CN110156888B - Application of CD96 recombinant protein in preparation of pharmaceutical composition for immune diseases - Google Patents

Application of CD96 recombinant protein in preparation of pharmaceutical composition for immune diseases Download PDF

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CN110156888B
CN110156888B CN201910392750.0A CN201910392750A CN110156888B CN 110156888 B CN110156888 B CN 110156888B CN 201910392750 A CN201910392750 A CN 201910392750A CN 110156888 B CN110156888 B CN 110156888B
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胡适
傅文燕
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Fengchao Medical Technology Shanghai Co ltd
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Abstract

The invention relates to the technical field of biomedical engineering, and provides a CD96 recombinant protein, a preparation method and application thereof, wherein the CD96 recombinant protein can be a full-length soluble fragment of a natural protein, a fragment containing the full length of an extracellular segment of the natural CD96 protein, a fragment containing the extracellular segment of the natural CD96 protein and retaining the biological activity thereof, a natural CD96 protein extracellular segment after removing one or more continuous amino acid residues at the N end, the C end or both of the N end and the C end, or a mutant of the CD96 protein, and has an amino acid sequence shown as SEQ ID No.1 or SEQ ID No. 3. Experiments prove that the two types of CD96 recombinant proteins have good treatment effects on immune-related diseases, can effectively treat the immune-related diseases by being combined with other anti-immune-related disease drugs, and has wide clinical application prospects.

Description

Application of CD96 recombinant protein in preparation of pharmaceutical composition for immune diseases
Technical Field
The invention relates to the technical field of biomedical engineering, in particular to a CD96 recombinant protein for treating immune diseases, a polynucleotide sequence for coding the CD96 recombinant protein, a vector and a medicament containing the same and application thereof.
Background
Activation of an immune response in the body requires signaling communication by several immune checkpoints (checkpoint). These immune checkpoint proteins can either transmit excitatory co-stimulatory signals to enhance immune responses or mediate co-inhibitory signaling to cause immune escape. The immunoglobulin superfamily, which is a major member of immunodetection proteins, occupies a central role in the coordination of immune responses. Some newly discovered targets in recent years, such as CD 28/cytotoxic T lymphocyte antigen-4 (CTLA-4); the combination of signal molecules such as B7.1/B7.2 receptor/ligand and the like has been widely noticed by academia at present, and especially plays an important role in tumor immunotherapy.
Recently, there have been a series of reports showing that immunoglobulin superfamily members can interact with fibronectin/fibronectin-like (Nectin-like) family members, thereby affecting NK cell and T cell function (Fuchs and Colonna, sensiars in cancer biology, 2006). Members of the immunoglobulin superfamily include CD226(DNAM-1) (Shibuya et al, Immunity,1996), CD96(TACTILE) (Wang et al, Journal of Immunity, 1992), TIGIT (T cell immunoglobulin and ITIM domains) (Boles et al, European Journal of Immunity, 2009; Yu et al, Nature Immunity, 2009), and CRTAM (class I restricted T cell related molecule) (Kennedy et al, Journal of leukemia biology,2000), among others. Among them, CD96 expression is mainly limited to NK cells, CD8+T cells, and CD4+T cells, the primary ligand of which is CD155, but CD96 has also been reported to be associated with CD111 (fibronectin-1) and to play a role in promoting NK and T cell adhesion (set et al, Biochemical and biological research communications, 2007).
The signal following CD96 activation is generally thought to negatively regulate the immune response, but the exact action and mechanism is not known. Due to the unique role of immune checkpoint in immune surveillance, it is possible to use CD96 protein as an active ingredient for the treatment of immune diseases, and similar research or report has not been found at present.
Disclosure of Invention
Based on the research background, the invention explores whether the CD96 recombinant protein can be used for treating immune-related diseases, and describes the specific structure, preparation method and application of the CD96 recombinant protein, namely provides the CD96 recombinant protein, and the preparation method and the application thereof.
In a first aspect of the invention, there is provided a CD96 recombinant protein, wherein the CD96 recombinant protein is selected from any one of the following cases:
A) the CD96 recombinant protein is a full-length soluble fragment of a natural protein, the soluble fragment reserves a structural domain which has the capacity of combining a natural ligand in the natural CD96 protein or part of an extracellular segment of the protein, and lacks part or all of a transmembrane segment and an intracellular segment of the protein; the CD96 protein is numbered P40200 (human), Q3U0X8 (murine) in the uniport database.
B) The CD96 recombinant protein is a fragment containing the full length of the extracellular segment of the natural CD96 protein;
C) the CD96 recombinant protein is a fragment which contains the extracellular segment of the natural CD96 protein and retains the biological activity;
D) the CD96 recombinant protein is a natural CD96 protein extracellular segment after removing one or more continuous amino acid residues at the N terminal, the C terminal or both of the N terminal and the C terminal;
E) the CD96 recombinant protein is a mutant of CD96 protein.
The CD96 recombinant protein also comprises one or more continuous amino acid residues of a transmembrane segment of a natural CD96 protein or one or more continuous amino acid residues in a signal peptide of the natural CD96 protein.
In preferred embodiments, the CD96 protein analog is derived from a human, mouse, or other mammal. The human CD96 protein analogue and the mouse CD96 protein analogue have amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.3 respectively, or consist of amino acid sequences in which any one or more amino acids in the amino acid sequences are deleted, added or substituted. The nucleotide sequence of the recombinant protein coding human CD96 is shown as SEQ ID NO.2, and the nucleotide sequence of the recombinant protein coding mouse CD96 is shown as SEQ ID NO. 4.
The CD96 recombinant protein can also be a fusion protein, the fusion partner of the recombinant protein is an IgG Fc region, the human CD96 fusion protein has an amino acid sequence shown as SEQ ID NO.5, the mouse CD96 fusion protein has an amino acid sequence shown as SEQ ID NO.7, and the nucleotide sequences for coding the fusion protein are respectively shown as SEQ ID NO.6 and SEQ ID NO. 8.
The IgG Fc region is an Fc region from an IgGl, IgG2, IgG3, or IgG4 antibody, preferably an Fc region of an IgGl antibody. The human IgG Fc region comprises the amino acid sequence shown in SEQ ID No.9, and further comprises some or all of the amino acids found in the wild-type IgG Fc sequence on the N-terminal side of the C residue at position 1 in the amino acid sequence of SEQ ID No. 9.
The amino acid sequence of the Fc region of the human IgG heavy chain (SEQ ID NO.9) is as follows:
EPKSCDKTHTCPPCPAPELLGGPSVX1LX2PPKPKDTLMISRTPEVTCX3VX4DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYX5STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX6
wherein X1Is F, Q or E; x2Is F, Q or E; x3Is V or T; x4Is V or T: x5Is N, D or Q: and X6Is K or absent.
The amino acid sequence and the nucleotide sequence of human IgG1Fc are respectively shown as SEQ ID NO.10 and SEQ ID NO.11, and the amino acid sequence and the nucleotide sequence of mouse IgG1Fc are respectively shown as SEQ ID NO.12 and SEQ ID NO. 13.
The term "human IgG FC region" of the invention has the meaning generally given by this term in the field of immunology. In particular, the term refers to a human IgG antibody fragment obtained by removing two antigen binding regions (Fab fragments) from an antibody. Specifically, the FC region includes the CH2 and CH3 constant region domains of an antibody, and may also include some or all of the hinge region.
There are four IgG subclasses in immunoglobulins (G1, G2, G3 and G4), each with different structures and biological functions known as effector functions. These effector functions are generally mediated by interaction with Fc receptors (Fc γ R) or by binding to complement factors Clq. Binding to Fc γ R results in antibody-dependent cell lysis, while binding to complement factors results in complement-mediated cell lysis. The structure and properties of the Fc region of the IgG subclasses are known in the art. The fusion protein of the invention may contain an Fc region from any IgG subclass, and is preferred because Gl and G3 have higher receptor binding and effector functional activity than the G2 and G4 antibodies.
In another preferred embodiment of the invention, the amino acid sequence of the CD96 recombinant protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity with the amino acid sequence depicted in SEQ ID No. 1; the polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity with the polynucleotide sequence shown in SEQ ID No. 2.
In another preferred embodiment of the invention, the amino acid sequence of the CD96 recombinant protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity with the amino acid sequence depicted in SEQ ID No. 3; the polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity with the polynucleotide sequence shown in SEQ ID No. 4.
