Tiangen Endotoxin-Free Plasmid Extraction Kit, and the ultraviolet spectrophotometer detects the OD260/OD280 of the plasmid solution. When the result is 1.8-2.0, measure the A value (OD ₂₆₀ ) and calculate according to the following formula Plasmid concentration:

Concentration (μg/μL) = A value × dilution factor × 50/1000

2. Preparation of cells to be transfected

About 24 hours before transfection, subculture the 293T cells again, try to ensure that the cells are in good condition and the density reaches 70-90% at the time of transfection; before transfection, replace the old medium with fresh serum-free DMEM medium.

3. PEI transient transfection

According to different culture devices, prepare a sufficient amount of transfection complex according to the ratio of PEI (μg):plasmid (μg) = 2:1 to 5:1: plasmid and PEI were diluted in an equal volume of opti-MEM, and then Add the PEI dilution to the DNA dilution, mix gently, and incubate at room temperature for 10-15 minutes to obtain a PEI/DNA transfection complex that is 1/10 times the volume of the medium. Evenly drop the transfection complex into the culture device, shake gently to mix it evenly with the fresh medium, replace with serum-free medium after 4-6 hours of transfection, culture for 3-5 days, and collect the culture supernatant.

Example 5 Screening of Stable Expression Cell Lines of IL-12Rβ1-CHR/Fc Fusion Protein

1. Preparation of Linearized Plasmids

Take 3 μg of DNA according to the concentration of the DNA sample, and add PvuI enzyme to digest in a 37°C water bath overnight. After digestion, take 100ng of the linearized plasmid for electrophoresis detection to check whether the linearization of the restriction enzyme digestion is complete (Figure 9). Add 1/10 volume of 3.0M NaOAC and 2 volumes of frozen absolute ethanol (-20°C) to the centrifuge tube where the sample is located, mix well and place at -80°C for 2 hours. Then centrifuge at 13200rpm for 10min, carefully suck off the supernatant, and dry for 10min.

2. Preparation of cells to be transfected

Remove the supernatant from the CHO cells that are in the logarithmic growth phase, add an appropriate amount of trypsin to digest at 37°C for 2-3min (the cells can be seen floating and dispersed under the microscope), then add an appropriate amount of serum-containing medium to stop the digestion, and the cells are blown away Then add to the centrifuge tube and centrifuge at 1000rpm for 5min. Discard the supernatant and flick the bottom of the centrifuge tube to float the cell pellet. Add an appropriate amount of 1×PBS to wash, record the liquid volume in the centrifuge tube and count the cells. Centrifuge at 1000rpm for 5min, remove the supernatant, flick the bottom of the centrifuge tube to float the cell pellet. According to the result of cell counting, add an appropriate amount of 1×PBS to make the cell density to 4× ¹⁰⁶ cells/mL.

3. Electrotransfection

Take 50 μL of 1×PBS solution containing 2.5 μg of linearized plasmid, add 50 μl of cell suspension, mix and add all into the pre-cooled electric shock cup. 2. Set the voltage of the electroporator to 160V, and shock once every 15ms. Pipette the cells to culture medium containing 3% FBS.

4. G418 screening of monoclonal

(1) After 24 hours of cell culture after electroporation, G418 was added at a concentration of 400 μg/mL, and the cell status was observed every day. If the number of dead cells increased, the concentration of G418 was appropriately reduced. If cell proliferation is obvious, increase the concentration of G418 to kill negative cells. The medium was changed every 2 days, and the resistance was screened for 10 days.

(2) Collect and count surviving cells, and dilute to 1 cell/200 μL with culture medium. 200 μL per well was added to a 96-well culture plate. After 8 days, a single clone was selected, and after 10 days, the cell culture solution of the single clone was taken out for ELISA detection. According to the ELISA results, the best 6 monoclonals were selected and transferred to 6-well plates for culture, and samples were taken for ELISA detection 5 days later. According to the ELISA results, the two cell lines with the best ELISA were selected and transferred to a 10cm culture dish to continue to expand the culture. After 48 hours, the supernatant was taken to detect the ELISA, and the cells were identified by the PCR method as the correct stable cell line (Figure 10). Freeze stable cell lines into liquid nitrogen tanks.

Example 6 Separation and purification of Fc fusion protein

Equilibrate the Protein A column with 10 times the column volume of binding buffer, centrifuge at 12000rpm for 20min to collect the culture supernatant, filter through a 0.45μm filter to remove impurities, mix and dilute with the binding buffer at a ratio of 1:1, and then use a flow rate of 1mL/min sample. After loading the sample, wash with 10 times the column volume of binding buffer. Add 0.06-0.2mL of 1M Tris in advance to the 1.5mL EP tube to collect samples to neutralize the pH of the eluent. The target protein was eluted with 2 to 5 times the column volume of citrate buffer, and the reduced and non-reduced forms of the protein were identified by SDS-PAGE and Western Blot, proving that the IL-12Rβ1-CHR/Fc fusion protein can dimerize in the non-reduced state It exists in body form (Fig. 11, Fig. 12).

