WO2018171411A1 - 一种融合蛋白及其制备方法和其在制备治疗眼科疾病、抗炎、抗肿瘤药物中的应用 - Google Patents

一种融合蛋白及其制备方法和其在制备治疗眼科疾病、抗炎、抗肿瘤药物中的应用 Download PDF

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WO2018171411A1
WO2018171411A1 PCT/CN2018/077968 CN2018077968W WO2018171411A1 WO 2018171411 A1 WO2018171411 A1 WO 2018171411A1 CN 2018077968 W CN2018077968 W CN 2018077968W WO 2018171411 A1 WO2018171411 A1 WO 2018171411A1
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fusion protein
protein
sequence
inflammation
neovascular
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PCT/CN2018/077968
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English (en)
French (fr)
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徐寒梅
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江苏融泰生物技术有限公司
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Priority to US16/496,397 priority Critical patent/US11542311B2/en
Priority to JP2020500947A priority patent/JP6895140B2/ja
Priority to EP18770915.9A priority patent/EP3604342B1/en
Publication of WO2018171411A1 publication Critical patent/WO2018171411A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5406—IL-4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention belongs to the technical field of biopharmaceuticals, and more particularly to a fusion protein having anti-inflammatory, anti-tumor and therapeutic effects on ophthalmic diseases, and a preparation method and application thereof.
  • vascular-related diseases diseases such as arthritis, tumors, bacteria-induced inflammation, and ophthalmic diseases such as AMD are called vascular-related diseases.
  • Arthritis-like inflammatory diseases refer to inflammatory diseases that occur in human joints and surrounding tissues, and can be divided into dozens. There are more than 100 million arthritis patients in China, and the number is increasing. The clinical manifestations are redness, swelling, heat, pain, dysfunction and joint deformity of the joints. In severe cases, joint disability and affecting the quality of life of patients. These include rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, gouty arthritis, ankylosing spondylitis, reactive arthritis, and infectious arthritis. Rheumatoid arthritis (RA) is one of the most common inflammatory joint diseases and major disabling factors in the clinic.
  • RA Rheumatoid arthritis
  • RA chronic systemic inflammatory disease
  • RA chronic systemic inflammatory disease
  • patients often have pain or swelling in the hands or wrists (especially swelling of the back of the wrist) as the first symptom, and the symptoms do not continue to be relieved.
  • ordinary symptomatic treatment can alleviate the symptoms, the symptoms are often repeated due to irregular or insufficient medication. .
  • the disease progresses, there may be obvious morning stiffness, usually up to 1 hour, and it is constantly increasing; at the same time, certain joint dysfunction occurs.
  • vasculitis and synovitis The synovial blood vessels in the joints proliferate, forming vasospasm, leading to thickening of the synovial membrane, increased exudation, secretion of various cytokines, invasion of cartilage and bone damage. It can also erode the muscle cavity, ligament, tendon sheath and muscles around it, which affects the stability of the joint, and is prone to joint malformation and dysfunction. Vasculitis can also invade all organs of the body and form systemic diseases. In the pathological process of arthritis, angiogenesis is a landmark histological change. Neovascularization is accompanied by synovial hyperplasia and inflammatory cell infiltration, which is the basis of vasospasm formation and joint destruction.
  • Neovascularization causes abnormal changes in synovial tissue in patients with rheumatoid arthritis. Therefore, inhibition of neovascularization can alleviate or cure arthritis inflammatory diseases to a certain extent.
  • Tumor angiogenesis is the morphological basis of tumor growth and metastasis. It not only provides nutrition to tumors, but also exports a large number of tumor cells to the host to cause tumor growth and metastasis. Most malignant solid tumors such as ovarian cancer, liver cancer, cervical cancer and breast cancer are vascular-dependent tumors. On the one hand, neovascularization provides nutrients and oxygen for tumor growth, and on the other hand, it is an important pathway for tumor metastasis. Therefore, inhibition of tumor angiogenesis is an important anticancer measure.
  • Iris neovascular eye disease, choroidal neovascular eye disease, retinal neovascular eye disease and corneal neovascular eye disease in ophthalmological diseases, the pathogenesis of which is related to the excessive formation of new blood vessels, inhibiting the formation of new blood vessels is the treatment of this
  • Angiogenesis inhibitors are a class of drugs that have attracted attention in the treatment of neovascular diseases in recent years, so blocking the formation of new blood vessels may become a new means of treating eye diseases in patients due to angiogenesis in the eye.
  • angiogenesis inhibitors angiostatin and endostatin are particularly attractive.
  • vascular inhibitors have very attractive prospects, their defects are also very obvious: the targets for inhibiting angiogenesis drugs such as endostatin and angiostatin are unclear, and their specificity to blood vessels And the selectivity is not good enough, the effect is limited, resulting in a large amount of drug used in the experiment. Therefore, a good anti-angiogenic drug should be selective for neovascular marker molecules to achieve a directional effect on neovascularization, and to increase the overall inhibition of angiogenesis by drugs: to use only low doses of drugs , the effect of inhibiting angiogenesis can be achieved efficiently.
  • Avastin has been successfully used in the treatment of ocular diseases, and there is still no such drug developed in China.
  • the integrin target of the present invention inhibits angiogenesis and will be a new option for the treatment of such ocular diseases.
  • arthritis, tumors, and eye diseases are diseases related to blood vessels. Tumor growth and metastasis depend on new blood vessels; inflammation and angiogenesis are two interrelated and common pathological processes; ophthalmic diseases such as age-related macular degeneration (AMD) are mainly characterized by choroidal neovascularization.
  • AMD age-related macular degeneration
  • Neovascularization under normal physiological conditions, is highly regulated and is an essential process in reproduction, embryonic development, tissue repair, and wound healing.
  • Angiogenesis also occurs under a variety of pathological conditions including: tumor growth and metastasis; inflammatory disorders such as rheumatoid arthritis, psoriasis, osteoarthritis, inflammatory bowel disease, Crohn's disease, Ulcerative colons and other inflammatory disorders.
  • Integrins are a class of receptors that are widely distributed on the cell surface and mediate the adhesion between cells and extracellular matrices and cells and cells. They participate in blood vessels by linking the interaction between intracellular cytoskeletal proteins and extracellular matrix molecules. generate. At least eight integrins ( ⁇ 1 ⁇ 1, ⁇ 2 ⁇ 1, ⁇ 3 ⁇ 1, ⁇ 6 ⁇ 1, ⁇ 6 ⁇ 4, ⁇ 5 ⁇ 1, ⁇ v ⁇ 3, ⁇ v ⁇ 5) are involved in angiogenesis, and ⁇ v ⁇ 3 plays an important role. Vv ⁇ 3 recognizes the arg-gly-asp (RGD) sequence in the ligand molecule.
  • RGD arg-gly-asp
  • Vv ⁇ 3 can be expressed in a variety of cell types and participates in physiological and pathological processes such as tumor angiogenesis, invasion, metastasis, inflammation, wound healing and coagulation in combination with multiple ligands during multicellular activity. Therefore, a polypeptide containing an RGD sequence has an integrin antagonist action, and the RGD sequence can be used as a carrier for targeted transport to the neovascular endothelium, thereby achieving a more efficient treatment for neovascular diseases. Therefore, the inhibition of angiogenesis by the vasopressor polypeptide not only prevents the delivery of oxygen and nutrients to the synovial membrane, but also directly causes the blood vessel to degenerate, thereby possibly inhibiting the synovial proliferation of the RA. Inhibition of neovascularization is key to the treatment of such diseases, and proliferation and migration of endothelial cells is a critical step in neovascularization.
  • the present invention provides a mammalian cell expression method, and the two different active polypeptides and proteins are linked to each other to be solved: 1. Increasing the affinity of the polypeptide molecule to the target, the cell Toxin, enhance the efficacy of peptide molecules. 2. Overcoming the shortcomings of short molecular half-life and frequent administration of polypeptide molecules. 3. Linking peptides targeting different targets through a linker to form a bifunctional protein molecule, and prolonging the half-life of the bifunctional protein, laying a foundation for long-acting drug development.
  • a fusion protein comprising a vascular inhibitory polypeptide HM-3 sequence, an interleukin 4 (IL-4) sequence and an Fc fragment of an antibody IgG1; wherein the protein is linked by a flexible (F) Linker, and the polypeptide at both ends can be The change moves, so that it has better ductility; the protein II is linked by a rigid (R) Linker, which is not easy to cause spatial displacement of the polypeptides at both ends, so that the functional domains at both ends do not affect each other.
  • the structure diagram of protein 1 is shown in Fig. 1; the structure diagram of protein 2 is shown in Fig. 2.
  • amino acid sequence corresponding to the protein one is SEQ ID NO. 1; the amino acid sequence corresponding to the protein two is SEQ ID NO.
  • the gene encoding the above fusion protein, the nucleic acid sequences of the coding sequences of SEQ ID NO. 1, and SEQ ID NO. 2 are SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
  • the above fusion protein is used in the preparation of a medicament for treating inflammation, antitumor and treating ophthalmic diseases.
  • the inflammation includes rheumatoid arthritis, osteoarthritis, gouty arthritis, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, infectious arthritis and traumatic arthritis, systemic Lupus erythematosus, psoriasis.
  • the tumor includes gastric cancer, lung cancer, liver cancer, breast cancer, colon cancer, glioma, melanoma, and cervical cancer, and originates from a person's head and neck, brain, thyroid, esophagus, pancreas, lung, liver. , stomach, breast, kidney, gallbladder, colon or rectum, ovary, cervix, uterus, prostate, bladder, testicular primary or secondary cancer, melanoma and sarcoma.
  • the ophthalmic diseases include iris neovascular eye disease, choroidal neovascular eye disease, retinal neovascular eye disease or corneal neovascular eye disease.