In another preferred embodiment of the invention, the amino acid sequence of the CD96 recombinant protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity with the amino acid sequence depicted in SEQ ID No. 5; the polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity with the polynucleotide sequence shown in SEQ ID No. 6.
In another preferred embodiment of the invention, the amino acid sequence of the CD96 recombinant protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity with the amino acid sequence depicted in SEQ ID No. 7; the polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to the polynucleotide sequence represented by SEQ ID NO 8.
Furthermore, the present invention relates to polynucleotides encoding any of the CD96 recombinant proteins of the present invention. The polynucleotide encoding the above-described CD96 recombinant protein may be in the form of RNA or DNA, which includes cDNA and synthetic DNA, and which may be double-stranded or single-stranded. The coding sequence encoding a protein of the invention may vary due to the redundancy or degeneracy of the genetic code.
The polynucleotide encoding the CD96 recombinant protein of the invention may include the following: coding sequence for protein only, coding sequence for protein and additional coding sequences (such as leader or secretory sequences or preprotein sequences): coding and non-coding sequences for a protein (such as introns or non-coding sequences 5 'and/or 3' to the coding sequence for a protein). Thus, the term "polynucleotide encoding a protein" encompasses not only polynucleotides that may include the coding sequence of the protein, but also polynucleotides that include additional coding and/or non-coding sequences.
In the embodiment of the invention, a small animal model of lupus nephritis, a peripheral PMBC cell population of a systemic lupus erythematosus patient, a small animal model of Collagen-induced arthritis (CIA) and a small animal model of graft-versus-host disease (GVHD) are taken as representatives of an immune disease model, and the curative effect of the CD96 recombinant protein is verified. The result shows that the CD96 recombinant protein can effectively improve the survival rate of lupus nephritis mice after 50 weeks and reduce the incidence rate of proteinuria; can effectively reduce the peripheral PMBC cell group IFN-gamma expression level of the systemic lupus erythematosus patient; can reduce the severity of arthritis by 50 to 66.6 percent at 40 days; can improve the survival rate of the mouse transplanted with spleen cells.
Accordingly, in a second aspect of the present invention, there is provided a pharmaceutical composition comprising a recombinant CD96 protein. The pharmaceutical composition comprises CD96 recombinant protein and a pharmaceutically acceptable carrier.
The CD96 recombinant protein and pharmaceutically acceptable auxiliary materials form a pharmaceutical preparation composition together, so that the pharmaceutical preparation composition can exert curative effects more stably, and the preparations can ensure the conformation integrity of the amino acid core sequence of the CD96 recombinant protein disclosed by the invention and simultaneously protect the multifunctional group of the protein to prevent the degradation (including but not limited to agglomeration, deamination or oxidation).
In general, liquid formulations can be stable for at least one year at 2 ℃ to 8 ℃ and lyophilized formulations can be stable for at least six months at 30 ℃. The preparation can be suspension, injection, or lyophilized preparation, preferably injection or lyophilized preparation.
For the hydro-acupuncture or freeze-dried preparation of the CD96 recombinant protein, pharmaceutically acceptable auxiliary materials comprise one or the combination of a surfactant, a solution stabilizer, an isotonic regulator and a buffer solution. Wherein the surfactant comprises nonionic surfactant such as polyoxyethylene sorbitol fatty acid ester (Tween-20 or Tween-80); poloxamer (such as poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; tetradecyl, oleyl, or octadecyl sarcosine; pluronics; monaquatm, etc., in an amount that minimizes the tendency of the bifunctional bispecific antibody protein to granulate; the solution stabilizer can be saccharides including reducing saccharides and non-reducing saccharides, amino acids including monosodium glutamate or histidine, alcohols including one of trihydric alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol or combinations thereof, and should be added in an amount such that the final formulation remains stable for a period of time deemed stable by one skilled in the art; the isotonic regulator can be one of sodium chloride and mannitol; the buffer may be one of TRIS, histidine buffer, and phosphate buffer.
When the recombinant CD96 protein and its composition are administered to animals including human, the dosage varies with the age and weight of the patient, the nature and severity of the disease, and the administration route, and the results of animal experiments and various conditions can be referred to, and the total dosage cannot exceed a certain range. In particular, the dosage of intravenous injection is 1-1800 mg/day.
In a third aspect of the invention, the invention provides an application of a CD96 recombinant protein, in particular an application in preparing a medicament for treating immune diseases.
Preferably, the application also refers to the combined use of the CD96 recombinant protein and other antitumor drugs.
Immune diseases include, but are not limited to, autoimmune diseases, inflammatory disorders, prevention of immune responses associated with rejection of donor tissue.
Autoimmune diseases include allograft rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, anti-neutrophil cytoplasmic autoantibodies (ANCA), autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's syndrome, sprue-dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarred pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, and Alzheimer's disease, Dermatomyositis, discoid lupus, idiopathic mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Graves ' disease, Guillain-Barre, Goodpasture's syndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic pulmonary fibrosis, Idiopathic Thrombocytopenic Purpura (ITP), IgA neuropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, juvenile arthritis, Kawasaki's disease, lichen planus (lichenplanus), lupus erythematosus (lupuxerathiosis), Meniere's disease, mixed connective tissue disease, multiple sclerosis, type I diabetes, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis (polychrondritis), polyadenylitis, polymyalgia syndrome, polymyositis, and dermatomyositis, Primary gammagroteinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Reynauld's phenomenon, reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, solid organ transplant rejection, stiff person syndrome, systemic lupus erythematosus, takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombotic thrombocytopenic purpura, ulcerative colitis, uveitis, vasculitis diseases such as dermatitis herpetiformis vasculitis, vitiligo, and Wegner's granulomatosis.
Inflammatory disorders including Acute Respiratory Distress Syndrome (ARDS), acute septic arthritis, allergic arthritis, juvenile idiopathic arthritis, allergic encephalomyelitis, allergic rhinitis, allergic vasculitis, allergy, asthma, atherosclerosis, chronic inflammation due to chronic bacterial or viral infection, Chronic Obstructive Pulmonary Disease (COPD), coronary heart disease, encephalitis, inflammatory bowel disease, inflammatory osteolysis, inflammation associated with acute and delayed hypersensitivity reactions, inflammation associated with tumors, peripheral nerve injury or demyelinating disease, inflammation associated with tissue trauma such as burns and ischemia, inflammation caused by meningitis, multiple organ injury syndrome, pulmonary fibrosis, sepsis and septic shock, Stevens-Johnson syndrome, undifferentiated joint suppuration, and undifferentiated spondyloarthropathy.
Graft-related diseases or conditions include graft-versus-host disease (GVDH), such as graft-versus-host disease associated with bone marrow transplantation, and immune conditions resulting from or associated with rejection of organ, tissue or cell graft transplantation (e.g., tissue or cell allografts or xenografts), including, for example, grafts of skin, muscle, neurons, pancreatic islets, organs, liver parenchymal cells, and the like. With respect to donor tissue, cells, transplants or solid organ transplants in a recipient, the pharmaceutical compositions provided herein are effective in preventing acute rejection of such transplants in the recipient and/or for long-term maintenance therapy to prevent rejection of such transplants in the recipient (e.g., inhibiting rejection of insulin-producing islet cell transplants from a donor in a recipient with diabetes).
In addition, the pharmaceutical compositions provided by the present invention may also be used to prevent or treat other diseases in which excessive immune responses are involved, including but not limited to cardiac conditions such as Congestive Heart Failure (CHF), myocarditis, and other myocardial conditions; skin conditions such as rosette (rosecea), acne and eczema; bone and tooth related diseases such as bone loss, osteoporosis, paget's disease, langerhans' cell histiocytosis, periodontal disease, disuse osteopenia, osteomalacia, fibrodysplasia ossifica ossicles, multiple fibrodysplasia, bone metastasis, bone pain management, humoral malignant hypercalcemia, periodontal reconstruction, spinal cord injury and bone fracture; metabolic conditions such as gaucher's disease; endocrine conditions such as cushing's syndrome; and neurological and neurodegenerative conditions such as alzheimer's disease.