Example item 7: Affinity detection of IL12Rβ1-CHR protein and its Fc fusion protein with IL-12 and IL-23 - direct ELISA

1. Enzyme-labeled strips were coated with 10ng hIL-12 or hIL-23 per well, and incubated overnight at 4°C.

2. Wash 3 times with PBST, each time for 10 minutes, add 100 μL of 5% BSA solution to each well, and incubate at 37°C for 2 hours.

3. Wash with PBST for 3 times, 10 min each time, add 1000, 500, 250, 125, 62.5, 31.3, 15.6 nM IL12Rβ1-CHR protein or IL12Rβ1-CHR/Fc fusion protein to each well, and incubate at 37°C for 2 hours.

4. Wash 3 times with PBST, 10 min each time, add 100 μL primary antibody solution against human IL12Rβ1 to each well, and incubate at 37°C for 2 h.

5. Wash 3 times with PBST, each time for 10 minutes, add 100 μL of HRP-labeled secondary antibody solution to each well, and incubate at 37°C for 40 minutes.

6. Wash with PBST 4 times, each time for 10 minutes, add 100 μL TMB solution to each well, place in the dark at 37°C for 20 minutes, immediately add 100 μL 2M sulfuric acid to terminate the color reaction, measure the OD ₄₅₀ value, use Graphpad software to draw a fitting curve, and calculate _EC50 value. The results showed that both IL12Rβ1-CHR protein and its Fc fusion protein could specifically bind to human IL-12 and IL-23 (Figure 13), which laid a good experimental foundation for the next in vivo and in vitro activity research.

Example 8: Pharmacokinetic study of receptor protein in rats

Wistar rats were randomly divided into 2 groups, 6 in each group, and 2 mg/kg IL12Rβ1-CHR protein and 4 mg/kg IL12Rβ1-CHR/Fc fusion protein were injected into the tail vein respectively. At 0.25, 0.5, 1, 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 48, 72, 96, 120 hours after administration, blood was taken from the orbit, and the blood samples were left at room temperature for 30 minutes, 3000r After centrifuging for 15 min at 100 min/min, the serum was carefully aspirated with a micro-sampler, the protein content in the serum was detected by ELISA, the blood drug concentration at different times was calculated, and the c-t curve was fitted using the pharmacokinetic analysis software PKSolver (Figure 14), and the IL12Rβ1- The plasma half-life of CHR protein drug is 4h, while the plasma half-life of IL12Rβ1-CHR/Fc fusion protein drug is much higher than that of IL12Rβ1-CHR, which is 40h.

Example 9: In vitro activity of receptor protein——inhibition of mouse Th1/Th17 cell differentiation in vitro

1. Preparation of Mouse Mixed Lymphocyte Suspension

C57BL/6 mice were killed by neck dislocation, sterilized by soaking in 75% ethanol for 3-5 minutes; the spleen was removed in an ultra-clean bench, washed with sterile PBS; the spleen was placed on a pre-wetted 70 μm cell strainer, and gently pressed with a syringe needle core , filter the cell suspension into a 50mL sterile plastic centrifuge tube, collect the spleen single-cell suspension; centrifuge at 1000rpm for 5min, discard the supernatant, add red blood cell lysate according to the amount of 1mL/spleen, lyse at room temperature for about 4min, add about 10 times the volume of PBS to stop the lysis reaction; centrifuge to discard the supernatant, then wash the cells twice with PBS, resuspend in IMDM, culture in a plastic plate for 8-24 hours, collect the suspended cells (ie mixed lymphocytes), count live cells, and set aside.

2. Enrichment of mouse CD4 ⁺ T cells

Aseptically isolate the spleen of the mouse, prepare a single-cell suspension of spleen cells, react the erythrocyte lysate at room temperature for 3-5 minutes, and stop the lysis reaction with 10 times the volume of sterile PBS; centrifuge to discard the supernatant, wash the cells once with 10 times the volume of sterile PBS, Add 90 μL magnetic bead sorting buffer for every ¹⁰⁷ cells, resuspend splenocytes; add 10 μL CD4(L3T4) magnetic beads, mix well, incubate at 4°C for 15 min; add 1-2 mL magnetic bead sorting solution, wash the cells once, After resuspension, centrifuge at 1000rpm for 10min, discard the supernatant, add 500μL magnetic bead sorting solution to resuspend the cell pellet, and mix evenly; take out the MS magnetic bead sorting column, place it on the sorting magnetic stand, and add 500μL magnetic bead sorting solution Run the column; after the sorting liquid flows out naturally under the action of gravity, transfer the cell suspension after incubation to the sorting column, and use the action of gravity until the liquid drains out naturally; add 500 μL of the sorting liquid to the sorting column , after draining off naturally, clean the separation column twice; remove the separation column and place it in a sterile 15mL centrifuge tube, add 1-2mL of separation liquid, push out the liquid quickly with the inner core of the separation column, and collect the effluent The CD4 ⁺ cells were washed once with culture medium and set aside.