  • the iris neovascular eye disease includes iris neovascular eye disease caused by neovascular glaucoma, diabetic retinopathy or central retinal vein thrombosis;
  • the choroidal neovascular eye disease includes age-related macular degeneration, centrality Exudative retinal choroiditis, ocular histoplasmosis syndrome or choroidal choroidal choroidal neovascular eye disease; said retinal neovascular eye disease including diabetes, tumor, retinal detachment, central retinal vein occlusion, retinal vein Retinal neovascular eye disease associated with peripheral inflammation, systemic lupus erythematosus, Eales disease or Coat disease; corneal neovascular eye disease including corneal neovascular disease caused by contact lens, alkali and other chemical burns, cornea Corneal neovascular eye disease caused by surgery, bacterial infection, chlamydial infection, viral infection or protozoal infection.
  • the pharmaceutical dosage form is a capsule, a tablet, a pill, an injection, a nasal spray or an aerosol.
  • the method for preparing the above fusion protein utilizes a method for expression and purification of prokaryotic cells or eukaryotic cells.
  • HM-3 has been well studied and found to have a good anti-angiogenic effect, but because it is a polypeptide, its half-life is shorter; IL-4 molecule can promote the proliferation of T cells, B cells and macrophages, thereby enhancing the body's Immune function, to achieve the role of killing tumors.
  • the invention combines the two on the one hand, and can play a dual anti-tumor effect, an anti-inflammatory effect and an anti-angiogenic effect, and at the same time improve the body immunity.
  • both HM-3 and interleukin 4 are polypeptides with a relatively small molecular weight, and the half-life in the body is relatively short, usually up to 30 minutes to 2 hours.
  • the Fc fragment sequence of IgG is introduced in the present invention, and the fusion protein construction technique is adopted.
  • the Fc fragments of HM-3, IL-4 and IgG1 are linked together by Linker to form a bifunctional fusion protein, which has the anti-angiogenic effect of HM-3 polypeptide and the anti-tumor effect of interleukin 4, and more importantly.
  • the addition of the Fc fragment significantly prolongs the half-life of HM-3 and interleukin-4, and it is expected that the product will provide patients with a long-acting bifunctional drug in the treatment of related diseases after the drug is formulated, for 1 week or 2 weeks, or even 1 month. Once administered, it effectively reduces the frequency of administration of the patient, increases the patient's drug compliance, and reduces the cost of treatment for the patient.
  • the invention solves the following problems: 1 synthetic molecular bottleneck of molecular weight and complex structure, especially macromolecular polypeptide molecules having secondary structure and high-order structure such as disulfide bond; 2 overcoming the difficulty in synthesis of chemical synthesis of large molecular weight polypeptide , low yield and other technical bottlenecks, significantly reduce the production cost of macromolecular peptides; 3 mammalian cells and other organisms express peptide molecules, can form the correct high-level structure, the affinity of peptide molecules and targets will be better than chemical synthesis a polypeptide molecule; a polypeptide molecule forms a fusion protein molecule with an Fc fragment of an antibody IgG1, IgG2 or IgG4, and an Fc fragment of IgG is prevented from being degraded by a Fc receptor-mediated (FcRn)-mediated recycling mechanism, while the molecular weight of the Fc fragment is Large, low renal clearance also ensures a significant increase in the half-life of the fusion protein over the polypeptid
  • the fusion protein formed by the fusion of the Fc fragment of IgG1 can also increase the cytotoxicity of ADCC and CDC, and can significantly increase the activity of anti-tumor molecules, and its anti-tumor effect is superior to that of polypeptide molecules; 5 using eukaryotic expression system, through linker
  • the Fc fragment of the antibody is ligated to the HM-3 and IL-4 sequences to prolong the half-life of the functional proteins HM-3 and IL-4, and the polypeptides of the two different targets are ligated to form a double-target fusion protein;
  • the protein one (SEQ ID NO. 1) and the protein two (SEQ ID NO. 2) of the present invention are a class of integrin blockers and immunomodulatory recombinant protein drugs, which not only inhibit vascular proliferation but also
  • the utility model has the functions of effectively regulating the immune system to inhibit tumor; the dual-function fusion protein drug can significantly enhance the effect of the drug on inhibiting and killing tumor cells and inhibiting vascular proliferation compared with a single polypeptide drug. More importantly, the fusion protein can significantly increase the half-life of the drug, improve the therapeutic effect of the drug, significantly improve the patient's drug compliance, and significantly reduce the cost of treatment.
  • the fusion protein drug achieves the function of treating arthritis, anti-tumor and inflammation-related ophthalmic diseases through the above bifunctional action and half-life extension.
  • the fusion protein sequence of the present invention includes an arginine-glycine-aspartate (RGD) sequence, and the RGD sequence is an important ligand of integrin, and the polypeptide containing the RGD sequence Gly-Gly-Gly-Gly- Arg-Gly-Asp can specifically recognize integrin, can effectively inhibit neovascularization, and can be used to treat tumor diseases, arthritis diseases and ophthalmic diseases.
  • the present invention adopts a flexible (S) or rigid (R) Linker combines two kinds of peptides to obtain protein one and protein two, which can improve the drug effect, prolong the half-life, enhance the stability, and has the characteristics of strong action and low toxicity;
  • the fusion protein of the present invention can be targeted to the neovascular endothelium, inhibit the formation of new blood vessels, and thereby achieve the effects of preventing or treating vascular and inflammation-related diseases;
  • the anti-tumor effect of the present invention has the effect of inhibiting various tumors.
  • the fusion protein I and the fusion protein II can effectively inhibit gastric cancer, lung cancer, liver cancer, breast cancer, melanoma,
  • the proliferation of colon cancer, glioma and cervical cancer, the inhibition rate of melanoma, gastric cancer and human glioma reached more than 50% at the concentration of 32 ⁇ g/mL;
  • the inhibition rate of colon cancer cells reached 64 ⁇ g/mL More than 40%, the inhibition rate of cervical cancer cells reached more than 50%;
  • the anti-inflammatory effect of the present invention can be obtained by a series of verification model experiments of Examples 4-10.
  • the fusion protein-1 and the fusion protein II can significantly inhibit lymphocyte proliferation and inhibit the production of IL-1 ⁇ inflammatory factors by macrophages. Inhibition of granuloma formation, reduction of capillary permeability in the model group, inhibition of ear swelling in the model group and swelling of the toes, and reduction of chronic inflammation of adjuvant arthritis in rats;
  • fusion protein I and fusion protein II can significantly inhibit proliferation of human retinal vascular endothelial cells, inhibit angiogenesis of chicken embryo chorioallantoic membrane, and inhibit corneal neovascularization. It inhibits the growth of iris neovascularization in rabbits, promotes the increase of choroidal blood flow in rabbit eyes, reduces the retinal neovascular plexus in OIR mice, inhibits the formation of neovascularization in neonatal rat retinopathy model, and has a certain therapeutic effect on diabetic retinopathy. .
  • Figure 1 is a schematic view showing the structure of a protein corresponding to SEQ ID NO. 1 in the present invention
  • Figure 2 is a schematic view showing the structure of a protein corresponding to SEQ ID NO. 2 in the present invention
  • 3 is an electrophoresis pattern of the fragment obtained by recovering Fc-IL4DM-HM3-1 and the expression vector pcDNA3.4/MCS(+) in the present invention
  • Figure 4 is a diagram showing the results of PCR verification of the bacterial liquid in the present invention.
  • Figure 5 is a diagram showing the results of capture of the fusion protein in the present invention.
  • Figure 6 is a diagram showing the fine purification of the fusion protein of the present invention.
  • Figure 7 is a graph showing the results of analysis of a fusion protein sample by the SDS-PAGE method of the present invention.
  • Figure 8 is a graph showing the results of analysis of a fusion protein sample by HPLC in the present invention.
  • vasopressor polypeptide HM-3 sequence is set forth in SEQ ID NO. 5
  • the interleukin 4 sequence is set forth in SEQ ID NO. 6
  • the human immunoglobulin IgG1-Fc region is passed through a different linker peptide Gly.
  • Fc-IL4DM-HM3 The DNA sequence of the above two fusion proteins Fc-IL4DM-HM3 was synthesized by a biotechnology company, ligated into a pUC57 vector to form a cloning vector, and stored in E. coli DH5 ⁇ to form a clonal strain. Both fusion proteins use pcDNA3.4/MCS(+) as an expression vector, and the vector construction process is completely identical. Therefore, Fc-IL4DM-HM3-1 is taken as an example, and the experimental procedure is as follows.
  • the Fc-IL4DM-HM3-1 clone strain sent by the biological company was provoked from the surface of the puncture bacteria, and connected to 2 tubes containing 5 mL of Amp-resistant LB medium, shaken at 37 ° C, 120 rpm. Train overnight.
  • the insert Fc-IL4DM-HM3-1 and the expression vector pcDNA3.4/MCS(+) obtained by gel recovery were carried out at 16 ° C according to the ratio of insert to vector molar ratio of 1:5. The reaction was ligated for 16 h.
  • Transient transfection is one of the ways to introduce DNA into eukaryotic cells.
  • transient transfection recombinant DNA is introduced into infectious cell lines to obtain transient but high levels of expression of the gene of interest. Sufficient protein can be obtained for experiments in a short period of time, saving cell screening time in stable transfection.
  • Fc-IL4DM-HM3 two novel fusion proteins, Fc-IL4DM-HM3, were expressed. Since the fusion protein expression process was completely consistent, the Fc-IL4DM-HM3-1 was used as an example. The experimental procedure was as follows.
  • the glycerol tube of the strain was stored by taking an Fc-IL4DM-HM3-1 expression vector from a -80 ° C refrigerator, and a 2 L shake flask containing 500 mL of Amp-resistant LB medium was placed, and shake-cultured at 37 ° C, 160 rpm overnight.
  • the cells were collected by centrifugation at 5000 g for 5 min, and the plasmid was extracted using a commercial endotoxin-free plasmid.
  • the plasmid concentration was controlled to be 1 mg/mL or more (if it is lower than this concentration, concentration is required), and then sterilized by filtration using a sterile 0.22 ⁇ m pore size filter to complete plasmid preparation.
  • the 293F cells used for transfection were passaged at a cell density of 0.4*10 6 cells/mL for four days from the day of resuscitation, and at least three passages were performed for transient transfection. During the passage, the passage volume was expanded as needed based on the volume of the final transfection medium.
  • Protein A is a cell wall protein isolated from S. aureus, which binds to mammalian IgG mainly through the Fc fragment and has high specificity and binding ability, and is widely used for purification of IgG antibodies and IgG-Fc fusion proteins.