In a fourth aspect of the present invention, a method for preparing a CD96 recombinant protein is provided, comprising the following steps:
(A) synthesizing a CD96 recombinant protein fragment and a CD96 extracellular Ig fusion protein fragment by using a whole gene;
(B) fusing the two fragments obtained in the step (A) with the CD96 recombinant protein fragment and the Ig fusion protein fragment by adopting an overlap PCR technology and loading the fused fragments into an expression vector to obtain an expression vector for encoding the CD96 recombinant protein;
(C) the expression vector is introduced into host cells to express the fusion protein, and an antibody affinity column is adopted for purification.
In the present invention, the expression vector includes a fusion DNA sequence linked to appropriate transcriptional and translational regulatory sequences, which may be one of pGEM-T, pcDNA3.1, pEE6.4, pEE12.4, pDFFR, pDR 1.
The fusion protein of the present invention can be easily produced in the following cells: mammalian cells such as CHO, NSO, HEK293, BHK or COS cells; bacterial cells such as E.coli, Bacillus subtilis or Pseudomonas fluorescens; insect cells, or fungal or yeast cells, which cells are cultured using techniques known in the art.
The CD96 recombinant protein disclosed in the present invention is prepared by culturing the above-mentioned host cell under expression conditions to express the fusion protein, and isolating or purifying the fusion protein.
Using the above method, the CD96 recombinant protein can be purified as a substantially homogeneous material, for example, as a single band on SDS-PAGE.
The fusion protein disclosed in the present invention can be isolated and purified by affinity chromatography, and the fusion protein polypeptide bound to the affinity column can be eluted by a conventional method such as high salt buffer, PH change, etc., depending on the characteristics of the affinity column used.
Various Protein purification Methods can be employed, and such Methods are known in the art and described, for example (Wilchek and Bayer,1990, Methods in enzymology) (Scopes,2013, Protein purification: principles and chromatography).
The invention has the following beneficial guarantee and effects:
the invention provides a CD96 recombinant protein, a preparation method and application thereof, which can promote the treatment of diseases or symptoms which are at least pathological bases due to excessive immune response and/or immune response imbalance, and the pharmaceutical composition has good treatment effect on immune diseases through experiments, can effectively treat the immune diseases through being combined with other anti-immune related disease drugs, and has wide clinical application prospect.
Drawings
FIG. 1 shows the result of the change of autoantibody level after a small animal model of lupus nephritis receives treatment by the CD96 recombinant protein provided by the invention;
FIG. 2 is the survival curve of small animal model of lupus nephritis after receiving CD96 recombinant protein treatment;
FIG. 3 shows the results of proteinuria incidence of small animal models of lupus nephritis treated with CD96 recombinant protein;
FIG. 4 shows the IFN-gamma expression level of peripheral mononuclear cells of a lupus patient after treatment with CD96 recombinant protein;
FIG. 5 is a graph of the effect of a small animal model of Collagen-induced arthritis (CIA) on the reduction of severity following treatment with recombinant CD96 protein;
FIG. 6 is a graph showing the effect of improved survival in a small animal model of Graft Versus Host Disease (GVHD) treated with recombinant CD96 protein.
Detailed Description
The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plastrons, methods of inserting genes encoding proteins into such vectors and plastrons, or methods of introducing plasmids into host cells. A Laboratory Manual, 2ndedition，Cold spring Harbor Laboratory Press。
Example 1 construction and expression of recombinant CD96 protein
(1) The whole gene is synthesized into an extracellular segment of human CD96 protein (hereinafter, hCD96) (the amino acid sequence and the nucleotide sequence are shown as SEQ ID NO.1 and 2), an extracellular segment of mouse CD96 protein (hereinafter, mCD96) (the amino acid sequence and the nucleotide sequence are shown as SEQ ID NO.3 and 4), an extracellular Ig fusion protein of human CD96 protein (hereinafter, hCD96-Ig) (the amino acid sequence and the nucleotide sequence are shown as SEQ ID NO.5 and 6), and an extracellular Ig fusion protein of mouse CD96 protein (hereinafter, mCD96-Ig) (the amino acid sequence and the nucleotide sequence are shown as SEQ ID NO.7 and 8).
(2) The sequences are fused and loaded into expression vectors by using an overlap PCR technology, and hCD96, mCD96, hCD96-Ig and mCD96-Ig expression vectors are respectively constructed and obtained.
(3) Fusion protein expression and purification
3X 10 inoculation in 3.5cm tissue culture dish5A CHO-K1 cells, transfected when cultured to 80% -85% confluence: mu.g of the expression vector plasmid and 20. mu.L of Lipofectamine2000Reagent (Invitrogen) were dissolved in 800. mu.L of serum-free DMEM medium, allowed to stand at room temperature for 5 minutes, mixed, and incubated at room temperature for 20 minutes to form a DNA-liposome complex, in which the serum-containing medium in the dish was replaced with 3mL of serum-free DMEM medium, and then the formed DNA-liposome complex was added to the plate, CO, and2after 4 hours of incubator culture, 2ml of DMEM complete medium containing 10% serum is supplemented and placed in CO2And (5) continuously culturing in an incubator. Cells were selected for resistant clones by changing to selection medium containing 500. mu.g/mL G418 and 300. mu.g/mL Zeocin 24h after transfection.
The high expression clone obtained by screening is subjected to amplification culture by a serum-free culture medium, corresponding antibody affinity columns are adopted for hCD96 and mCD96 for purification, Protein A affinity columns (products of GE company) are adopted for hCD96-Ig and mCD96-Ig for separation and purification of recombinant proteins respectively, then PBS is used for dialysis, and finally the quantification is carried out by an ultraviolet absorption method.
Example 2 therapeutic Effect of CD96 recombinant protein on lupus mouse model
Lupus nephritis, as a representative immune system disease, has a morbidity rate of about 50/10 ten thousand, which accounts for about 0.7 per thousand of the population in China. More than 90% lupus nephritis is seen in women, mainly young and middle-aged women, generally speaking, the kidney of patients under 30 years old has high incidence rate, about 70% of patients have kidney damage clinical manifestations with different degrees, proteinuria and microscopic hematuria with different degrees are common, cast urine and kidney function damage are often accompanied, and the normal life of the patients is seriously influenced.
The lupus mouse model is mainly generated by crossing NZB female mice and NZW male mice, and the first-generation hybrid (NZB multiplied by NZW) F1 can generate typical lupus symptoms including lupus nephritis, and is one of the currently accepted animal models for researching lupus nephritis. The model was established in non-patent literature (Zhou et al, Clinical immunology, 2016). Mice were then grouped into mCD 96-treated, mCD 96-Ig-treated and control groups, each group n-10.
The mCD96 and mCD96-Ig were injected in 100 μ g of each, twice a week intraperitoneally for ten consecutive weeks. Control group was injected with control IgG.
The mCD96 and mCD96-Ig in the serum of the mice are detected by an ELISA method, and the serum content is higher than 10 mug/ml.
Autoantibody levels, survival curves and a comparison of incidence of proteinuria for each treatment group are shown in fig. 1-3, respectively.
As can be seen from fig. 1, the antibody levels in the three groups of mice were comparable in the first 30 weeks, and after 30 weeks, the antibody levels in the mCD96 and mCD 96-Ig-treated groups did not increase much, but increased significantly in the control group of mice.
As shown in figure 2, the survival rate of the control group mice starts to slide down in a cliff manner after 50 weeks, the survival rate of the mice is less than 10% after 65 weeks, the survival rate of the mice treated by mCD96 and mCD96-Ig starts to slide down after 70 weeks, and the survival rate is still about 25% after 85 weeks, which shows that the survival rate of the mice can be remarkably improved by mCD96 and mCD 96-Ig.
As shown in fig. 3, the control mice developed proteinuria from 15 weeks and the incidence of proteinuria gradually increased with the passage of time to 100% at 40 weeks; whereas the mice treated with mCD96 and mCD96-Ig began to develop proteinuria after 40 weeks and increased much less than the control mice.
Example 3 Effect of CD96 recombinant protein on peripheral PMBC in Lupus patients
Peripheral PMBC cell populations of 10 patients with systemic lupus erythematosus were collected, isolation method references (Singh et al, Frontiers in immunology,2017) and the PMBC population cells obtained by isolation were treated at 1X 105The density of each well was plated in a well plate in RPMI 1640 medium with 10% FBS.
Appropriate number of wells were divided into hCD96 and hCD96-Ig treated groups, control treated group (control IgG) as different treatment groups, and the protein concentration was 5. mu.g/mL. At 5% CO2After culturing at 37 ℃ for 24 hours in an incubator, the expression level of IFN-. gamma.in each cell population was measured by flow cytometry, a specific method reference (Singh et al, Frontiers in immunology, 2017).