3. In vitro stimulation and differentiation of mouse Th1/Th17 cells

At a density of 10 ⁶ cells per well, add the mixed lymphocyte suspension or CD4 ⁺ T cell suspension to the 24-well plate pre-coated with anti-CD3 antibody, and then add the mouse Th17 cell in vitro polarization stimulating factor IL- 23. IL-6 and TGF-β (or IL-12, anti-IL-4), which stimulate Th1 cell polarization in vitro, and set up blank group, Th1/Th17 induction group, IL12Rβ1-CHR protein intervention group and IL12Rβ1-CHR /Fc fusion protein intervention group (adding different concentrations of receptor protein to investigate its inhibitory effect), p40 antibody positive drug group, cultured in 37°C CO ₂ incubator for 72 hours.

4. ELISA detection of cytokines in cell supernatant

After mouse lymphocytes were stimulated in vitro for 72 hours, they were centrifuged at 1000rpm for 5 minutes to separate the cell pellet and culture supernatant. The cell pellet was washed once with PBS before use. The cell culture supernatant was used to detect inflammatory factors IFN-γ and IL-17A by ELISA The secretory expression of the protein was operated according to the instructions of the ELISA kit. As shown in Figure 15, IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein can inhibit the secretion and expression of Th1/Th17-specific cytokines IFN-γ and IL-17A, and the Fc fusion protein has a higher inhibition efficiency.

5. Flow cytometry detection of differentiation level of mouse Th1/Th17 cells

After the mouse lymphocytes were polarized by Th1/Th17, add PMA with a concentration of 10ng/mL and ionomycin with a final concentration of 1μM for 4-6 hours, and add Golgistop for 1 hour according to the ratio of 1:1500; centrifuge to collect the cell pellet, stain the buffer The cells were washed once with liquid; the supernatant was discarded by centrifugation, and the blocking solution containing anti-mouse CD16/CD32 antibody was added to each tube of cells, and incubated on ice in the dark for 30 min. Wash once with staining buffer, centrifuge to discard the supernatant, add CD4 surface marker antibody, and incubate on ice in the dark for 30min. After the cells were fixed and ruptured, intracellular labeled anti-IL-17A or IFN-γ was added, and incubated for 30 min at room temperature in the dark. Finally, the cells were resuspended in 500 μL staining buffer and tested on the machine. As shown in Figure 16, IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein can inhibit the differentiation and development of Th17 cells, and the Fc fusion protein has a higher inhibition efficiency.

6. Real-time quantitative PCR analysis of changes in gene expression levels of transcription factors in Th1/Th17 cells

According to the instructions of the total RNA extraction kit, extract the total RNA of each group of cells; then use it as a template, use reverse transcriptase, random primers, etc. to synthesize the first strand of cDNA; design for fluorescent quantitative PCR detection of related genes (such as mβ- Actin, mT-bet and RORγt etc.) upstream and downstream primers. Using β-actin as an internal reference gene, SYBR Green labeled fluorescence, and using the △△Ct value method to quantify the relative expression level of the target gene in each group of samples, the results showed that IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein can reduce T- bet and RORγt gene expression levels, and the Fc fusion protein had a better reducing effect ( FIG. 17 ).

Example 10: Receptor protein in vitro activity - inhibiting human Th1/Fh17 cell differentiation in vitro

1. Extraction of Human Peripheral Blood Lymphocytes

Mix normal saline for injection with fresh human anticoagulated blood at a ratio of 1:1, slowly add it into a centrifuge tube containing an equal volume of lymphocyte separation solution along the wall of the centrifuge tube with a dropper, keep the interface clear, and centrifuge at 2000rpm for 20-30min. The liquid in the centrifuge tube is divided into three layers, from top to bottom are the plasma layer, lymphocyte separation liquid, red blood cell and granulocyte sedimentation, there is an obvious milky white cloudy narrow band at the junction of the middle and upper liquid levels, carefully absorb the cells there, and transfer into another new centrifuge tube. Add physiological saline for injection according to the ratio of 1:10 (V/V), mix thoroughly, centrifuge horizontally at 1000 rpm for 10 minutes, discard the supernatant, wash the cell pellet twice in the same way, count the cells, and set aside.

2. In vitro stimulation and differentiation of human Th1/Th17 cells

At a density of 10 ⁶ cells per well, add the isolated mononuclear cell fluid into a 24-well plate pre-coated with amCD3 antibody, and then add human Th17 cell polarization stimulation factors IL-1β, IL-23, IL -6 and TGF-β (or IL-12, anti-IL-4), which stimulate Th1 cell polarization in vitro, and set up blank group, Th1/Th17 induction group, IL12Rβ1-CHR protein intervention group and IL12Rβ1-CHR/Fc fusion Protein intervention group (adding different concentrations of receptor protein to investigate its inhibitory effect), cultured in 37°C CO ₂ incubator for 96 hours.

3. Detection and analysis by ELISA, flow cytometry, and Q-PCR methods found that IL12Rβ1-CHR protein and IL12Rβ1-CHR/Fc fusion protein can also inhibit the differentiation and development of human Th1/Th17 cells and human IFN-γ, IL-17A cytokines The secreted expression of Fc fusion protein has higher inhibition efficiency (Fig. 18, Fig. 19, Fig. 20).