  • Two novel fusion proteins, Fc-IL4DM-HM3-1 have IgG-Fc fragments and the purification process is completely identical. Therefore, Fc-IL4DM-HM3-1 was transiently transfected on a 1.6L scale as an example. The experimental procedure is as follows.
  • Sample pretreatment The transiently transfected cell culture medium at the end of 1.6 L culture was centrifuged at 7500 rpm for 20 min at 4 ° C, and about 1.46 L of the supernatant was obtained for the next protein A capture.
  • the column was washed with 420 mM PB, 0.15 M NaCl, pH 7.0, approximately 800 mL, flow rate 20 mL/min.
  • the target protein was eluted with 550 mM citric acid-sodium citrate, 0.15 M NaCL pH 3.0, and the collection was started at a peak of 20 mAu, and collection was stopped at 20 mAu after the peak; the flow rate was 20 mL/min.
  • sample is proteinA eluent, loading amount 40mL; 4) collecting sample, peak 3 is the target protein peak, collecting peak 3, starting peak 10mAu collection, 10mAu after the peak stops collecting.
  • Sample ultrafiltration concentration The sample of peak 3 was combined and concentrated by ultrafiltration.
  • the ultrafiltration membrane was selected to be 10 kDa, and the sample was concentrated to a protein concentration of more than 5 mg/mL, and then the sample was dispensed and stored in a refrigerator at -80 °C.
  • the initial concentration was about 0.29 mg/mL, and finally concentrated to 27 mL, and the concentration was about 5.53 mg/mL; the sample was dispensed and stored frozen.
  • samples were subjected to release detection by SDS-PAGE and HPLC (Fig. 7 and Fig. 8), and then used for drug-based evaluation studies.
  • the MTT assay was used to detect the inhibitory effect of the integrin blocker fusion protein obtained in Example 1 on the proliferation of various tumor cells, including melanoma cell B16F10, gastric cancer cell MGC-803, lung cancer cell A549, and liver cancer cell Hep-G2.
  • Tumor cells were cultured in a 37 ° C, 5% CO 2 incubator to a density of 90% or more, and collected by trypsinization. The cells were resuspended in the culture medium and counted under a microscope to adjust the cell concentration to 3.0 ⁇ 104 cells. In mL, the cell suspension was inoculated into a 96-well plate at 100 ⁇ L per well and cultured overnight at 37 ° C in a 5% CO 2 incubator. The fusion protein I, the fusion protein II, and the positive drug paclitaxel Taxol were diluted with the culture solution to each predetermined concentration. After the cells were fully adhered, each dilution was separately added to a 96-well plate at 100 ⁇ L per well.
  • the integrin blocker fusion protein I and protein II were added as the administration group, Taxol was used as the positive control group, and the culture medium without any drug was used as the blank control group, and incubated at 37 ° C in a 5% CO 2 incubator for 48 hours. . 20 ⁇ L of 5 mg/mL of MTT was added to each well of a 96-well plate, and incubation was continued for 4 hours. The medium was aspirated and dissolved in 100 ⁇ L of DMSO per well. The absorbance was measured at 570 nm with a microplate reader, and the growth inhibition (PI) was calculated.
  • the formula is as follows:
  • N test is the OD value of the test group and N control is the OD value of the blank control group.
  • the fusion protein can effectively inhibit gastric cancer, lung cancer, liver cancer, breast cancer, melanoma, colon cancer, and glue.
  • Proliferation of stromal and cervical cancer at the concentration of 32 ⁇ g/mL, the inhibition rate of melanoma, gastric cancer and human glioma reached more than 50%; at the concentration of 64 ⁇ g/mL, the inhibition rate of colon cancer cells reached more than 40%, and the inhibition rate of cervical cancer cells It can reach more than 50%; it needs high concentration for lung cancer, liver cancer and breast cancer cells to achieve effective inhibition.
  • Human umbilical vein endothelial cells were cultured with endothelial cells containing 5% fetal bovine serum and 1 ⁇ ECGS in a 37 ° C, 5% CO 2 incubator to a confluence of more than 90%, using the transwell method.
  • the activity of fusion protein I and protein II to inhibit endothelial cell migration was detected.
  • the endothelial cell HUVEC was only used for the 2nd to 8th generation. The specific operation was as follows:
  • HUVEC cells cultured to logarithmic growth phase were digested with 0.2% EDTA, collected, washed twice with PBS, resuspended in endothelial cell culture medium containing 0.1% BSA, counted under a microscope, and the cell concentration was adjusted to 1 ⁇ 105 / mL;
  • test liquid of each group diluted to 100 ⁇ L with a cell culture solution containing 0.1% BSA;
  • Blank control group a cell culture medium containing no drug
  • Endo group To dilute 5mg/mL Ente's solution to a predetermined concentration with a drug-free cell culture solution;
  • Fusion protein group the fusion protein was diluted to 10 ⁇ g/mL with a drug-free cell culture medium
  • the cells were seeded into a transwell chamber at 100 ⁇ L per well, and each group of test solutions was added to the chamber.
  • a transwell chamber Into a 24-well plate, 0.6 mL of endothelial cell culture medium containing 5% fetal bovine serum and 1 ⁇ ECGS was added to stimulate cell migration, and incubated at 5% CO 2 for 24 h at 37 ° C;
  • N test was the number of cell migration in the test group
  • N control was the number of cell migration in the blank control group.
  • mice The spleen of the mice was taken out under aseptic conditions, washed 3 times in empty 1640 medium, ground in 5 mL syringe core, filtered through a 200-mesh sieve, and prepared into a single cell suspension, centrifuged (1000 rpm, 5 min), and the supernatant was discarded. Tris-NH 4 Cl was used to break the red blood cells, and the cells were allowed to stand in an ice water bath for 4 min, centrifuged (1000 rpm, 5 min), the supernatant was discarded, and the cells were washed twice with sterile PBS.
  • cells were suspended in RPMI 1640 medium (5 mL) supplemented with 10% fetal calf serum, counted, adjusted to a cell concentration of 5 ⁇ 10 6 /mL, and cultured in a 96-well culture plate.
  • mice were intraperitoneally injected with 1 mL of broth medium (containing 6%) starch. After three days, the mouse peritoneal macrophages were aseptically taken and washed twice with 1640 medium to adjust the cell concentration to 2 ⁇ . 106 cells/mL were injected into a 24-well culture plate, 1 mL per well, and incubated in a cell culture incubator for 3 hours, shaking once every 30 minutes to allow the cells to adhere sufficiently. Then, it was washed twice with the culture solution to remove unattached cells. The blank group was added with PBS, the positive group was added with the positive drug dexamethasone Dex, and the control group was the low, medium and high concentrations of the fusion protein one and two. After the administration, the culture was continued for 48 hours and centrifuged at 1000 r/min for 15 minutes. The supernatant was taken as a sample for the activity of IL-1 ⁇ to be tested.
  • the model group dexamethasone-positive group (10mg/kg), fusion protein I and protein II were effective doses of 64mg/kg in the experimental group.
  • the rats were anesthetized with sodium pentobarbital (40 mg/kg), and the abdominal coat was cut off. The skin of the lower abdomen was cut under sterile conditions. The incision was about 1 cm long, and the vascular clamp was used to expand the subcutaneous tissue. A sterile dry cotton ball was implanted subcutaneously into one side of the groin, the incision was sutured, and an appropriate amount of amoxicillin was applied to the incision to prevent infection.
  • the drug was administered once every 5 days from the day after surgery.
  • the rats were sacrificed by cervical dislocation at the 24th day after the seventh day of administration, the inguinal skin was cut, the cotton ball was taken out together with the surrounding granulation tissue, and the surrounding tissues were removed. After continuous drying for 48 hours in an oven at 60 ° C, the weight was accurately weighed.
  • Table 12 The experimental results are shown in Table 12.
  • mice Eighty Kunming mice were randomly divided into 8 groups, 10 in each group, which were empty model group, dexamethasone-positive group (10 mg/kg), fusion protein- and protein II high, medium and low doses. (128, 32, 8 mg/kg) was the experimental group. The drug was administered once every 5 days, and the blank model group was given the same volume of physiological saline for 5 days and fed normally. On the 5th day after the administration, 5 g/L of Evans blue physiological saline solution 10 kg/mL was injected into the tail vein, followed by intraperitoneal injection of 10 kg/mL HAc solution (6 mL/L) to cause inflammation. After 20 minutes, the mice were sacrificed by cervical dislocation.
  • mice Effects of fusion protein-1 and protein para-xylene on ear swelling in mice
  • mice Eighty Kunming mice were divided into 8 groups, 10 in each group, numbered.
  • the saline group was used as the blank control group, the aspirin group (200 mg/kg) was used as the positive control group, and the high, medium and low doses of fusion protein I and protein II (128, 32, and 8 mg/kg) were used as the experimental group.
  • Mice were injected once every 5 days.
  • the blank control group was given an equal volume of physiological saline.
  • 0.05 mL of xylene was applied to both sides of the right ear of the mice to cause inflammation, and the left ear was not coated as normal ears. After 2 hours, the mice were sacrificed by dislocation, and the ears were cut along the auricle.
  • the experimental results show that the high doses of fusion protein I and protein II can significantly inhibit the xylene-induced ear swelling in mice, and the inhibition can be enhanced with the increase of dose.
  • Eighty SD rats were randomly divided into 8 groups, 10 in each group, which were blank model group, dexamethasone-positive group (5 mg/kg) and high, medium and low doses of fusion protein-1 and protein 2.
  • Groups (128, 32, 8 mg/kg) were experimental groups. The drug was administered once every 5 days, and the model group was given the same volume of physiological saline for 3 days and fed normally.
  • 0.1 mL of carrageenan was injected subcutaneously into the right hind paw of the rat to induce inflammation, and the paw volume was measured at 1 h, 3 h, 5 h, and 7 h after the inflammation.
  • the experimental results showed that the toes of the rats in each group were swollen rapidly, reaching the peak of swelling at about 3 to 5 hours, and disappeared at 7 hours.