As shown in FIG. 4, the ratio of IFN-. gamma.positive cells was decreased in both hCD96 and hCD96-Ig treated groups compared to the control group, and the hCD96-Ig treated group was most effective, with the ratio of IFN-. gamma.positive cells being only 1/3 of the control group.
Example 4 attenuation of collagen-induced arthritis by recombinant CD96 protein
Mouse collagen-induced arthritis models reference non-patent literature (Ohmi et al, Nature communications, 2016). Arthritis began to appear three to four weeks after the DBA/1 mice were immunized with 100 micrograms of type II collagen. Three to five days after the onset of arthritis, the mouse feet were swollen and inflammatory arthritis lasted for more than three to four weeks. Although the inflammation subsides, the joint is permanently stiff. The severity of arthritis was assessed by examining the onset of erythema and swelling of the joints two or three times a week based on a visual scoring system (table 1) that assesses the severity of arthritis (the average of all mice severity scores in the experimental group was calculated). Mice that had (developping) CIA were randomly assigned to each group of 10 into mCD 96-treated, mCD 96-Ig-treated and control groups.
The dose of mCD96 and mCD96-Ig was 50 μ g per dose, and was administered intraperitoneally every other day from day 20 to day 40. Control groups were injected with control IgG and the severity of arthritis was assessed.
As shown in fig. 5, the severity of arthritis measured on a severity basis was reduced by 50% -66.6% at 40 days compared to the control group.
TABLE 1 visual Scoring System for severity of arthritis
Figure BDA0002057118790000101
Example 5 therapeutic Effect of CD96 recombinant protein on GVHD
Female week-old (C57BL/6 XBALB/C) BCF1 mice, aged 8-12 weeks and weighing 20-25 grams, were used in this experiment. The day before transplantation with spleen cells from the recipient mice, the recipient mice received compound sulfamethoxazole (bactrim) treatment and were irradiated with 700cGy γ rays. Spleen cells from female parental BALB/c mice were treated with a composition containing 10% RPMI and 1% penicillin/streptomycinThe medium of (4) was prepared, and then the cells were collected by centrifugation at 400g for 10 minutes. To induce Graft Versus Host Disease (GVHD), 1X 10 was used7Live splenocytes from allogeneic BALB/c mice were transplanted into gamma-irradiated BCF1 recipient mice by reverse injection, and then 10 model mice were randomly assigned into mCD 96-treated, mCD 96-Ig-treated, and control groups.
mCD96 and mCD96-Ig were injected at 200 μ g/dose intraperitoneally on days 0, 2, 4, and 6 after transplantation into recipient mice with GVHD, control groups were given control IgG, and mice were weighed every two days to monitor survival of recipient mice, as shown in fig. 6.
As shown in fig. 6, all control mice in this experiment eventually died due to rapid loss of GVHD in the control recipient mice, whereas the mortality rate was reduced in the mCD96 group and the mCD96-Ig treated group showed the longest survival rate.
In conclusion, in lupus models, arthritis models or graft-versus-host disease models, the CD96 recombinant protein has a good treatment effect on immune system diseases, and is beneficial to the development of subsequent clinical tests.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Sequence listing
<110> Feng tide pharmaceutical technology (Shanghai) Co Ltd
Application of <120> CD96 recombinant protein in preparation of pharmaceutical composition for immune diseases
<130> specification of claims
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 498
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 1
Val Trp Glu Lys Thr Val Asn Thr Glu Glu Asn Val Tyr Ala Thr Leu
1 5 10 15
Gly Ser Asp Val Asn Leu Thr Cys Gln Thr Gln Thr Val Gly Phe Phe
20 25 30
Val Gln Met Gln Trp Ser Lys Val Thr Asn Lys Ile Asp Leu Ile Ala
35 40 45
Val Tyr His Pro Gln Tyr Gly Phe Tyr Cys Ala Tyr Gly Arg Pro Cys
50 55 60
Glu Ser Leu Val Thr Phe Thr Glu Thr Pro Glu Asn Gly Ser Lys Trp
65 70 75 80
Thr Leu His Leu Arg Asn Met Ser Cys Ser Val Ser Gly Arg Tyr Glu
85 90 95
Cys Met Leu Val Leu Tyr Pro Glu Gly Ile Gln Thr Lys Ile Tyr Asn
100 105 110
Leu Leu Ile Gln Thr His Val Thr Ala Asp Glu Trp Asn Ser Asn His
115 120 125
Thr Ile Glu Ile Glu Ile Asn Gln Thr Leu Glu Ile Pro Cys Phe Gln
130 135 140
Asn Ser Ser Ser Lys Ile Ser Ser Glu Phe Thr Tyr Ala Trp Ser Val
145 150 155 160
Glu Asn Ser Ser Thr Asp Ser Trp Val Leu Leu Ser Lys Gly Ile Lys
165 170 175
Glu Asp Asn Gly Thr Gln Glu Thr Leu Ile Ser Gln Asn His Leu Ile
180 185 190
Ser Asn Ser Thr Leu Leu Lys Asp Arg Val Lys Leu Gly Thr Asp Tyr
195 200 205
Arg Leu His Leu Ser Pro Val Gln Ile Phe Asp Asp Gly Arg Lys Phe
210 215 220
Ser Cys His Ile Arg Val Gly Pro Asn Lys Ile Leu Arg Ser Ser Thr
225 230 235 240
Thr Val Lys Val Phe Ala Lys Pro Glu Ile Pro Val Ile Val Glu Asn
245 250 255
Asn Ser Thr Asp Val Leu Val Glu Arg Arg Phe Thr Cys Leu Leu Lys
260 265 270
Asn Val Phe Pro Lys Ala Asn Ile Thr Trp Phe Ile Asp Gly Ser Phe
275 280 285
Leu His Asp Glu Lys Glu Gly Ile Tyr Ile Thr Asn Glu Glu Arg Lys
290 295 300
Gly Lys Asp Gly Phe Leu Glu Leu Lys Ser Val Leu Thr Arg Val His
305 310 315 320
Ser Asn Lys Pro Ala Gln Ser Asp Asn Leu Thr Ile Trp Cys Met Ala
325 330 335
Leu Ser Pro Val Pro Gly Asn Lys Val Trp Asn Ile Ser Ser Glu Lys
340 345 350
Ile Thr Phe Leu Leu Gly Ser Glu Ile Ser Ser Thr Asp Pro Pro Leu
355 360 365
Ser Val Thr Glu Ser Thr Leu Asp Thr Gln Pro Ser Pro Ala Ser Ser
370 375 380
Val Ser Pro Ala Arg Tyr Pro Ala Thr Ser Ser Val Thr Leu Val Asp
385 390 395 400
Val Ser Ala Leu Arg Pro Asn Thr Thr Pro Gln Pro Ser Asn Ser Ser
405 410 415
Met Thr Thr Arg Gly Phe Asn Tyr Pro Trp Thr Ser Ser Gly Thr Asp
420 425 430
Thr Lys Lys Ser Val Ser Arg Ile Pro Ser Glu Thr Tyr Ser Ser Ser
435 440 445
Pro Ser Gly Ala Gly Ser Thr Leu His Asp Asn Val Phe Thr Ser Thr
450 455 460
Ala Arg Ala Phe Ser Glu Val Pro Thr Thr Ala Asn Gly Ser Thr Lys
465 470 475 480
Thr Asn His Val His Ile Thr Gly Ile Val Val Asn Lys Pro Lys Asp
485 490 495
Gly Met
<210> 2
<211> 1494
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
gtgtgggaga aaaccgtgaa caccgaggag aacgtgtatg cgaccctggg cagcgatgtg 60