Example 11: Receptor protein activity in vivo—improving EAE symptoms in animal models of multiple sclerosis

1. Establishment of EAE model

C57BL/6 female mice at 6-8 weeks were randomly divided into model group, normal group, IL12Rβ1-CHR protein group, IL12Rβ1-CHR/Fc fusion protein group and cyclosporine CsA positive drug group. Mix equal volumes of mMOG diluted in sterile PBS and TB diluted in complete Freund's adjuvant until it becomes water-in-oil. On the day of immunization, 200 μL of emulsified antigen was subcutaneously injected into the abdomen of each mouse, using a two-point injection method, 100 μL per point. At the same time, on the day of immunization (day 0) and day 2, each mouse was orbitally injected with 200 μL of PTX dilution (2.5 μg/mL). On the day of onset, IL12Rβ1-CHR or IL12Rβ1-CHR/Fc or ddH ₂ O was administered via caudal vein, or CSA was administered intragastrically, daily for 10 consecutive days, and changes in clinical scores were observed.

2. Model Evaluation

From the 0th day of immunization to the end of the experiment, 2 people use a double-blind method every day to measure the weight of the mice at the same time, and observe the mental state, hair, diet and behavior, etc. According to the Benson scoring standard, the severity of EAE Conduct a clinical evaluation:

0 points: Normal mice with shiny hair, strong limbs and tail, no clinical signs of EAE;

1 point: The tension in the proximal end of the tail disappears, and mild gait can be seen;

2 points: one hind limb is weak, crawling forward, and can recover after turning over passively;

3 points: Both hind limbs are weak, passive turning over can not recover, but can move after being stimulated;

4 points: paralysis of both hind limbs, movement, difficulty in eating and drinking, accompanied by incontinence of urine and feces;

5 points: Dying state or death.

(1) The IL12Rβ1-CHR/Fc administration group and the IL12Rβ1-CHR/Fc administration group had significant therapeutic effects on EAE, both of which were better than the positive administration group. The highest score reached 3.5 points, the positive drug was about 2.5, while the IL12Rβ1-CHR administration group had a maximum score of 2 points, and the IL12Rβ1-CHR/Fc administration group had a maximum score of 1.5 points, which was significantly different from the model group (Figure 21).

(2) At the same time, the monitoring of the body weight of each experimental group found that the body weight of the positive drug group decreased significantly on the day of drug administration, and continued until the drug was stopped, with a maximum rate of decline of 18%, while the mice administered with the receptor protein There was no significant weight loss; and the study found that the administration of positive drugs seriously reduced the number of red blood cells and the level of hemoglobin, while the hematological indicators of the mice in the receptor protein administration group did not change, suggesting that the receptor protein was compared with these non-specific Specific immunosuppressants may have better clinical efficacy and better safety.

3. Pathological index detection:

1) Separate the brain and spinal cord of the mouse, soak them in formalin and fix them for 24-48 hours immediately, make paraffin sections, stain with hematoxylin-eosin (HE), and analyze the demyelinating injury and inflammation of the central nervous system of EAE mice cell infiltration. Compared with the model group, the treatment of IL12Rβ1-CHR and IL12Rβ1-CHR/Fc can alleviate the infiltration of inflammatory cells in brain and spinal cord tissues and the symptoms of spinal cord demyelination ( FIG. 22 ).

2) At the peak of the disease after immunization, blood was collected from the orbit of the mice, and the serum was centrifuged to separate the serum. The expression levels of cytokines such as IFN-γ and IL-17A were detected by ELISA. The expression levels of sex factors were significantly lower than those of the model group (Figure 23).

3) Flow cytometric detection of Th1/Th17 cell differentiation: Isolate mouse spleen at the peak of onset, sort CD4 ⁺ cells with magnetic beads, add PMA at a concentration of 10 ng/mL and ionomycin at a final concentration of 1 μM for 4-6 hours, follow 1 Golgistop was added at a ratio of :1500 for 1 hour, extracellular and intracellular fluorescent staining was used to detect the differentiation rate of Th1/Th17 by flow cytometry. Compared with the model group, the administration of IL12Rβ1-CHR and IL12Rβ1-CHR/Fc greatly inhibited Th1 / Differentiation of Th17 cells ( FIG. 24 ).

Example 11: In vivo activity of receptor protein—improving CIA symptoms of rheumatoid arthritis animal model

1. Establishment of CIA model

Wistar rats aged 6-8 weeks were randomly divided into model group, normal control group, IL12Rβ1-CHR, IL12Rβ1-CHR/Fc and positive drug CsA group. Chicken type II collagen peptide dissolved in 100mM acetic acid was mixed with Freund's complete adjuvant in equal volume at low temperature to prepare immune antigen. On the 0th day, 400 μg antigen was injected intradermally at the base of the tail, and the same dose was boosted 7 days later to induce arthritis in Wistar rats. . The IL12Rβ1-CHR and IL12Rβ1-CHR/Fc groups were given IL12Rβ1-CHR and IL12Rβ1-CHR/Fc proteins by tail vein on the 0th day of immunization, and the positive drug group were given CsA by intragastric administration.