  • the high doses of fusion protein I and protein II can significantly inhibit carrageenan-induced rat toe swelling, and the low dose inhibition is not obvious.
  • Rats in each group were anesthetized with ether, then subcutaneously injected with 0.1 mL of inactivated M. tuberculosis complete Freund's adjuvant in the left hind toe of the rat.
  • Primary arthritis occurs in the left hind paw of the mouse, and secondary arthritis occurs in the right hind paw around 13d.
  • the blank control group was injected with an equal volume of physiological saline.
  • the drug was administered after 13 days of modeling, wherein the methotrexate group was administered once every 5 days for 15 days for a total of 4 times; the high, medium and low doses of fusion protein 1 and protein 2 (128 mg/kg) 32 mg/kg, 8 mg/kg) was administered once every 5 days for 15 days.
  • a fat-soluble marker was used as a marker in the left and right posterior ankle joint of each rat, and the left and right hind legs of the animal were respectively immersed in the volume measuring device.
  • the immersion depth is bounded by the mark, and the reading value at the scale pipette of the device is the initial volume of the animal's left and right hind legs.
  • the day of modeling is counted as the 0th day, recorded as d0, and the volume of the left hind foot (modeling foot) is measured from the first day of modeling, every 2 days until the contralateral non-inflammatory foot (right hind foot)
  • the swelling occurs that is, secondary arthritis occurs
  • the volume of the left and right hind paws is measured every 2 days, and the degree of primary and secondary toe swelling is determined.
  • Arthritis index score After the appearance of secondary inflammation, the arthritis index score was performed every 2 days.
  • the initial body weight of each group of rats was weighed before modeling, and the body weight was measured every 2 days from the day of modeling, and the initial body weight was subtracted, which is the weight gain of each group of rats.
  • the experimental results are shown in Table 16.
  • the experimental results showed that after the model was established, the left hind paw was swollen rapidly (primary inflammation), and on the 13th day, the hind paw (non-contralateral inflammatory foot) began to be red and swollen (that is, secondary inflammation occurred), joint The inflammatory index and the whole body score began to rise, reaching the highest value on the 19th day, and gradually decreased with the swelling degree and score of each group.
  • the primary toe swelling degree was used to reflect the therapeutic effect of each treatment group on primary arthritis.
  • the high and medium doses of each administration group were able to treat primary arthritis to a certain extent compared with the model group.
  • the positive drug methotrexate had the best effect, and the fusion protein 1 and protein 2 had a good effect in the high dose group, which had a significant difference (**P ⁇ 0.01).
  • the treatment of secondary toe swelling reflected each treatment.
  • the activity of the integrin blocker polypeptide to inhibit proliferation of human retinal vascular endothelial cells was examined by MTT assay.
  • HRCEC cells were cultured in a 37 ° C, 5% CO 2 incubator to a density of 90% or more, and collected by trypsinization. The cells were resuspended in the culture medium and counted under a microscope to adjust the cell concentration to 3.0 ⁇ 10 4 /mL. The cell suspension was inoculated into a 96-well plate at 100 ⁇ L per well and cultured overnight at 37 ° C in a 5% CO 2 incubator. The polypeptide I, the polypeptide II, the polypeptide III, and the Avastin were diluted with the culture solution to respective predetermined concentrations.
  • each dilution was separately added to a 96-well plate at 100 ⁇ L per well.
  • the integrin blocker polypeptide was added as the administration group, and Avastin was used as a positive control group, and the culture medium without any drug was used as a blank control group, and incubated at 37 ° C, 5% CO 2 incubator for 48 hours. 20 ⁇ L of 5 mg/mL of MTT was added to each well of a 96-well plate, and incubation was continued for 4 hours.
  • the medium was aspirated and dissolved in 100 ⁇ L of DMSO per well.
  • the absorbance was measured at 570 nm with a microplate reader, and the growth inhibition (PI) was calculated.
  • the formula is as follows:
  • N test is the OD value of the test group and N control is the OD value of the blank control group.
  • fusion protein I and protein II significantly inhibited the proliferation inhibition of human retinal vascular endothelial cells (HRCEC) in a dose-dependent manner. At 64 ⁇ g/mL, the inhibition rate reached more than 50%.
  • CAM Chicken embryo chorioallantoic membrane
  • CAM assay was used to investigate the activity of fusion protein I and protein II in inhibiting angiogenesis in vivo.
  • biosynthesis rate of collagen reaches the maximum on the 8th to 11th day of chicken embryo development, which is the most vigorous phase of angiogenesis, and the body's immune system has not yet been fully established, so the development is selected to the 8th day.
  • the chicken embryos began to be administered.
  • the polypeptide on the drug-loaded paper has a certain diffusion range limitation on the chicken embryo chorioallantoic membrane, only the number of new blood vessels within a radius of 5 mm from the edge of the paper is counted in the test. Take the following steps:
  • Chicken embryo chorioallantoic membrane (CAM) analysis of fusion protein in vivo inhibition of angiogenic activity results negative control was treated with PBS, positive control Avastin dose was 10 ⁇ g, fusion protein I, protein two set high, medium and low dose treatment Chicken embryos were 128 ⁇ g, 32 ⁇ g, and 8 ⁇ g, respectively. The results are shown in Table 18.
  • mice Fifteen healthy BALB/c mice, male, weighing 20-25 g, were examined under the slit lamp microscope for the anterior segment of the eye and the appendage to exclude ocular lesions.
  • the alkali burn model was given to 0.3% ofloxacin eye drops 1 day before the preparation, 2 times a day. After the mice were anesthetized by intraperitoneal injection of 1.8% Avertin, the single-layer filter paper with a diameter of 2 mm was clamped with tweezers, immersed in a 1 mol/L sodium hydroxide solution to make it saturated, the excess liquid was removed, and the filter paper was placed.
  • the filter paper was discarded, and the burned area and the conjunctival sac were thoroughly washed with 15 mL of PBS for 1 min.
  • the cotton swab wipes away too much water, and under the operating microscope, the corneal epithelium is scraped off by the corneal scraping knife parallel to the limbus. Be careful not to damage the subepithelial stromal layer and the limbus, and apply the erythromycin eye ointment in the conjunctival sac. Prevent infection.
  • mice Fifteen mice were randomly divided into two groups: fusion protein group, fusion protein group 2 and control group, with 5 rats in each group. After alkali burn, 64 ⁇ g of fusion protein, 64 ⁇ g of fusion protein II and saline were injected intravitreally. The inflammatory reaction and neovascularization of the corneas in each group were observed under slit lamp microscope on the 7th, 7th, and 14th after every 7 days. On the 14th day after the alkali burn, the corneal neovascularization was recorded under the slit lamp microscope in the anterior segment of the eye. All the mice were sacrificed by cervical dislocation and the eyeballs were removed. The saline was washed with 4% paraformaldehyde. h, dehydrated in PBS containing 30% sucrose overnight, embedded in OCT frozen section embedding agent, stored in -80 ° C refrigerator, frozen section 8 ⁇ m, immunocytochemical detection of CD31 expression.
  • Microvessel density is an indicator for evaluating angiogenesis.
  • Standards for counting microvessels Microscopically, the endothelial cells or cell clusters that are clearly demarcated from adjacent tissues in the corneal tissue and stained brown or brown are counted in the neovascularization. The number of new blood vessels in the whole section was counted under a 10 ⁇ 20 microscope. After the corneal tissue was photographed, the area of the entire corneal tissue was calculated by image processing software Image J, and the density of new blood vessels in the whole section of the sample was determined. The results are shown in Table 19.
  • CD31 was used as a microvascular marker, which was mainly expressed in the cytoplasm of vascular endothelial cells.
  • the staining positive cells were stained brownish or brown by vascular endothelial cells without background staining.
  • the CD31-positive neovascularization of the fusion protein I and protein II experimental groups was significantly reduced compared with the control group. Fusion protein I and protein 2 were significantly different from the control group.
  • the experimental results show that both fusion protein and protein II can inhibit the growth of corneal neovascularization and can be used as a drug for the treatment of corneal neovascular diseases.
  • the 577nm argon ion laser was used to coagulate the main branch vein of rabbit retina, and venous occlusion was confirmed by fundus fluorescein angiography (FFA). After 5-12 days, the iris fluorescein angiography (IFA) showed that the fluorescein leakage was obvious in the iris vessels compared with the normal control group, confirming the formation of the iris neovascularization animal model (NVI).
  • FFA fundus fluorescein angiography
  • RESULTS Under the optical microscope, it was observed that the anterior surface of the iris was a fibrous vascular membrane residue mainly composed of fibrous tissue, and there were few open vascular lumens. Vascular residues can be seen in the iris matrix, which are necrotic cells and cell debris. The iris surface of the control eye under light microscopy is a fibrous vascular membrane with branched and potential lumens.
  • the ultrastructure of the iris in the treatment group is a series of degenerative changes.
  • the endothelial cells of the large blood vessels in the middle of the iris matrix have normal nucleus, cytoplasm and cell connections.
  • Capillary capillaries and degenerated wall cells without potential lumens indicate regression of neovascularization.
  • fusion protein-I and fusion protein II can inhibit neovascularization and degenerate formed blood vessels.
  • New Zealand white rabbits weighing 2.5-3.0 kg were randomly divided into 3 groups, which were labeled as control group, fusion protein group and protein two group.
  • White rabbits in each group were anesthetized with intramuscular injection after mixing with 35 mg/kg xylazine, and then anesthesia was maintained with an initial amount of half an intramuscular injection per hour. Increase the intraocular pressure of the left eye to 40 mmHg, which can reduce the blood flow to 1/3 of the normal value.
  • the right carotid artery was cannulated to the left ventricle for injection of microspheres (calculation of ocular blood flow), and the femoral artery cannula was used for blood collection.
  • Each group was intravitreally injected with physiological saline, 128 ⁇ g of fusion protein-128 ⁇ g of fusion protein 2, and the ocular blood flow of rabbit eyes with high intraocular pressure was measured by color microsphere technique at 0, 30, and 60 minutes after administration. At each time point, 0.2 mL (about 2 million) microspheres were injected. Immediately after the microspheres were injected, blood was collected through the femoral artery for 60 seconds, and placed in a heparinized anticoagulant tube, and the amount of blood collected was recorded.