aacctgacct gccagaccca gaccgtgggc ttttttgtgc agatgcagtg gagcaaagtg 120
accaacaaaa ttgatctgat tgcggtgtat cacccgcagt atggctttta ttgcgcgtat 180
ggccgcccgt gcgagagcct ggtgaccttt accgagaccc cggagaacgg cagcaaatgg 240
accctgcacc tgcgcaacat gagctgcagc gtgagcggcc gctatgagtg catgctggtg 300
ctgtatccgg agggcattca gaccaaaatt tataacctgc tgattcagac ccacgtgacc 360
gcggatgagt ggaacagcaa ccacaccatt gagattgaga ttaaccagac cctggagatt 420
ccgtgctttc agaacagcag cagcaaaatt agcagcgagt ttacctatgc gtggagcgtg 480
gagaacagca gcaccgatag ctgggtgctg ctgagcaaag gcattaaaga ggataacggc 540
acccaggaga ccctgattag ccagaaccac ctgattagca acagcaccct gctgaaagat 600
cgcgtgaaac tgggcaccga ttatcgcctg cacctgagcc cggtgcagat ttttgatgat 660
ggccgcaaat ttagctgcca cattcgcgtg ggcccgaaca aaattctgcg cagcagcacc 720
accgtgaaag tgtttgcgaa accggagatt ccggtgattg tggagaacaa cagcaccgat 780
gtgctggtgg agcgccgctt tacctgcctg ctgaaaaacg tgtttccgaa agcgaacatt 840
acctggttta ttgatggcag ctttctgcac gatgagaaag agggcattta tattaccaac 900
gaggagcgca aaggcaaaga tggctttctg gagctgaaaa gcgtgctgac ccgcgtgcac 960
agcaacaaac cggcgcagag cgataacctg accatttggt gcatggcgct gagcccggtg 1020
ccgggcaaca aagtgtggaa cattagcagc gagaaaatta cctttctgct gggcagcgag 1080
attagcagca ccgatccgcc gctgagcgtg accgagagca ccctggatac ccagccgagc 1140
ccggcgagca gcgtgagccc ggcgcgctat ccggcgacca gcagcgtgac cctggtggat 1200
gtgagcgcgc tgcgcccgaa caccaccccg cagccgagca acagcagcat gaccacccgc 1260
ggctttaact atccgtggac cagcagcggc accgatacca aaaaaagcgt gagccgcatt 1320
ccgagcgaga cctatagcag cagcccgagc ggcgcgggca gcaccctgca cgataacgtg 1380
tttaccagca ccgcgcgcgc gtttagcgag gtgccgacca ccgcgaacgg cagcaccaaa 1440
accaaccacg tgcacattac cggcattgtg gtgaacaaac cgaaagatgg catg 1494
<210> 3
<211> 515
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 3
Val Trp Glu Glu Leu Phe Asn Val Gly Asp Asp Val Tyr Ala Leu Pro
1 5 10 15
Gly Ser Asp Ile Asn Leu Thr Cys Gln Thr Lys Glu Lys Asn Phe Leu
20 25 30
Val Gln Met Gln Trp Ser Lys Val Thr Asp Lys Asn Asp Met Ile Ala
35 40 45
Leu Tyr His Pro Gln Tyr Gly Leu Tyr Cys Gly Gln Glu His Ala Cys
50 55 60
Glu Ser Gln Val Ala Ala Thr Glu Thr Glu Lys Gly Val Thr Asn Trp
65 70 75 80
Thr Leu Tyr Leu Arg Asn Ile Ser Ser Ala Leu Gly Gly Lys Tyr Glu
85 90 95
Cys Ile Phe Thr Leu Tyr Pro Glu Gly Ile Lys Thr Thr Val Tyr Asn
100 105 110
Leu Ile Val Glu Pro Tyr Thr Gln Asp Glu His Asn Tyr Thr Ile Glu
115 120 125
Ile Glu Thr Asn Arg Thr Leu Glu Ile Pro Cys Phe Gln Asn Thr Ser
130 135 140
Ser Glu Ile Pro Pro Arg Phe Thr Phe Ser Trp Leu Val Glu Lys Asp
145 150 155 160
Gly Val Glu Glu Val Leu Phe Thr His His His His Val Asn Asn Ser
165 170 175
Thr Ser Phe Lys Gly Arg Ile Arg Leu Gly Gly Asp Tyr Arg Leu His
180 185 190
Leu Ser Pro Val Gln Ile Gln Asp Asp Gly Arg Thr Phe Ser Cys His
195 200 205
Leu Thr Val Asn Pro Leu Lys Ala Trp Lys Met Ser Thr Thr Val Lys
210 215 220
Val Phe Ala Lys Pro Glu Ile Leu Met Thr Val Glu Asn Ser Thr Met
225 230 235 240
Asp Val Leu Gly Glu Arg Val Phe Thr Cys Leu Leu Lys Asn Val Phe
245 250 255
Pro Lys Ala Asn Ile Thr Trp Phe Ile Asp Gly Arg Phe Leu Gln Gly
260 265 270
Asn Glu Glu Gly Ile Tyr Ile Thr Asn Glu Glu Lys Asn Cys Ser Ser
275 280 285
Gly Phe Trp Glu Leu Lys Ser Val Leu Thr Arg Met His Ser Gly Pro
290 295 300
Ser Gln Ser Asn Asn Met Thr Ala Trp Cys Met Ala Leu Ser Pro Gly
305 310 315 320
Pro Arg Asn Lys Met Trp Asn Thr Ser Ser Gln Pro Ile Thr Val Ser
325 330 335
Phe Asp Ser Val Ile Ala Pro Thr Lys His Leu Pro Thr Val Thr Gly
340 345 350
Ser Thr Leu Gly Thr Gln Pro Phe Ser Asp Ala Gly Val Ser Pro Thr
355 360 365
Gly Tyr Leu Ala Thr Pro Ser Val Thr Ile Val Asp Glu Asn Gly Leu
370 375 380
Thr Pro Asp Ala Thr Pro Gln Thr Ser Asn Ser Ser Met Thr Thr Lys
385 390 395 400
Asp Gly Asn Tyr Leu Glu Ala Ser Ser Gly Thr Asp Ala Lys Asn Ser
405 410 415
Ser Arg Ala Ala Ala Ser Ser Lys Ser Gly Ser Trp Pro Phe Pro Phe
420 425 430
Thr Ser Pro Pro Glu Trp His Ser Leu Pro Gly Thr Ser Thr Gly Pro
435 440 445
Gln Glu Pro Asp Ser Pro Val Ser Trp Ile Pro Ser Glu Val His Thr
450 455 460
Ser Ala Pro Leu Asp Ala Ser Leu Ala Pro His Asp Thr Ile Ile Ser
465 470 475 480
Thr Thr Thr Glu Phe Pro Asn Val Leu Thr Thr Ala Asn Gly Thr Thr
485 490 495
Lys Ile Asp His Gly Pro Ile Thr Ser Ile Ile Val Asn Gln Pro Ser
500 505 510
Asp Gly Met
515
<210> 4
<211> 1545
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
gtgtgggagg agctgtttaa cgtgggcgat gatgtgtatg cgctgccggg cagcgatatt 60
aacctgacct gccagaccaa agagaaaaac tttctggtgc agatgcagtg gagcaaagtg 120
accgataaaa acgatatgat tgcgctgtat cacccgcagt atggcctgta ttgcggccag 180
gagcacgcgt gcgagagcca ggtggcggcg accgagaccg agaaaggcgt gaccaactgg 240
accctgtatc tgcgcaacat tagcagcgcg ctgggcggca aatatgagtg catttttacc 300
ctgtatccgg agggcattaa aaccaccgtg tataacctga ttgtggagcc gtatacccag 360
gatgagcaca actataccat tgagattgag accaaccgca ccctggagat tccgtgcttt 420
cagaacacca gcagcgagat tccgccgcgc tttaccttta gctggctggt ggagaaagat 480
ggcgtggagg aggtgctgtt tacccaccac caccacgtga acaacagcac cagctttaaa 540
ggccgcattc gcctgggcgg cgattatcgc ctgcacctga gcccggtgca gattcaggat 600
gatggccgca cctttagctg ccacctgacc gtgaacccgc tgaaagcgtg gaaaatgagc 660
accaccgtga aagtgtttgc gaaaccggag attctgatga ccgtggagaa cagcaccatg 720
gatgtgctgg gcgagcgcgt gtttacctgc ctgctgaaaa acgtgtttcc gaaagcgaac 780
attacctggt ttattgatgg ccgctttctg cagggcaacg aggagggcat ttatattacc 840
aacgaggaga aaaactgcag cagcggcttt tgggagctga aaagcgtgct gacccgcatg 900
cacagcggcc cgagccagag caacaacatg accgcgtggt gcatggcgct gagcccgggc 960
ccgcgcaaca aaatgtggaa caccagcagc cagccgatta ccgtgagctt tgatagcgtg 1020
attgcgccga ccaaacacct gccgaccgtg accggcagca ccctgggcac ccagccgttt 1080
agcgatgcgg gcgtgagccc gaccggctat ctggcgaccc cgagcgtgac cattgtggat 1140
gagaacggcc tgaccccgga tgcgaccccg cagaccagca acagcagcat gaccaccaaa 1200
gatggcaact atctggaggc gagcagcggc accgatgcga aaaacagcag ccgcgcggcg 1260
gcgagcagca aaagcggcag ctggccgttt ccgtttacca gcccgccgga gtggcacagc 1320
ctgccgggca ccagcaccgg cccgcaggag ccggatagcc cggtgagctg gattccgagc 1380
gaggtgcaca ccagcgcgcc gctggatgcg agcctggcgc cgcacgatac cattattagc 1440
accaccaccg agtttccgaa cgtgctgacc accgcgaacg gcaccaccaa aattgatcac 1500
ggcccgatta ccagcattat tgtgaaccag ccgagcgatg gcatg 1545
<210> 5
<211> 730
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 5
Val Trp Glu Lys Thr Val Asn Thr Glu Glu Asn Val Tyr Ala Thr Leu