2. Model Evaluation

From the 0th day of immunization to the end of the experiment, 2 people use a double-blind method every day to measure the weight of the rats at the same time, and observe the changes in mental state, hair, diet and behavior, etc., according to the arthritis index (AI) Scoring criteria for clinical assessment of CIA severity:

0 points: normal, no arthritis;

1 point: red spots or mild swelling (ankle is obviously red and swollen or red and swollen only limited to a single toe)

2 points: Moderate swelling of joints;

3 points: Severe swelling (redness and swelling of the whole foot, including severe redness and swelling of the toes);

4 points: Severe swelling and unable to bear weight (redness and swelling involving the whole limb, including inflammation of multiple joints);

(1) The AI of each rat is the sum of hindlimb AI, with a maximum of 8 points. After the initial immunization, the rats were scored for AI every two days. The data are shown in Figure 25. The average AI score of the IL12Rβ1-CHR group was no more than 4 points, and the average AI score of the IL12Rβ1-CHR/Fc group was no more than 2 points. The model group showed a significant difference.

(2) At the same time, the body weight of each experimental group was monitored daily, and it was found that the positive drug group lost weight on the day of drug administration, with a maximum rate of 22%, while the mice administered with the receptor protein did not appear to lose weight significantly.

3. Detection of pathological indicators

1) Imaging examination: the first immunization, and 46 days after the initial immunization, X-ray examinations were performed on the ankle joints of the hindlimbs of the experimental rats, and the imaging changes of the ankle joints were observed and compared. The X-ray films of the normal group showed no swelling of the cartilage tissue, no destruction of the bone, and a clear space between the toe facet joints, while the model group showed changes in soft tissue swelling and bone erosion around the ankle joint. IL12Rβ1-CHR and IL12Rβ1-CHR/Fc administration The lesions in the group were mild (Fig. 26).

2) H&E stained pathological sections: 46 days after the initial immunization, the rats were sacrificed, the ankle joints of the hindlimbs were separated, fixed in formalin, and decalcified, and embedded in paraffin. Severe hyperplasia, infiltration of a large number of inflammatory cells, formation of pannus in the joint cavity, release of various inflammatory mediators and proteolytic enzymes, etc., damage to the cartilage surface, and severe bone involvement; synovial hyperplasia was not obvious in the IL12Rβ1-CHR administration group, and inflammatory The degree of cell infiltration was low, the surface of cartilage was smooth, no destruction, and the degree of bone involvement was low; in the IL12Rβ1-CHR/Fc administration group, there was almost no inflammatory cell infiltration, the surface of cartilage was smooth, no destruction, and the degree of bone involvement was low (Figure 27).

3) ELISA method was used to detect the expression levels of relevant cytokines in rat serum: blood was collected from the orbit of the rats at the peak of the onset period, the serum was separated, and the expression levels of TNF-α, IFN-γ, IL-17A and other cytokines in the serum were detected by ELISA method. The secretion and expression of inflammatory factors in the administration group were significantly lower than those in the model group ( FIG. 28 ).

Example 12: Receptor protein activity in vivo - improving symptoms of chronic graft-versus-host disease (cGVHD)-induced systemic lupus erythematosus (SLE) model

1. Establishment of SLE model:

The 8-10 week-old (DAB/2×C57BL/6J) F1 hybrid mice were randomly divided into model group, CsA group, IL12Rβ1-CHR/Fc prophylactic administration group, IL12Rβ1-CHR/Fc treatment administration group and normal control group. 8-10 weeks old DBA\2 mice were killed by neck dislocation, splenocytes were separated to make single cell suspension, red blood cells were removed with ammonium chloride lysate, cell pellet was resuspended in normal saline, and cell count was used; each BDF1 mouse tail 5x10 ⁷ cells were injected intravenously, and the normal control group was injected with the same amount of normal saline. The IIL12Rβ1-CHR and IL12Rβ1-CHR/Fc administration groups were given corresponding proteins by tail vein at the second week.

2. Pathological index detection and model evaluation

1) Blood samples: Blood was collected from the orbits of the mice in each group every week, and the content of anti-ssDNA antibodies in the serum was detected by indirect ELISA. After 2 weeks, the content of anti-ssDNA antibody in the model group increased rapidly, while the increase in the content of anti-ssDNA antibody in the IL12Rβ1-CHR administration group was significantly smaller than that in the model group, and the anti-ssDNA antibody content in the IL12Rβ1-CHR/Fc administration group did not increase until the third week. Anti-ssDNA antibodies increased, and the peak antibody levels in the administration group were significantly lower than those in the model group (Figure 29).