  • fusion protein 1 and protein 2 doses were 64 ⁇ g or physiological saline (negative group), and retinal blood vessels were evaluated on the 17th day ( To label the unclosed blood vessels, 50 mL of Texas Red-labeled tomato lectin was injected into the left ventricle and circulated for 5 minutes). The experimental results are shown in Table 21.
  • a fluctuating oxygen-inducing animal model was adopted, and newborn rats (within 12 hours) spontaneously delivered on the same day were randomly divided into three groups: an oxygen model group, an oxygen-treated group, and a normal control group.
  • the oxygenation model was subdivided into three subgroup models and the treatment group was placed in a semi-closed oxygen chamber made of plexiglass.
  • the chamber was connected to medical oxygen, and the oxygen meter was adjusted to a concentration of 80% ⁇ 2%. After 24 hours, the oxygen was passed. Nitrogen gas was introduced into the cabin, and the oxygen concentration was adjusted to 10% ⁇ 2% and maintained for 24 hours. Repeatedly, the oxygen concentration in the oxygen chamber is maintained between 80% and 10% every 24 hours, and then transferred to the air for 7 days.
  • the oxygen concentration was monitored 8 times a day, and the ambient temperature in the control cabin was 23 °C ⁇ 2 °C.
  • the litter was replaced, fed, changed, and replaced.
  • the normal control group was placed in an animal house feeding environment. Compared with the control group, if the ADP staining of the retinal patch showed obvious vascular changes, the number of vascular endothelial cells that broke through the retinal inner membrane into the vitreous was increased, and the difference was statistically significant.
  • the oxygen-treated components were divided into two sub-groups. On the 7th day of modeling, the intravitreal injection was administered, and the fusion protein-1 and protein 2 were administered at a dose of 100 ⁇ g. The oxygen-administered model group and the control group were given only normal saline. Dosing for 1 week.
  • the eyeballs were removed and fixed in a 40 g/L paraformaldehyde solution for 24 hours.
  • the gradient alcohol is dehydrated and the xylene is transparent.
  • the slices were continuously cut to a thickness of 4 ⁇ m, and the surrounding of the optic disc was avoided as much as possible.
  • the slices are parallel to the sagittal plane of the cornea to the optic disc.
  • Ten eyes were randomly selected from each eyeball to be stained with hematoxylin and eosin, and the number of vascular endothelial cells that broke through the inner limiting membrane of the retina was counted (only the vascular endothelial cells nucleus closely related to the inner limiting membrane were counted), and the average per eyeball was counted. The number of cells sliced.
  • RESULTS No vascular endothelial cell nuclei that broke through the intraretinal membrane into the vitreous were found in the control group. In the model group, there were more vascular endothelial cell nuclei that broke through the inner limiting membrane of the retina. Some of them appeared alone, some clustered, and some vascular endothelial cells near the deep retinal vessels were also seen on some sections, confirming that they originated from the retina instead of the vitreous or eye. Other organizations. Only a few vascular endothelial cell nuclei that broke through the inner limiting membrane of the retina were seen in the treatment group. The experimental results are shown in Table 22.
  • mice were modeled with streptozotocin STZ.
  • STZ was dissolved in 0.1 mol/L, pH 4.5 citrate buffer to prepare a 2% solution. All experimental Wistar rats were fasted for 12 h before injection, and each rat was intraperitoneally injected with 2% STZ solution at a dose of 65 mg/kg. After the injection, the animals were kept in a single cage, and urine sugar and blood sugar were detected at 48 hours. Urine sugar is above +++, and blood sugar is higher than 16.7mmol/L.
  • the model of diabetic retinopathy was successfully modeled by blood glucose, urine glucose, urine output and retinal VEGF immunohistochemistry.
  • mice Fifteen rats were randomly divided into three groups, which were labeled as control group, fusion protein-treatment group and fusion protein two treatment group.
  • the control group was injected with normal saline (0.1 mL), and the fusion protein I and the fusion protein were all administered with 100 ⁇ g (0.1 mL) once every 7 days for 2 weeks, 4 weeks, 8 weeks. Observed on the 12th week.
  • the experimental results are shown in Table 23.
  • the retinal tissue of the experimental group increased the thickness of each layer of retinal tissue compared with the control group. Compared with the control group, the number of retinal ganglion cells in the experimental group was increased compared with the control group. It indicated that both fusion protein and protein II could have a certain therapeutic effect on diabetic retinopathy at 100 ⁇ g dose.

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Abstract

本发明公开了一种融合蛋白、其制备方法及其在治疗眼科疾病、抗炎、抗肿瘤药物中的应用，属于生物制药技术领域。所述融合蛋白采用一种柔性(F)或刚性(R)的Linker将两种血管生成抑制多肽HM-3、IL-4与免疫球蛋白Fc片段通过氨基酸Linker连接而成，通过基因工程的方法在真核细胞表达，并通过亲和层析等方法纯化获得。