1 5 10 15
Gly Ser Asp Val Asn Leu Thr Cys Gln Thr Gln Thr Val Gly Phe Phe
20 25 30
Val Gln Met Gln Trp Ser Lys Val Thr Asn Lys Ile Asp Leu Ile Ala
35 40 45
Val Tyr His Pro Gln Tyr Gly Phe Tyr Cys Ala Tyr Gly Arg Pro Cys
50 55 60
Glu Ser Leu Val Thr Phe Thr Glu Thr Pro Glu Asn Gly Ser Lys Trp
65 70 75 80
Thr Leu His Leu Arg Asn Met Ser Cys Ser Val Ser Gly Arg Tyr Glu
85 90 95
Cys Met Leu Val Leu Tyr Pro Glu Gly Ile Gln Thr Lys Ile Tyr Asn
100 105 110
Leu Leu Ile Gln Thr His Val Thr Ala Asp Glu Trp Asn Ser Asn His
115 120 125
Thr Ile Glu Ile Glu Ile Asn Gln Thr Leu Glu Ile Pro Cys Phe Gln
130 135 140
Asn Ser Ser Ser Lys Ile Ser Ser Glu Phe Thr Tyr Ala Trp Ser Val
145 150 155 160
Glu Asn Ser Ser Thr Asp Ser Trp Val Leu Leu Ser Lys Gly Ile Lys
165 170 175
Glu Asp Asn Gly Thr Gln Glu Thr Leu Ile Ser Gln Asn His Leu Ile
180 185 190
Ser Asn Ser Thr Leu Leu Lys Asp Arg Val Lys Leu Gly Thr Asp Tyr
195 200 205
Arg Leu His Leu Ser Pro Val Gln Ile Phe Asp Asp Gly Arg Lys Phe
210 215 220
Ser Cys His Ile Arg Val Gly Pro Asn Lys Ile Leu Arg Ser Ser Thr
225 230 235 240
Thr Val Lys Val Phe Ala Lys Pro Glu Ile Pro Val Ile Val Glu Asn
245 250 255
Asn Ser Thr Asp Val Leu Val Glu Arg Arg Phe Thr Cys Leu Leu Lys
260 265 270
Asn Val Phe Pro Lys Ala Asn Ile Thr Trp Phe Ile Asp Gly Ser Phe
275 280 285
Leu His Asp Glu Lys Glu Gly Ile Tyr Ile Thr Asn Glu Glu Arg Lys
290 295 300
Gly Lys Asp Gly Phe Leu Glu Leu Lys Ser Val Leu Thr Arg Val His
305 310 315 320
Ser Asn Lys Pro Ala Gln Ser Asp Asn Leu Thr Ile Trp Cys Met Ala
325 330 335
Leu Ser Pro Val Pro Gly Asn Lys Val Trp Asn Ile Ser Ser Glu Lys
340 345 350
Ile Thr Phe Leu Leu Gly Ser Glu Ile Ser Ser Thr Asp Pro Pro Leu
355 360 365
Ser Val Thr Glu Ser Thr Leu Asp Thr Gln Pro Ser Pro Ala Ser Ser
370 375 380
Val Ser Pro Ala Arg Tyr Pro Ala Thr Ser Ser Val Thr Leu Val Asp
385 390 395 400
Val Ser Ala Leu Arg Pro Asn Thr Thr Pro Gln Pro Ser Asn Ser Ser
405 410 415
Met Thr Thr Arg Gly Phe Asn Tyr Pro Trp Thr Ser Ser Gly Thr Asp
420 425 430
Thr Lys Lys Ser Val Ser Arg Ile Pro Ser Glu Thr Tyr Ser Ser Ser
435 440 445
Pro Ser Gly Ala Gly Ser Thr Leu His Asp Asn Val Phe Thr Ser Thr
450 455 460
Ala Arg Ala Phe Ser Glu Val Pro Thr Thr Ala Asn Gly Ser Thr Lys
465 470 475 480
Thr Asn His Val His Ile Thr Gly Ile Val Val Asn Lys Pro Lys Asp
485 490 495
Gly Met Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
500 505 510
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
515 520 525
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
530 535 540
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
545 550 555 560
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
565 570 575
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
580 585 590
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
595 600 605
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
610 615 620
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
625 630 635 640
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
645 650 655
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
660 665 670
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
675 680 685
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
690 695 700
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
705 710 715 720
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
725 730
<210> 6
<211> 2190
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
gtgtgggaga aaaccgtgaa caccgaggag aacgtgtatg cgaccctggg cagcgatgtg 60
aacctgacct gccagaccca gaccgtgggc ttttttgtgc agatgcagtg gagcaaagtg 120
accaacaaaa ttgatctgat tgcggtgtat cacccgcagt atggctttta ttgcgcgtat 180
ggccgcccgt gcgagagcct ggtgaccttt accgagaccc cggagaacgg cagcaaatgg 240
accctgcacc tgcgcaacat gagctgcagc gtgagcggcc gctatgagtg catgctggtg 300
ctgtatccgg agggcattca gaccaaaatt tataacctgc tgattcagac ccacgtgacc 360
gcggatgagt ggaacagcaa ccacaccatt gagattgaga ttaaccagac cctggagatt 420
ccgtgctttc agaacagcag cagcaaaatt agcagcgagt ttacctatgc gtggagcgtg 480
gagaacagca gcaccgatag ctgggtgctg ctgagcaaag gcattaaaga ggataacggc 540
acccaggaga ccctgattag ccagaaccac ctgattagca acagcaccct gctgaaagat 600
cgcgtgaaac tgggcaccga ttatcgcctg cacctgagcc cggtgcagat ttttgatgat 660
ggccgcaaat ttagctgcca cattcgcgtg ggcccgaaca aaattctgcg cagcagcacc 720
accgtgaaag tgtttgcgaa accggagatt ccggtgattg tggagaacaa cagcaccgat 780
gtgctggtgg agcgccgctt tacctgcctg ctgaaaaacg tgtttccgaa agcgaacatt 840
acctggttta ttgatggcag ctttctgcac gatgagaaag agggcattta tattaccaac 900
gaggagcgca aaggcaaaga tggctttctg gagctgaaaa gcgtgctgac ccgcgtgcac 960
agcaacaaac cggcgcagag cgataacctg accatttggt gcatggcgct gagcccggtg 1020
ccgggcaaca aagtgtggaa cattagcagc gagaaaatta cctttctgct gggcagcgag 1080
attagcagca ccgatccgcc gctgagcgtg accgagagca ccctggatac ccagccgagc 1140
ccggcgagca gcgtgagccc ggcgcgctat ccggcgacca gcagcgtgac cctggtggat 1200
gtgagcgcgc tgcgcccgaa caccaccccg cagccgagca acagcagcat gaccacccgc 1260
ggctttaact atccgtggac cagcagcggc accgatacca aaaaaagcgt gagccgcatt 1320
ccgagcgaga cctatagcag cagcccgagc ggcgcgggca gcaccctgca cgataacgtg 1380
tttaccagca ccgcgcgcgc gtttagcgag gtgccgacca ccgcgaacgg cagcaccaaa 1440
accaaccacg tgcacattac cggcattgtg gtgaacaaac cgaaagatgg catggagccg 1500
aaaagctgcg ataaaaccca cacctgcccg ccgtgcccgg cgccggagct gctgggcggc 1560
ccgagcgtgt ttctgtttcc gccgaaaccg aaagataccc tgatgattag ccgcaccccg 1620
gaggtgacct gcgtggtggt ggatgtgagc cacgaggatc cggaggtgaa atttaactgg 1680
tatgtggatg gcgtggaggt gcacaacgcg aaaaccaaac cgcgcgagga gcagtataac 1740
agcacctatc gcgtggtgag cgtgctgacc gtgctgcacc aggattggct gaacggcaaa 1800
gagtataaat gcaaagtgag caacaaagcg ctgccggcgc cgattgagaa aaccattagc 1860
aaagcgaaag gccagccgcg cgagccgcag gtgtataccc tgccgccgag ccgcgaggag 1920
atgaccaaaa accaggtgag cctgacctgc ctggtgaaag gcttttatcc gagcgatatt 1980
gcggtggagt gggagagcaa cggccagccg gagaacaact ataaaaccac cccgccggtg 2040
ctggatagcg atggcagctt ttttctgtat agcaaactga ccgtggataa aagccgctgg 2100
cagcagggca acgtgtttag ctgcagcgtg atgcacgagg cgctgcacaa ccactatacc 2160
cagaaaagcc tgagcctgag cccgggcaaa 2190
<210> 7
<211> 742
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 7
Val Trp Glu Glu Leu Phe Asn Val Gly Asp Asp Val Tyr Ala Leu Pro
1 5 10 15
Gly Ser Asp Ile Asn Leu Thr Cys Gln Thr Lys Glu Lys Asn Phe Leu
20 25 30
Val Gln Met Gln Trp Ser Lys Val Thr Asp Lys Asn Asp Met Ile Ala