2) Kidney tissue specimens: mice in each group were killed by neck dislocation at 10 weeks, bilateral kidneys were taken out, part of them were frozen and sectioned, immunofluorescent stained directly, labeled with FITC-goat anti-mouse IgG, and immune deposits were detected by laser scanning fluorescence microscope , as shown in Figure 30, diffuse particle-like fluorescent substance deposition can be seen in the model group, the fluorescent deposits in the IL12Rβ1-CHR administration group are significantly reduced, and only a small amount of fluorescent substance deposition can be seen in the IL12Rβ1-CHR/Fc administration group; the other part is 10% formaldehyde After 48 hours of fixation, paraffin sections were prepared, stained with HE and PAS, and observed under an ordinary optical microscope. As shown in Figure 31, the kidney tissue of the model group showed a significant increase in glomerular volume, severe mesangial hyperplasia and cellular crescents , a large number of inflammatory cell infiltration in the interstitium, partial sclerosis of the glomerulus, and deposition of a large number of immune complexes; while the renal tissue lesions in the IL12Rβ1-CHR administration group improved, only mild mesangial hyperplasia was seen, and a small amount of neutral mesangial area appeared. In the granulocytes, a small amount of immune complex deposition can be seen; in the IL12Rβ1-CHR/Fc administration group, the renal tissue lesions improved, and a small amount of neutrophils appeared in the mesangial area, without immune complex deposition.

3) Determination of expression of Th17 cell-related factors: ELISA was used to detect the expression levels of cytokines such as IFN-γ and IL-17A in serum, and the secretion and expression of inflammatory factors in the administration group were significantly lower than those in the model group (Figure 32)

Example 13: In vivo activity of receptor protein—improving symptoms of ulcerative colitis animal model

1. Establishment of animal model of acute DSS enteritis

Male C57BL mice aged 6-8 weeks were randomly divided into model group, IL12Rβ1-CHR administration group, IL12Rβ1-CHR/Fc administration group and normal control group. Except for the control group, all the other groups drank freely with 5% DSS solution instead of ordinary drinking water for 7 days. After 7 days, all experimental mice continued to drink distilled water freely. The IL12Rβ1-CHR administration group and the IL12Rβ1-CHR/Fc administration group were intraperitoneally injected with the fusion protein once a day for 7 consecutive days.

2. Model evaluation

During the modeling process, observe the general situation of the mice such as diet and activities every day, monitor the body weight, observe the properties of the feces and whether there is occult blood, observe the general condition of the mice, focus on observing and recording the changes in body weight, stool properties and blood in the stool, The severity of colitis was assessed by disease activity index (DAI). The DAI score includes three aspects: stool properties, fecal occult blood, and weight loss. Integral 0 to 4 points. 0 points: normal stool pattern, no fecal occult blood, no weight loss; 1 point: normal stool pattern, no fecal occult blood, body weight loss 1% to 5%; 2 points: loose stool, positive fecal occult blood, weight loss > 5% to 10%; 3 points: loose stools, positive fecal occult blood, weight loss > 10% to 15%; 4 points: loose stools, gross bloody stools, weight loss > 15% (normal stools: formed stools; loose stools : Pasty, semi-formed stools that do not adhere to the anus; Loose stools: Unformed stools that can adhere to the anus).

The disease attack occurred in the model group and the protein administration group on the second day after modeling, and the inflammation in the model group continued throughout the experiment without obvious self-healing tendency; The CHR/Fc fusion protein administration group began to slow down the increase of DAI on the 5th and 4th day after treatment. On the 7th day after modeling, compared with the model group, the DAI of the administration group was significantly lower than that of the model group (P<0.05), suggesting that IL12Rβ1-CHR and IL12Rβ1-CHR/Fc fusion protein can improve the degree of inflammation in mice with ulcerative colitis ( Figure 33).

3. Detection of pathological indicators

1) H&E stained pathological sections: mice were sacrificed on the 7th day of modeling, about 1 cm of mouse colon tissue was aseptically taken, fixed in 10% formalin, dehydrated, embedded, sectioned, HE stained, and observed under a microscope Histopathological changes of the colon. Multifocal superficial ulcers can be seen in the colon tissue of the mice in the model group, most of the glands are destroyed, the normal structure of the glands is lost, the arrangement is disordered, the gland cavity disappears, and dense inflammatory cell infiltration can be seen; the colonic glands of the mice in the fusion protein administration group The body was slightly damaged, the structure basically returned to normal, the depth of inflammatory cell infiltration was slightly lower than that of the model group, the number of inflammatory cells was also significantly reduced, and the degree of inflammation was significantly reduced (Figure 34).

2) Take 1cm colon tissue from mice in each experimental group, make tissue homogenate under ice bath condition, take supernatant after centrifugation, use ELISA method to measure the concentration of IFN-γ and IL-17A, the secretion of inflammatory factors in the administration group The expression level was significantly reduced compared with the model group (Figure 35).

Example 14: In vivo activity of receptor protein—improving the symptoms of ConA-induced acute liver injury animal model

1. Establishment of liver injury model

Female BALB/c mice aged 6-8 weeks were randomly divided into model group, IL12Rβ1-CHR administration group, IL12Rβ1-CHR/Fc administration group and normal control group. ConA was diluted with sterile normal saline, and mice were injected into the tail vein at a dose of 20 mg/kg to induce the model, and the control group was injected with an equal volume of PBS. The fusion protein administration group was given IL12Rβ1-CHR and IL12Rβ1-CHR/Fc fusion protein by tail vein before modeling. Serum and liver tissue samples were collected 12 hours later for the determination of transaminase ALT and pathological histochemical analysis.