Description

一种融合蛋白及其制备方法和其在制备治疗眼科疾病、抗炎、抗肿瘤药物中的应用 技术领域
本发明属于生物制药技术领域，更具体地说，涉及一种具有抗炎、抗肿瘤、治疗眼科疾病功能的融合蛋白及其制备方法和应用。
背景技术
关节炎、肿瘤、细菌引起的炎症、眼科疾病(如AMD)等疾病均被称为血管相关性疾病。
关节炎类炎症疾病，是指发生在人体关节及其周围组织的炎性疾病，可分为数十种。我国的关节炎患者有1亿以上，且人数在不断增加。临床表现为关节的红、肿、热、痛、功能障碍及关节畸形，严重者导致关节残疾、影响患者生活质量。其中主要包括风湿性关节炎、类风湿关节炎、骨关节炎、痛风性关节炎、强直性脊柱炎、反应性关节炎、感染性关节炎等。这其中类风湿关节炎(Rheumatoid arthritis，RA)是临床最常见的炎性关节病和主要致残因素之一。在全世界约为0.5％-1.0％，RA的发病在我国约为0.4％，是一种病因尚未明了的慢性全身性炎症性疾病，以慢性、对称性、多滑膜关节炎和关节外病变为主要临床表现，属于自身免疫炎性疾病。患者常以手部或腕部疼痛及肿胀(特别是腕背部的肿胀)为首发症状，症状持续不缓解，普通的对症治疗虽可以缓解症状，但常常由于用药不规则或不足量而导致症状反复。病情进展时可以出现明显的晨僵，通常可达1小时以上，并不断加重；同时出现一定的关节功能障碍。其基本病理特点是血管炎和滑膜炎。关节内滑膜血管增生，形成血管翳，导致滑膜增厚，渗出增多，分泌多种细胞因子，侵犯软骨并引起骨质损害。对其周围的肌腔、韧带、腱鞘以及肌肉等组织也均可侵蚀，从而影响关节的稳定，容易发生关节畸形而出现功能障碍。血管炎亦可侵犯周身各脏器组织，形成***性疾病。关节炎的病理过程中，血管新生是一种标志性的组织学改变，新生血管形成伴随滑膜增生和炎细胞浸润，是其中血管翳形成及关节破坏的基础。原来应该没有血管存在的关节软骨因某种异常变化，而形成新的血管，使得软骨受到侵蚀，造成关节变形或疼痛。新生血管引起类风湿关节炎患者滑膜组织发生异常变化。所以抑制新生血管形成在一定程度上可以缓解或治愈关节炎类炎症疾病。
近年我国肿瘤的发病率和病死率不断增长。无限制生长、侵袭和转移是肿瘤的恶性标志和特征，也是导致治疗失败和死亡的主要原因。因此，控制肿瘤的生长、侵袭和转移是改善预后，提高生存率的主要措施。1971年Folkman首先提出肿瘤生长依赖血管新生的理论，肿瘤血管生成是肿瘤生长和转移的形态学基础，它不仅向肿瘤提供营养，也向宿主输出大量的 肿瘤细胞导致肿瘤的生长和转移。绝大多数恶性实体肿瘤如卵巢癌、肝癌、***和乳腺癌等都是血管依赖性肿瘤。新生血管一方面为肿瘤生长提供营养和氧气，另一方面还是肿瘤转移的重要途径。因此，抑制肿瘤血管形成是重要的抗癌措施。
眼科类疾病中的虹膜新生血管性眼病、脉络膜新生血管性眼病、视网膜新生血管性眼病及角膜新生血管性眼病等，其发病机理均与新生血管的过度形成有关，抑制新生血管的形成是治疗这类疾病的关键，而内皮细胞的增殖和迁移是新生血管形成的关键步骤。血管生成抑制剂是近年在治疗新生血管性疾病中引起重视的一类药物，因此阻断新生血管形成可能成为治疗由于眼中血管生成导致的患者眼病的新手段。在这些血管生成抑制剂中，尤其以血管抑素和内皮抑素最引人注目。尽管这些血管抑制剂呈现出非常诱人的前景，但其缺陷也非常明显：迄今为止的抑制血管生成药物，如内皮抑素、血管抑素等作用靶点不明确，它们对血管的专一性和选择性还不够好，效果有限，导致实验中用药量较大。因此，一个好的抗血管生成药物应该对新生血管的标记分子具有选择性，以达到对新生血管的导向性作用，从整体上提高药物对血管生成的抑制作用：做到只使用低剂量的药物，就能达到高效的抑制血管生成的效果。Avastin目前已经成功用于眼部疾病的治疗，而我国尚缺乏自主研制的此类药物。而本发明的整合素靶点抑制血管生成将成为治疗这类眼病的新选择。
此外，关节炎类炎症、肿瘤、眼病都是与血管相关的疾病。肿瘤的生长和转移依赖新生血管；炎症与血管新生是两个相互联系,共同发展的病理过程；眼科疾病如年龄相关性黄斑变性(AMD)主要表现是脉络膜新生血管的生成。
新生血管生成，在正常生理条件下，受到高度调节，在生殖、胚胎发育、组织修复和创伤愈合中是必不可少的过程。血管生成在多种病理条件下也会发生，所述病理条件包括：肿瘤生长和转移；炎性障碍，例如类风湿性关节炎、银屑病、骨关节炎、炎性肠病、克隆病、溃疡性结肠类以及其它炎性障碍。
整合素是一类广泛分布于细胞表面的受体，能介导细胞与细胞外基质及细胞与细胞之间的相互黏附，它们通过连接胞内细胞骨架蛋白和细胞外基质分子的相互作用参与血管生成。目前至少有8种的整合素(α1β1、α2β1、α3β1、α6β1、α6β4、α5β1、αvβ3、αvβ5)参与血管生成，其中αvβ3发挥着重要作用。αvβ3能识别配体分子中的精-甘-天冬序列(arg-gly-asp，RGD)。αvβ3可以表达于多种细胞类型，并与多细胞活动过程中的多种配体结合参与肿瘤的血管生成、侵袭、转移、炎症、伤口愈合和凝血等生理和病理过程。因此，含有RGD序列的多肽具有整合素拮抗剂作用，RGD序列可以作为一种载体，靶向运输到新生血管内皮，从而对新生血管性疾病达到更高效率的治疗。因此，血管抑制多肽通过抑制血管生成不仅可阻止向滑膜输送氧气和营养物质，且直接导致血管退化，从而可能抑制RA滑膜增生。抑制新生 血管的形成是治疗这类疾病的关键，而内皮细胞的增殖和迁移是新生血管形成的关键步骤。
发明内容
1.要解决的问题
针对现有多肽半衰期短，靶点单一的问题，本发明提供一种哺乳动物细胞表达方法，将两种不同的活性多肽及蛋白质进行连接期望解决：1、提高多肽分子与靶点的亲和力、细胞毒，增强多肽分子疗效。2、克服多肽分子半衰期短，频繁给药的缺点。3、将针对不同靶点的多肽通过linker链接，形成双功能蛋白分子，同时延长双功能蛋白的半衰期，为长效药物开发打下基础。
2.技术方案
为了解决上述问题，本发明所采用的技术方案如下：
一种融合蛋白，融合蛋白分子中含有血管抑制多肽HM-3序列，白介素4(IL-4)序列以及抗体IgG1的Fc片段；其中蛋白一由一种柔性(F)Linker链接，两端多肽可变化移动，从而有更好的延展性；蛋白二由一种刚性(R)Linker链接，不易使两端的多肽形成空间位移，从而使两端功能域不相互影响。蛋白一的结构示意图如附图1所示；蛋白二的结构示意图如附图2所示。
进一步地，蛋白一对应的氨基酸序列为SEQ ID NO.1；蛋白二对应的氨基酸序列为SEQ ID NO.2。
编码上述的融合蛋白的基因，编码序列SEQ ID NO.1、SEQ ID NO.2的核酸序列分别为SEQ ID NO.3、SEQ ID NO.4。
上述的融合蛋白在制备治疗炎症、抗肿瘤和治疗眼科疾病药物中的应用。
进一步地，所述的炎症包括类风湿关节炎、骨关节炎、痛风性关节炎、强直性脊柱炎、银屑病关节炎、反应性关节炎、感染性关节炎和创伤性关节炎、***性红斑狼疮、银屑病。
进一步地，所述的肿瘤包括胃癌、肺癌、肝癌、乳腺癌、结肠癌、胶质瘤、黑色素瘤和***以及起源于人的头颈部、脑部、甲状腺、食管、胰腺、肺脏、肝脏、胃、乳腺、肾脏、胆囊、结肠或直肠、卵巢、子宫颈、子宫、***、膀胱、睾丸的原发或继发的癌、黑色素瘤以及肉瘤。
进一步地，所述的眼科疾病包括虹膜新生血管性眼病、脉络膜新生血管性眼病、视网膜新生血管性眼病或角膜新生血管性眼病。
进一步地，所述的虹膜新生血管性眼病包括新生血管性青光眼、糖尿病视网膜病变或视网膜中央静脉栓塞引起的虹膜新生血管性眼病；所述的脉络膜新生血管性眼病包括年龄相关性黄斑变性、中心性渗出性视网膜脉络炎、眼组织胞浆菌病综合征或葡行性脉络膜病变脉络 膜新生血管性眼病；所述的视网膜新生血管性眼病包括糖尿病、肿瘤、视网膜脱落、视网膜中央静脉阻塞、视网膜静脉周围炎、全身性红斑狼疮、Eales病或Coat病相关的视网膜新生血管性眼病；所述的角膜新生血管性眼病包括角膜接触镜所致角膜新生血管性疾病，以及碱及其他化学物质烧伤、角膜手术、细菌感染、衣原体感染、病毒感染或原虫感染引起的角膜新生血管性眼病。
进一步地，药物的剂型为胶囊、片剂、药丸、注射剂、鼻喷剂或气雾剂。
上述的融合蛋白的制备方法，利用原核细胞或真核细胞表达及纯化方法。
3.有益效果
相比于现有技术，本发明的有益效果为：
(1)HM-3经过大量研究，发现具有很好的抗血管新生效果，但因为是多肽，半衰期较短；IL-4分子可以促进T细胞、B细胞以及巨噬细胞增殖，从而增强机体的免疫功能，达到杀伤肿瘤的作用。本发明一方面将二者结合，可以起到双重抗肿瘤作用，抗炎作用及抗血管新生作用，同时提高机体免疫力。另外HM-3和白介素4均为分子量较小的多肽，在体内半衰期比较短，通常均能达到30分钟-2个小时左右，本发明引入了IgG的Fc片段序列，通过融合蛋白构建技术，将HM-3、IL-4和IgG1的Fc片段通过Linker链接在一起构成了双功能融合蛋白，该融合蛋白具有HM-3多肽的抗血管新生和白介素4的抗肿瘤的双重效果，更为重要的是加入了Fc片段显著延长了HM-3和白介素4的半衰期，预期产品成药后在相关领域疾病治疗中为患者提供一种长效的双功能药物，达到1周或2周，甚至1个月给药一次，有效的降低了患者的用药频率，增加了患者的药物顺应性、降低了患者的治疗成本。
(2)本发明解决了：①分子量大、结构复杂的多肽分子合成瓶颈，特别是具有二硫键等二级结构和高级结构的大分子多肽分子；②克服了大分子量多肽化学合成的合成困难、收率低等技术瓶颈，显著降低大分子多肽的生产成本；③哺乳动物细胞等生物体细胞表达多肽分子，可以形成正确的高级结构，多肽分子与靶点的亲和力等将优于化学合成的多肽分子；④多肽分子与抗体IgG1、IgG2或IgG4的Fc片段形成融合蛋白分子，IgG的Fc片段通过与Fc受体(FcRn)介导的再循环机制避免被降解，与此同时Fc片段分子量较大，肾清除率低，也保证了融合蛋白比多肽半衰期显著增长。；与此同时IgG1的Fc片段融合形成的融合蛋白还可以增加ADCC和CDC细胞毒，可显著提高抗肿瘤分子的活性，其抗肿瘤药效优于多肽分子；⑤采用真核表达***，通过linker对抗体Fc片段与HM-3、IL-4序列进行连接，延长功能蛋白HM-3、IL-4的半衰期，同时将两种不同靶点的多肽进行连接，形成了双靶点融合蛋白；
(3)本发明的蛋白一(SEQ ID NO.1)和蛋白二(SEQ ID NO.2)是一类整合素阻断剂 类和免疫调节的重组蛋白药物，不仅具有抑制血管增生，同时还具有有效调节免疫***抑制肿瘤的功能；该双功能融合蛋白药物相比较单个多肽药物可显著增强药物对抑制和杀伤肿瘤细胞，并抑制血管增生的作用。更为重要的是融合蛋白可以显著增加药物的半衰期，可以提高药物的治疗效果，更能显著提高患者的药物顺应性，并显著降低患者的治疗费用。该融合蛋白药物通过以上双功能作用和半衰期延长达到治疗关节炎、抗肿瘤和炎症相关的眼科疾病的功能。
(4)本发明中的融合蛋白序列包括精氨酸-甘氨酸-天冬氨酸(RGD)序列，RGD序列是整合素的一个重要配体，含有RGD序列的多肽Gly-Gly-Gly-Gly-Arg-Gly-Asp能够特异性的识别整合素，能有效抑制新生血管生成，可用于***疾病、关节炎类疾病与眼科类疾病；本发明采用一种柔性(S)或刚性(R)的Linker将两种多肽融合分别得到蛋白一和蛋白二，能提高药效，延长半衰期，增强稳定性，并且具有作用效果强，毒性小等特点；
(5)本发明的融合蛋白可以靶向到新生血管内皮，抑制新生血管形成，进而达到预防或治疗血管及炎症相关疾病的效果；
(6)本发明在抗肿瘤方面，具有抑制多种肿瘤的功效，通过实施例2中MTT实验结果，融合蛋白一、融合蛋白二能有效的抑制胃癌、肺癌、肝癌、乳腺癌、黑色素瘤、结肠癌、胶质瘤和***的增殖，在32μg/mL浓度下，对黑色素瘤、胃癌、人脑胶质瘤抑制率达到50％以上；64μg/mL浓度下，对结肠癌细胞抑制率达到40％以上，对***细胞抑制率达到50％以上；
(7)本发明在抑制新生血管形成方面，通过实施例3细胞迁移实验可明显看到2μg/mL浓度下对HUVEC的迁移抑制有显著作用，抑制率为70％以上；
(8)本发明在抗炎症效果方面，通过实施例4-10的一系列验证模型实验可得，融合蛋白一和融合蛋白二能显著抑制淋巴细胞增殖、抑制巨噬细胞产生IL-1β炎症因子、抑制肉芽肿形成、降低模型组毛细血管通透性、抑制模型组耳肿胀及足趾肿胀、并降低大鼠佐剂关节炎慢性炎症程度；