35 40 45
Leu Tyr His Pro Gln Tyr Gly Leu Tyr Cys Gly Gln Glu His Ala Cys
50 55 60
Glu Ser Gln Val Ala Ala Thr Glu Thr Glu Lys Gly Val Thr Asn Trp
65 70 75 80
Thr Leu Tyr Leu Arg Asn Ile Ser Ser Ala Leu Gly Gly Lys Tyr Glu
85 90 95
Cys Ile Phe Thr Leu Tyr Pro Glu Gly Ile Lys Thr Thr Val Tyr Asn
100 105 110
Leu Ile Val Glu Pro Tyr Thr Gln Asp Glu His Asn Tyr Thr Ile Glu
115 120 125
Ile Glu Thr Asn Arg Thr Leu Glu Ile Pro Cys Phe Gln Asn Thr Ser
130 135 140
Ser Glu Ile Pro Pro Arg Phe Thr Phe Ser Trp Leu Val Glu Lys Asp
145 150 155 160
Gly Val Glu Glu Val Leu Phe Thr His His His His Val Asn Asn Ser
165 170 175
Thr Ser Phe Lys Gly Arg Ile Arg Leu Gly Gly Asp Tyr Arg Leu His
180 185 190
Leu Ser Pro Val Gln Ile Gln Asp Asp Gly Arg Thr Phe Ser Cys His
195 200 205
Leu Thr Val Asn Pro Leu Lys Ala Trp Lys Met Ser Thr Thr Val Lys
210 215 220
Val Phe Ala Lys Pro Glu Ile Leu Met Thr Val Glu Asn Ser Thr Met
225 230 235 240
Asp Val Leu Gly Glu Arg Val Phe Thr Cys Leu Leu Lys Asn Val Phe
245 250 255
Pro Lys Ala Asn Ile Thr Trp Phe Ile Asp Gly Arg Phe Leu Gln Gly
260 265 270
Asn Glu Glu Gly Ile Tyr Ile Thr Asn Glu Glu Lys Asn Cys Ser Ser
275 280 285
Gly Phe Trp Glu Leu Lys Ser Val Leu Thr Arg Met His Ser Gly Pro
290 295 300
Ser Gln Ser Asn Asn Met Thr Ala Trp Cys Met Ala Leu Ser Pro Gly
305 310 315 320
Pro Arg Asn Lys Met Trp Asn Thr Ser Ser Gln Pro Ile Thr Val Ser
325 330 335
Phe Asp Ser Val Ile Ala Pro Thr Lys His Leu Pro Thr Val Thr Gly
340 345 350
Ser Thr Leu Gly Thr Gln Pro Phe Ser Asp Ala Gly Val Ser Pro Thr
355 360 365
Gly Tyr Leu Ala Thr Pro Ser Val Thr Ile Val Asp Glu Asn Gly Leu
370 375 380
Thr Pro Asp Ala Thr Pro Gln Thr Ser Asn Ser Ser Met Thr Thr Lys
385 390 395 400
Asp Gly Asn Tyr Leu Glu Ala Ser Ser Gly Thr Asp Ala Lys Asn Ser
405 410 415
Ser Arg Ala Ala Ala Ser Ser Lys Ser Gly Ser Trp Pro Phe Pro Phe
420 425 430
Thr Ser Pro Pro Glu Trp His Ser Leu Pro Gly Thr Ser Thr Gly Pro
435 440 445
Gln Glu Pro Asp Ser Pro Val Ser Trp Ile Pro Ser Glu Val His Thr
450 455 460
Ser Ala Pro Leu Asp Ala Ser Leu Ala Pro His Asp Thr Ile Ile Ser
465 470 475 480
Thr Thr Thr Glu Phe Pro Asn Val Leu Thr Thr Ala Asn Gly Thr Thr
485 490 495
Lys Ile Asp His Gly Pro Ile Thr Ser Ile Ile Val Asn Gln Pro Ser
500 505 510
Asp Gly Met Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr
515 520 525
Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
530 535 540
Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp
545 550 555 560
Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp
565 570 575
Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn
580 585 590
Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp
595 600 605
Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro
610 615 620
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala
625 630 635 640
Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp
645 650 655
Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile
660 665 670
Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn
675 680 685
Thr Gln Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys
690 695 700
Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys
705 710 715 720
Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu
725 730 735
Ser His Ser Pro Gly Lys
740
<210> 8
<211> 2226
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
gtgtgggagg agctgtttaa cgtgggcgat gatgtgtatg cgctgccggg cagcgatatt 60
aacctgacct gccagaccaa agagaaaaac tttctggtgc agatgcagtg gagcaaagtg 120
accgataaaa acgatatgat tgcgctgtat cacccgcagt atggcctgta ttgcggccag 180
gagcacgcgt gcgagagcca ggtggcggcg accgagaccg agaaaggcgt gaccaactgg 240
accctgtatc tgcgcaacat tagcagcgcg ctgggcggca aatatgagtg catttttacc 300
ctgtatccgg agggcattaa aaccaccgtg tataacctga ttgtggagcc gtatacccag 360
gatgagcaca actataccat tgagattgag accaaccgca ccctggagat tccgtgcttt 420
cagaacacca gcagcgagat tccgccgcgc tttaccttta gctggctggt ggagaaagat 480
ggcgtggagg aggtgctgtt tacccaccac caccacgtga acaacagcac cagctttaaa 540
ggccgcattc gcctgggcgg cgattatcgc ctgcacctga gcccggtgca gattcaggat 600
gatggccgca cctttagctg ccacctgacc gtgaacccgc tgaaagcgtg gaaaatgagc 660
accaccgtga aagtgtttgc gaaaccggag attctgatga ccgtggagaa cagcaccatg 720
gatgtgctgg gcgagcgcgt gtttacctgc ctgctgaaaa acgtgtttcc gaaagcgaac 780
attacctggt ttattgatgg ccgctttctg cagggcaacg aggagggcat ttatattacc 840
aacgaggaga aaaactgcag cagcggcttt tgggagctga aaagcgtgct gacccgcatg 900
cacagcggcc cgagccagag caacaacatg accgcgtggt gcatggcgct gagcccgggc 960
ccgcgcaaca aaatgtggaa caccagcagc cagccgatta ccgtgagctt tgatagcgtg 1020
attgcgccga ccaaacacct gccgaccgtg accggcagca ccctgggcac ccagccgttt 1080
agcgatgcgg gcgtgagccc gaccggctat ctggcgaccc cgagcgtgac cattgtggat 1140
gagaacggcc tgaccccgga tgcgaccccg cagaccagca acagcagcat gaccaccaaa 1200
gatggcaact atctggaggc gagcagcggc accgatgcga aaaacagcag ccgcgcggcg 1260
gcgagcagca aaagcggcag ctggccgttt ccgtttacca gcccgccgga gtggcacagc 1320
ctgccgggca ccagcaccgg cccgcaggag ccggatagcc cggtgagctg gattccgagc 1380
gaggtgcaca ccagcgcgcc gctggatgcg agcctggcgc cgcacgatac cattattagc 1440
accaccaccg agtttccgaa cgtgctgacc accgcgaacg gcaccaccaa aattgatcac 1500
ggcccgatta ccagcattat tgtgaaccag ccgagcgatg gcatggtgcc gcgcgattgc 1560
ggctgcaaac cgtgcatttg caccgtgccg gaggtgagca gcgtgtttat ttttccgccg 1620
aaaccgaaag atgtgctgac cattaccctg accccgaaag tgacctgcgt ggtggtggat 1680
attagcaaag atgatccgga ggtgcagttt agctggtttg tggatgatgt ggaggtgcac 1740
accgcgcaga cccagccgcg cgaggagcag tttaacagca cctttcgcag cgtgagcgag 1800
ctgccgatta tgcaccagga ttggctgaac ggcaaagagt ttaaatgccg cgtgaacagc 1860
gcggcgtttc cggcgccgat tgagaaaacc attagcaaaa ccaaaggccg cccgaaagcg 1920
ccgcaggtgt ataccattcc gccgccgaaa gagcagatgg cgaaagataa agtgagcctg 1980
acctgcatga ttaccgattt ttttccggag gatattaccg tggagtggca gtggaacggc 2040
cagccggcgg agaactataa aaacacccag ccgattatga acaccaacgg cagctatttt 2100
gtgtatagca aactgaacgt gcagaaaagc aactgggagg cgggcaacac ctttacctgc 2160
agcgtgctgc acgagggcct gcacaaccac cacaccgaga aaagcctgag ccacagcccg 2220
ggcaaa 2226
<210> 9
<211> 232
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 9
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Gln Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Thr Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asp Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 10