2. Detection of pathological indicators

1) Determination of serum transaminases: 12 hours after ConA treatment, blood was collected from the orbits of experimental mice, centrifuged to obtain supernatants, and ALT levels in each group were measured with alanine aminotransferase (ALT) kits. Compared with the normal group, the serum ALT level in the model group was significantly increased, suggesting that the injection of Con A caused severe damage to the mouse liver cells. The levels of serum transaminases in mice administered with IL12Rβ1-CHR and IL12Rβ1-CHR/Fc fusion protein were significantly lower than those in the model group, suggesting that the receptor protein has a therapeutic effect on liver cell injury (Figure 36).

2) Histopathological analysis of the liver: the liver tissues of the mice in each experimental group were aseptically separated, fixed with 10% formaldehyde, embedded in paraffin, and sectioned to observe the liver damage by HE staining. In the model group, the swelling and degeneration of liver cells were obvious, with scattered necrosis or even large sheets of necrosis, and a large number of inflammatory cell infiltration in the portal area and around the necrotic tissue. However, the pathological sections of the administration group showed improvements in hepatic cell swelling and necrosis ( FIG. 37 ).

3) Detection of serum cytokines: ELISA kits were used to detect two important hepatitis inflammatory indicators, TNF-α and IL-6, in the blood supernatants of different groups. Compared with the model group, the levels of TNF-α and IL-6 in the serum of the mice in the protein administration group were significantly reduced ( FIG. 38 ).

Example 15: In vivo activity of receptor protein—improving the symptoms of psoriasis-like inflammation model

1. Establishment of psoriasis-like inflammation model

Five-week-old female BALB/c mice were randomly divided into normal control group, model group, IL12Rβ1-CHR administration group and IL12Rβ1-CHR/Fc fusion protein administration group. The back of the mice was shaved, with an area of about 3cm×5cm. Except for the control group, the external base cream was given, and the rest of the groups were given imiquimod cream, once a day, for 14 consecutive days, and massaged about 60 times with single finger light pressure . The administration group was injected intraperitoneally with IL12Rβ1-CHR or IL12Rβ1-CHR/Fc fusion protein at regular intervals every day from the first day.

2. Model Evaluation

1) Corrected PASI score: observe local skin changes every day, take pictures and record, and calculate the corrected PASI score for psoriasis-like inflammation. Since the affected area is relatively fixed, PASI is corrected according to the degree of erythema, scale, and skin hypertrophy. The corresponding scores for each index are as follows, and the final PASI score is obtained by adding the three.

Score rating severity 0 1 asymptomatic 1 2 Mild (spot color light red; bran-like desquamation) 2 3 Moderate (spotted red; thinly covered with flaky scales) 3 4 Severe (bright or dark red macules; thick, partially covered scales) 4 5 Very severe (purplish red spots; thick scales, full-thickness skin lesions)

Judging from the PASI score trend graph, the back skin of the mice in the normal group did not change significantly during the entire medication process, while the back skin of the mice in the model group gradually appeared red macules and plaques after 3 days of topical application of imiquimod cream And scales, the most obvious on the 8th day, severe erythema on the back skin, covered with thick silvery white scales, showing typical psoriasis-like changes. In the IL12Rβ1-CHR administration group and the IL12Rβ1-CHR/Fc administration group, slight erythema and thin-layer scales gradually appeared on the 9th day and 11th day, respectively, but there was no obvious change in the degree of erythema and scale after that.

2) HE pathological section: On the seventh day, the back skin of the mice was taken for H&E staining. Under the microscope, the skin tissue of the mice in the control group was normal, and there were only 2 to 3 layers of keratinocytes; the skin lesions of the mice in the model group showed obvious thickening ( Hyperkeratosis), residual keratinocyte nuclei (parakeratosis), acanthosis and dermal inflammatory cell infiltration; the administration group had mild hyperkeratosis, acanthosis and inflammatory infiltration, but compared with the psoriasis group Significantly lessened (Figure 39).

3. Detection of pathological indicators

The contents of cytokines IL-17A, IL-22 and IFN-γ in the serum of mice in each group were detected by ELISA, and the study showed that the contents of cytokines in the administration group were significantly lower than those in the model group (Figure 40).

Example 16: Receptor activity in vivo—improving the symptoms of a mouse orthotopic liver cancer model

1. Establishment of mouse orthotopic liver cancer model

Female BALB/c mice aged 6-8 weeks were randomly divided into normal control group, liver cancer model group, IL12Rβ1-CHR administration group and IL12Rβ1-CHR/Fc administration group. Take the fresh H22 cell line passaged in the peritoneal cavity of mice, and adjust the cell concentration to 1×10 ⁶ cells/mL with normal saline after routine washing. After mice were anesthetized, 0.1 mL of cancer cell suspension was injected into the liver under direct vision. The normal control group was injected with the same dose of normal saline. After the administration group was inoculated with H22 liver cancer cells, in addition to daily intraperitoneal injection of IL12Rβ1-CHR or IL12Rβ1-CHR/Fc fusion protein, it continued for 13 days.