(9)本发明在治疗眼科疾病方面，通过实施例11-19可知，融合蛋白一和融合蛋白二能显著抑制人视网膜血管内皮细胞增殖、抑制鸡胚***新生血管生成、抑制角膜新生血管生长、抑制家兔虹膜新生血管生长、促进兔眼脉络膜血流量增加、减少OIR小鼠视网膜新生血管丛、抑制氧诱导新生鼠视网膜病变模型新生血管的形成、并对糖尿病视网膜病变产生一定的治疗作用。
附图说明
图1为本发明中SEQ ID NO.1对应的蛋白的结构示意图；
图2为本发明中SEQ ID NO.2对应的蛋白的结构示意图；
图3为本发明中Fc-IL4DM-HM3-1和表达载体pcDNA3.4/MCS(+)胶回收获得片段电泳图；
图4为本发明中菌液PCR验证结果图；
图5为本发明中融合蛋白捕获结果图；
图6为本发明中融合蛋白的精细纯化图；
图7为本发明中SDS-PAGE方法对融合蛋白样品的分析结果图；
图8为本发明中HPLC对融合蛋白样品的分析结果图。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
实施例1
(一)融合蛋白基因获得及表达载体的构建
血管抑制多肽HM-3序列如SEQ ID NO.5所示，白介素4序列如SEQ ID NO.6所示，将人免疫球蛋白IgG1-Fc区(SEQ ID NO.7)通过不同的连接肽Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser柔性(F)linker、AlaGluAlaAlaAlaLysGluAlaAlaAlaLysGluAlaAlaAlaLysGluAlaAlaAlaLysAla刚性(R)linker，与IL4DM-HM3蛋白连接，设计出2种新型Fc融合蛋白Fc-IL4DM-HM3，以柔性(F)linker构建的蛋白一的氨基酸序列如SEQ ID NO.1所示，以刚性(R)linker构建的蛋白二的氨基酸序列如SEQ ID NO.2所示，根据CHO细胞密码子偏爱，对2种新型Fc融合蛋白Fc-IL4DM-HM3的编码序列进行优化，并全部在3`端引入NheI酶切位点、Kozak序列、信号肽，在5`端引入XhoI酶切位点，通过全基因合成的方法获得如下DNA序列SEQ ID NO.3、SEQ ID NO.4。
委托生物公司合成上述2种融合蛋白Fc-IL4DM-HM3的DNA序列，连接在pUC57载体上形成克隆载体，保存于E.coli DH5α中形成克隆菌株。2种融合蛋白均使用pcDNA3.4/MCS(+)作为表达载体，载体构建过程完全一致，因此以Fc-IL4DM-HM3-1为例，实验过程如下。
1、在无菌条件下，将生物公司寄送的Fc-IL4DM-HM3-1克隆菌株从穿刺菌表面挑起，接入2支含5mL Amp抗性LB培养基的试管，37℃，120rpm震荡过夜培养。
2、经过培养的2支试管的菌液，1支试管加入2.5mL无菌60％甘油，混匀后，按1mL/支分装至无菌离心管，制成甘油管，-80℃冻存。另1支试管中的菌液，12000rpm离心1min收集菌体，使用常规的商业质粒小量提取试剂盒提取Fc-IL4DM-HM3-1的克隆载体。
3、使用限制性内切酶NheI/XhoI，对Fc-IL4DM-HM3-1克隆载体和表达载体pcDNA3.4/MCS(+)进行双酶切，通过水平核酸电泳分离具有粘性末端的***片段Fc-IL4DM-HM3-1和表达载体pcDNA3.4/MCS(+)，使用商业DNA胶回收试剂盒进行回收。 DNA片段回收结果如图3所示。
4、使用T4连接酶，按照***片段与载体摩尔比为1：5的比例，将胶回收获得的***片段Fc-IL4DM-HM3-1和表达载体pcDNA3.4/MCS(+)在16℃进行连接反应，持续时间16h。
5、取20uL DNA连接，加入100uL刚刚融化的E.coli TOP10感受态细胞中，轻轻混匀，冰浴30min。随后混合物在42℃热激45s后，迅速冰浴2～3min。向混合物中加入900uL无抗性LB培养基，37℃震荡培养1h。在4℃条件下，4500rpm离心1min，无菌条件下弃900uL上清液，将剩余菌液同沉淀菌体轻轻吹吸混匀，移液枪全部吸出，涂布于Amp抗性LB固体平板，37℃静止培养12h。
6、挑取20个单菌落，接种于含5mL Amp抗性LB培养基的试管，37℃，120rpm震荡过夜培养。
7、将上一步接种的菌株中，正常生长的菌株，各保存3支甘油管。同时对每一株菌进行菌液PCR验证(见图4)，筛选出阳性克隆，将保存的甘油管寄送生物技术公司进行测序验证。最终获得正确的表达载体。
8、取出测序正确的菌株保存的甘油管，接入含30mL Amp抗性LB培养基的250mL摇瓶中，37℃，120rpm过夜培养，保存20支甘油管，-80℃冻存。至此完成Fc-IL4DM-HM3-1表达载体构建工作。
(二)融合蛋白表达
瞬时转染是将DNA导入真核细胞的方式之一。在瞬时转染中，重组DNA导入感染性强的细胞系以获得目的基因暂时但高水平的表达。可以在较短的时间内获得足够的蛋白用于实验，节约了稳定转染中细胞筛选时间。使用Expi293 Expression System瞬时转染表达***，表达2种新型融合蛋白Fc-IL4DM-HM3，由于融合蛋白表达过程完全一致，因此以Fc-IL4DM-HM3-1为例，实验过程如下。
1、质粒制备。
从-80℃冰箱中取一支Fc-IL4DM-HM3-1表达载体保存菌株甘油管，接入含500mL Amp抗性LB培养基的2L摇瓶，37℃，160rpm过夜震荡培养。
培养结束后，5000g离心5min收集菌体，使用商业化的除内毒素质粒大提试剂盒提取质粒。将质粒浓度控制在1mg/mL以上(若低于此浓度，需要浓缩)，再使用无菌0.22μm孔径滤膜过滤除菌，完成质粒制备。
2、瞬时转染细胞前期准备工作
用于转染的293F细胞，从复苏当天算起，每培养四天按照0.4*10 6cells/mL细胞密度进行传代，至少进行三次传代再进行瞬时转染。传代过程中，根据最终转染培养基体积，按需 扩大传代体积。
3、瞬时转染(以30mL转染体积为例，根据需要成倍增加)
(1)实验前一天，将6*10 7的活细胞接入30mL Expi293 Expression Medium，37℃，8％CO 2，125rpm震荡培养。
(2)实验当天，先对前一天培养的细胞计数，细胞密度应为3-5*10 6cells/mL，活率大于95％。
(3)将7.5*10 7cells吸入新的125mL锥形瓶，补加预热的Expi293 Expression Medium至25.5mL。
(4)准备质粒-转染试剂混合液
①将30μg质粒DNA复溶于1.5mL的Opti-MEM I Reduced Serum Medium，轻柔混匀。
②将81μL的ExpiFectamine 293 Reagent加入Opti-MEM I Reduced Serum Medium，定容至1.5mL。轻柔混匀，室温孵育5min(长时间孵育影响转化效率)。
③将上述两种溶液混合，轻柔混匀，室温孵育20-30min。完成质粒-转染试剂混合液准备工作。
(5)将3mL质粒-转染试剂混合液加入第3步的细胞培养液中，共28.5mL。
(6)37℃，8％CO 2，125rpm震荡培养20h。
(7)加入150μL的ExpiFectamine 293 Transfection Enhancer 1和1.5mL的ExpiFectamine 293 Transfection Enhancer 2。至此，总体积为30mL。
(8)37℃，8％CO2，125rpm震荡培养。在6天结束培养，进行蛋白纯化。
(三)融合蛋白纯化
Protein A是一种分离自金黄色葡萄球菌的细胞壁蛋白，主要通过Fc片段结合哺乳动物IgG，具有极高的特异性和结合能力，广泛用于IgG抗体和IgG-Fc融合蛋白的纯化。2种新型融合蛋白Fc-IL4DM-HM3-1，由于都具有IgG-Fc片段，纯化过程完全一致，因此以1.6L规模瞬时转染生产Fc-IL4DM-HM3-1为例，实验过程如下。
1、样品预处理：将1.6L结束培养的瞬时转染细胞培养液在4℃条件下，以7500rmp离心20min，所获得上清液约1.46L用于下一步protein A捕获。
2、目的蛋白亲和捕获(如图5)
色谱柱信息如下
Figure PCTCN2018077968-appb-000001
Figure PCTCN2018077968-appb-000002
①首先用500mL的0.2M NaOH进行灭菌，流速10mL/min；
②20mM PB，0.15M NaCl，pH7.0平衡色谱柱，体积约为1000mL，流速20mL/min；
③上样：样品预先调节pH至中性，流速20mL/min；
④20mM PB，0.15M NaCl，pH7.0冲洗色谱柱，约800mL，流速20mL/min。
⑤50mM柠檬酸-柠檬酸钠，0.15M NaCL pH3.0洗脱目的蛋白，起峰20mAu开始收集，峰后20mAu停止收集；流速20mL/min。
⑥色谱柱最后用0.2M NaOH清洗500mL，用水冲洗至中性后，用20％乙醇保存色谱柱。
⑦色谱柱最后用0.2M NaOH清洗500mL，用水冲洗至中性后，用20％乙醇保存色谱柱。
3、凝胶层析进行进一步的分离纯化(如图6)
色谱柱参数：
Figure PCTCN2018077968-appb-000003
①用0.5M NaOH 300mL进行灭菌，流速10mL/min，后用超纯水冲洗至约中性。
②用PBS缓冲液，pH7.4平衡色谱柱，平衡体积约为1500mL，流速10mL/min。
③上样，样品为proteinA洗脱液，上样量40mL；4)收集样品，峰3为目的蛋白峰，收集峰3，起峰10mAu开始收集，峰后10mAu停止收集。
④最后用0.1M NaOH保存色谱柱，流速10mL/min。
4、样品超滤浓缩：对峰3的样品进行合并超滤浓缩，超滤膜选用10kDa，样品浓缩至目的蛋白浓度大于5mg/mL，然后分装样品，于-80℃冰箱保存。初始浓度约0.29mg/mL，最终浓缩至27mL，浓度约为5.53mg/mL；分装样品，冻存。与此同时，采用SDS-PAGE和HPLC等方法对样品进行放行检测(图7和图8)，然后用于成药性评价研究。
实施例2
融合蛋白对多种肿瘤细胞增殖抑制作用
采用MTT法检测实施例1中得到的整合素阻断剂融合蛋白对多种肿瘤细胞增殖的活性抑制作用，包括黑色素瘤细胞B16F10、胃癌细胞MGC-803、肺癌细胞A549、肝癌细胞Hep-G2、乳腺癌细胞MDA-MB-231、结肠癌细胞HCT-116、人脑胶质瘤U87、***细胞Hela。
将肿瘤细胞在37℃、5％CO 2的培养箱中培养至密度90％以上时用胰蛋白酶消化收集，用培养液重悬细胞并在显微镜下计数，将细胞浓度调整为3.0×104个/mL，将细胞悬液接种到96孔板中，每孔100μL，并于37℃，5％CO 2培养箱中培养过夜。将融合蛋白一、融合蛋白二、阳性药紫杉醇Taxol用培养液稀释到各个预定浓度。待细胞完全贴壁后，将各个稀释液分别加入96孔板中，每孔100μL。以加入整合素阻断剂融合蛋白一、蛋白二作为给药组，Taxol作为阳性对照组，以不加任何药物的培养液作为空白对照组，在37℃，5％CO 2培养箱孵育48小时。向96孔板中每孔加入20μL 5mg/mL的MTT，继续培养4小时。吸去培养基，每孔加入100μL DMSO溶解。用酶标仪在570nm下检测，参比波长为630nm处测定吸光值，并计算生长抑制率(proliferation inhibition，PI)，公式如下：
Figure PCTCN2018077968-appb-000004
其中N test为测试组的OD值，N control为空白对照组的OD值。
数据统计：
试验独立重复5次，试验得到的结果计算mean±SD，并进行统计t-test检验，P<0.05为显著性差异，P<0.01为极显著性差异。实验结果见表1-8。
表1 蛋白一、蛋白二对黑色素瘤细胞B16F10增殖抑制作用
Figure PCTCN2018077968-appb-000005
Figure PCTCN2018077968-appb-000006
*P<0.05，**P<0.01vs control.