<211> 232
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 10
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 11
<211> 696
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
gagccgaaaa gctgcgataa aacccacacc tgcccgccgt gcccggcgcc ggagctgctg 60
ggcggcccga gcgtgtttct gtttccgccg aaaccgaaag ataccctgat gattagccgc 120
accccggagg tgacctgcgt ggtggtggat gtgagccacg aggatccgga ggtgaaattt 180
aactggtatg tggatggcgt ggaggtgcac aacgcgaaaa ccaaaccgcg cgaggagcag 240
tataacagca cctatcgcgt ggtgagcgtg ctgaccgtgc tgcaccagga ttggctgaac 300
ggcaaagagt ataaatgcaa agtgagcaac aaagcgctgc cggcgccgat tgagaaaacc 360
attagcaaag cgaaaggcca gccgcgcgag ccgcaggtgt ataccctgcc gccgagccgc 420
gaggagatga ccaaaaacca ggtgagcctg acctgcctgg tgaaaggctt ttatccgagc 480
gatattgcgg tggagtggga gagcaacggc cagccggaga acaactataa aaccaccccg 540
ccggtgctgg atagcgatgg cagctttttt ctgtatagca aactgaccgt ggataaaagc 600
cgctggcagc agggcaacgt gtttagctgc agcgtgatgc acgaggcgct gcacaaccac 660
tatacccaga aaagcctgag cctgagcccg ggcaaa 696
<210> 12
<211> 227
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 12
Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu
1 5 10 15
Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr
20 25 30
Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys
35 40 45
Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val
50 55 60
His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
65 70 75 80
Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val
115 120 125
Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser
130 135 140
Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu
145 150 155 160
Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro
165 170 175
Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val
180 185 190
Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu
195 200 205
His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser
210 215 220
Pro Gly Lys
225
<210> 13
<211> 681
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
gtgccgcgcg attgcggctg caaaccgtgc atttgcaccg tgccggaggt gagcagcgtg 60
tttatttttc cgccgaaacc gaaagatgtg ctgaccatta ccctgacccc gaaagtgacc 120
tgcgtggtgg tggatattag caaagatgat ccggaggtgc agtttagctg gtttgtggat 180
gatgtggagg tgcacaccgc gcagacccag ccgcgcgagg agcagtttaa cagcaccttt 240
cgcagcgtga gcgagctgcc gattatgcac caggattggc tgaacggcaa agagtttaaa 300
tgccgcgtga acagcgcggc gtttccggcg ccgattgaga aaaccattag caaaaccaaa 360
ggccgcccga aagcgccgca ggtgtatacc attccgccgc cgaaagagca gatggcgaaa 420
gataaagtga gcctgacctg catgattacc gatttttttc cggaggatat taccgtggag 480
tggcagtgga acggccagcc ggcggagaac tataaaaaca cccagccgat tatgaacacc 540
aacggcagct attttgtgta tagcaaactg aacgtgcaga aaagcaactg ggaggcgggc 600
aacaccttta cctgcagcgt gctgcacgag ggcctgcaca accaccacac cgagaaaagc 660
ctgagccaca gcccgggcaa a 681

Claims (5)

  1. The application of CD96 recombinant protein in preparing medicine for treating immunological diseases,
    the amino acid sequence of the CD96 recombinant protein is shown in SEQ ID NO.1 or SEQ ID NO. 3; or the CD96 recombinant protein is a fusion protein, the amino acid sequence of the fusion protein is shown as SEQ ID NO.5 or SEQ ID NO.7,
    the immune disorders include lupus erythematosus, arthritis, prevention of an immune response associated with rejection of donor tissue.
  2. 2. The use of a CD96 recombinant protein according to claim 1 in the preparation of a medicament for the treatment of an immune disorder, wherein:
    wherein, when the amino acid sequence of the CD96 recombinant protein is shown as SEQ ID NO.1 or SEQ ID NO.3, the nucleotide sequence of the coded CD96 recombinant protein is shown as SEQ ID NO.2 or SEQ ID NO. 4.
  3. 3. The use of a CD96 recombinant protein according to claim 1 in the preparation of a medicament for the treatment of an immune disorder, wherein:
    wherein, when the CD96 recombinant protein is a fusion protein, the fusion partner is an IgG Fc region, and the nucleotide sequence for coding the fusion protein is shown as SEQ ID NO.6 or SEQ ID NO. 8.
  4. 4. Use of a CD96 recombinant protein according to claim 3 in the preparation of a medicament for the treatment of an immune disorder, wherein:
    wherein the IgG Fc region is an Fc region from an IgGl, IgG2, IgG3 or IgG4 antibody,
    the IgG FC region comprises the amino acid sequence shown in SEQ ID NO. 9.
  5. 5. The use of a CD96 recombinant protein according to claim 1 in the preparation of a medicament for the treatment of an immune disorder, wherein:
    wherein, the CD96 recombinant protein is combined with other anti-immune related disease drugs.
CN201910392750.0A 2019-05-13 2019-05-13 Application of CD96 recombinant protein in preparation of pharmaceutical composition for immune diseases Active CN110156888B (en)

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CN114762723A (en) * 2020-12-31 2022-07-19 吴伯骥 Application of HrpZpst protein in pharmacy for recognizing and activating multiple types of receptors and/or membrane proteins and signal paths thereof

Citations (3)

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Publication number Priority date Publication date Assignee Title
CN102057272A (en) * 2008-04-09 2011-05-11 健泰科生物技术公司 Novel compositions and methods for the treatment of immune related diseases
CN102204901A (en) * 2010-03-30 2011-10-05 中国科学院上海生命科学研究院 Reagent and method for regulating immune molecules
CN107982538A (en) * 2017-12-26 2018-05-04 深圳市体内生物医药科技有限公司 A kind of pharmaceutical composition and its application

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Publication number Priority date Publication date Assignee Title
CN102057272A (en) * 2008-04-09 2011-05-11 健泰科生物技术公司 Novel compositions and methods for the treatment of immune related diseases
CN102204901A (en) * 2010-03-30 2011-10-05 中国科学院上海生命科学研究院 Reagent and method for regulating immune molecules
CN107982538A (en) * 2017-12-26 2018-05-04 深圳市体内生物医药科技有限公司 A kind of pharmaceutical composition and its application

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Fc IgG1 heavy chain constant region, partial [Homo sapiens];Niranjana,K.R.P.;《GenBank》;20160725;第1页 *
T-cell surface protein tactile precursor;Martint L;《NCBI》;20180729;第1页 *

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