2. Detection of pathological indicators

1) HE pathological sections: mice were sacrificed on the 14th day after modeling, and liver tumor tissues were fixed for routine HE staining for histopathological diagnosis. Large tumor foci can be seen in the model group, with diffuse infiltration of cancer cells, no obvious borders, tight arrangement, less interstitium, and a small amount of tumor giant cells and necrotic foci; And the border with normal liver tissue is clear (Figure 41).

2) Trizol method was used to extract the total RNA of the liver tissue of each experimental group, reverse transcription to synthesize cDNA, and Q-PCR to detect the expression levels of IL-17A and IFN-γmRNA. The expression levels of IL-17A and IFN-γmRNA in the liver tissue of the treatment group were significantly lower than those of the model group (Figure 42).

Claims (20)

1. a people IL12R β1receptor extracellular region cytokine is in conjunction with homologous region albumen, i.e. people IL12R β 1-CHR albumen, it is characterized in that, described IL12R β 1-CHR is the critical area that IL-12R β1receptor extracellular region is combined with people 1L-12/IL-23, there is as shown in SEQ ID.1 gene order and aminoacid sequence as shown in SEQ ID.2, and according to codon degenerate, same acid sequence of encoding, but the gene order that Nucleotide is replaced.

2. a people IL12R β 1-CHR/Fc fusion rotein, it is characterized in that, described IL12R β 1-CHR is except having as shown in SEQ ID.1 gene order and aminoacid sequence as shown in SEQ ID.2, also there is as shown in SEQ ID.3 gene order and the N end signal peptide sequence of one section of 69 nucleotide coding of aminoacid sequence as shown in SEQ ID.4, and according to codon degenerate, to encode same acid sequence, but the gene order that Nucleotide is replaced.

3. fusion rotein according to claim 2, it is characterized in that, described Fc fragment comprises hinge area, CH2 district and CH3 district, described Fc fragment is selected from the immunoglobulin IgG of human or animal, IgM, IgD, IgA or their hypotype, described Fc fragment be natural type Fc or to acceptor and/or the complement saltant type Fc in conjunction with relevant amino acid change.

4. fusion rotein according to claim 2, is characterized in that, described Fc fragment is preferable over and derives from people 1gG1, including but not limited to SEQ ID.5 and SEQ ID.6; Simultaneously including but not limited to aminoacid sequence SEQ ID7 corresponding with it and SEQ ID8.

5. fusion rotein according to claim 2, is characterized in that, for directly to merge or to be merged by catenation sequence between described IL12R β 1-CHR and Fc sequence.

6. fusion rotein according to claim 2, is characterized in that, described antigen-4 fusion protein gene sequence codon is optimized, obtain two kinds can inside Chinese hamster ovary celI the gene order of high expression, be respectively SEQ ID 9 and SEQ ID10.

7. comprise a carrier for any one protein coding DNA sequence in claim 1-6, it is characterized in that, described is eukaryotic vector and prokaryotic vector.

8. carrier according to claim 7, is characterized in that, described eukaryotic vector is mammalian cell expression vector.

9. comprise a host cell for the carrier of any one protein coding DNA sequence in claim 1-6, it is characterized in that, described host cell is selected from eukaryotic cell or prokaryotic cell prokaryocyte.

10. fusion rotein according to claim 9, is characterized in that, described eukaryotic cell is Chinese hamster ovary celI, and described prokaryotic cell prokaryocyte is Bacillus coli cells.

The autoimmune disorder purposes that protein for treatment in 11. claim 1-6 described in any one is feature with Th1/Th17 dysfunction and IL-12/IL-23 high expression level, include but not limited to rheumatoid arthritis, Ke Luoshi is sick, multiple sclerosis and systemic lupus erythematous etc.

Albumen in 12. claim 1-6 described in any one improves multiple sclerosis animal model EAE disease symptoms.

Albumen in 13. claim 1-6 described in any one improves rheumatoid arthritis animal model CIA disease symptoms.

Albumen in 14. claim 1-6 described in any one improves systemic lupus erythematous animal model disease symptoms.

Albumen in 15. claim 1-6 described in any one improves enteritis animal model disease symptoms.

Albumen in 16. claim 1-6 described in any one improves hepatitis animal disease symptoms.

Albumen in 17. claim 1-6 described in any one improves psoriasiform inflammatory model disease symptoms.

Albumen in 18. claim 1-6 described in any one improves mouse original position liver cancer model disease symptoms.

Albumen in 19. claim 1-6 described in any one improves the application of the autoimmune disorder animal model disease symptoms that other are feature with Th1/Th17 dysfunction and IL-12/IL-23 high expression level.

20. pharmaceutical composition and the application thereof comprising the albumen in claim 1-6 described in any one.