结果显示，融合蛋白一、蛋白二能有效抑制黑色素瘤细胞B16F10，在8μg/mL浓度下，抑制率达到40％以上。
表2 蛋白一、蛋白二对胃癌细胞MGC-803增殖抑制作用
Figure PCTCN2018077968-appb-000007
Figure PCTCN2018077968-appb-000008
*P<0.05，**P<0.01vs control.
结果显示，融合蛋白一、蛋白二能有效抑制胃癌细胞MGC-803，在32μg/mL浓度下，抑制率达到50％以上。
表3 蛋白一、蛋白二对肺癌细胞A549增殖抑制作用
Figure PCTCN2018077968-appb-000009
*P<0.05，**P<0.01vs control.
结果显示，融合蛋白一、蛋白二能有效抑制肺癌细胞A549，在128μg/mL浓度下，抑制率达到45％以上。
表4 蛋白一、蛋白二对肝癌细胞Hep-G2增殖抑制作用
Figure PCTCN2018077968-appb-000010
*P<0.05，**P<0.01vs control.
结果显示，融合蛋白一、蛋白二对肝癌细胞Hep-G2有一定抑制作用，抑制率随浓度增加而提高。
表5 蛋白一、蛋白二对乳腺癌细胞MDA-MB-231增殖抑制作用
Figure PCTCN2018077968-appb-000011
Figure PCTCN2018077968-appb-000012
*P<0.05，**P<0.01vs control.
结果显示，融合蛋白一、蛋白二能有效抑制乳腺癌细胞MDA-MB-231，在128μg/mL浓度下，抑制率达到40％以上。
表6 蛋白一、蛋白二对结肠癌细胞HCT-116增殖抑制作用
Figure PCTCN2018077968-appb-000013
Figure PCTCN2018077968-appb-000014
*P<0.05，**P<0.01vs control.
结果显示，融合蛋白一、蛋白二能有效抑制结肠癌细胞HCT-116，在64μg/mL浓度下，抑制率达到40％以上。
表7 蛋白一、蛋白二对人脑胶质瘤U87增殖抑制作用
Figure PCTCN2018077968-appb-000015
Figure PCTCN2018077968-appb-000016
*P<0.05，**P<0.01vs control.
结果显示，融合蛋白一、蛋白二能显著抑制人脑胶质瘤U87，在32μg/mL浓度下，抑制率达到50％以上。
表8 蛋白一、蛋白二对***细胞Hela增殖抑制作用
Figure PCTCN2018077968-appb-000017
*P<0.05，**P<0.01vs control.
结果显示，融合蛋白一、蛋白二能显著抑制***细胞Hela，在64μg/mL浓度下，抑制率达到55％左右。
综合以上，融合蛋白一、蛋白二整合素阻断剂对多种肿瘤细胞增殖抑制作用见表1-8，融合蛋白能有效的抑制胃癌、肺癌、肝癌、乳腺癌、黑色素瘤、结肠癌、胶质瘤和***的增殖。其中，在32μg/mL浓度下，对黑色素瘤、胃癌、人脑胶质瘤抑制率达到50％以上；64μg/mL浓度下，对结肠癌细胞抑制率达到40％以上，对***细胞抑制率达到50％以上；对肺癌、肝癌、乳腺癌细胞需要较高浓度才能达到有效抑制。
实施例3
三维transwell法检测融合蛋白一、蛋白二抑制人脐静脉内皮细胞迁移的活性
人脐静脉内皮细胞(HUVEC)用含5％胎牛血清和1×ECGS的内皮细胞培养液，在37℃、5％CO 2的培养箱中培养至90％以上的汇合度时，采用transwell法检测融合蛋白一、蛋白二的抑制内皮细胞迁移的活性，内皮细胞HUVEC只用第2-8代，具体操作如下：
(1)用10mg/mL Matrigel用DMEM培养基以1:4稀释，涂布于transwell小室膜上，室温风干；
(2)将培养到对数生长期的HUVEC细胞用0.2％EDTA消化，收集，用PBS洗涤两次后用含有0.1％BSA的内皮细胞培养液重悬，在显微镜下计数，将细胞浓度调整到1×105个/mL；
(3)配制各组的试验用液，用含0.1％BSA的细胞培养液稀释到100μL；
分组如下：
空白对照组：为不含药物的细胞培养液；
恩度组：为用不含药物的细胞培养液将5mg/mL的恩度药液稀释到预定浓度；
融合蛋白组：为用不含药物的细胞培养液将融合蛋白稀释到10μg/mL；
(4)将细胞接种到transwell小室中，每孔100μL，并且将各组试验用液加入小室中。24孔板中加入0.6mL含5％胎牛血清和1×ECGS的内皮细胞培养液刺激细胞迁移，于5％CO 2，37℃孵育24h；
(5)弃去孔中培养液，用90％酒精常温固定30min，0.1％结晶紫常温染色10min，清水漂净，用棉签轻轻擦掉上层未迁移细胞，显微镜下观察并选择四个视野拍照计数，按照公式计算迁移抑制率(migration inhibition，MI)：
Figure PCTCN2018077968-appb-000018
其中N test为测试组的细胞迁移数，N control为空白对照组的细胞迁移数。
数据统计：
试验独立重复3次，试验得到的结果计算mean±SD，并进行统计t-test检验，P<0.05为显著性差异，P<0.01为极显著性差异。实验结果见表9。
表9 蛋白一、蛋白二对HUVEC的迁移抑制
Figure PCTCN2018077968-appb-000019
*P<0.05，**P<0.01vs control.
由实验结果可见在融合蛋白一、蛋白二的作用下，迁移的内皮细胞数与阴性对照相比显著减少，2μg/mL浓度下对HUVEC的迁移抑制有显著作用，抑制率为70％以上，对细胞迁移的抑制率与阴性对照相比有极显著性的差异(P<0.01)，浓度在0.5μg/mL～4μg/mL之间时抑制效果最好。
实施例4
融合蛋白一、蛋白二对小鼠脾脏淋巴细胞增殖的影响
无菌条件下取出小鼠脾脏，空1640培养基清洗3次，5mL注射器芯研磨，200目筛网过滤，制成单细胞悬液，离心(1000rpm、5min)，弃上清，Tris-NH 4Cl破解红细胞，冰水浴静置4min，离心(1000rpm、5min)，弃上清，用无菌PBS洗涤细胞两次。最后加入10％小牛血清的RPMI 1640培养液(5mL)悬浮细胞，细胞计数，调整细胞浓度为5×106个/mL，于96孔培养板中培养。
实验设空白对照组、刀豆蛋白A(ConA)组、***(Dex)组(0.02mg/mL)、蛋白一和蛋白二为实验组。各组分别加入脾脏淋巴细胞悬液100μL/孔后，空白对照组加入空1640培养液100μL，ConA组加入ConA(终浓度为5μg/mL)，Dex组加入Dex，蛋白一和蛋白二在加入不同浓度提取物的基础上加ConA(终浓度为5μg/mL)。37℃细胞培养箱静止培养48h，培养结束后每孔加入20μL MTT，继续培养4h，最后弃掉每孔所有溶液，每孔加入100μL DMSO，震荡，用酶标仪检测570nm处OD值，每孔设5个平行。实验结果见表10。
表10蛋白一、蛋白二对小鼠脾脏淋巴细胞增殖的影响
Figure PCTCN2018077968-appb-000020
*P<0.05，**P<0.01vs control.
结果显示，与ConA组比较，融合蛋白一、蛋白二均能在一定程度抑制小鼠脾脏淋巴细胞。
实施例5
融合蛋白一、蛋白二对小鼠腹腔巨噬细胞产生IL-1β的影响
(1)IL-1β产生：小鼠腹腔注射1mL肉汤培养基(含6％)淀粉，三天后无菌取小鼠腹腔巨噬细胞，用1640培养基洗2次，调整细胞浓度为2×106个/mL，注入24孔培养板，每孔1mL，置细胞培养箱中孵育3h，每隔30min振动一次，使细胞充分贴壁。然后用培养液洗2次，除去未贴壁细胞。空白组加入PBS，阳性组加入阳性药***Dex，对照组为低中高三种浓度的融合蛋白一和二，给药后连续培养48h，1000r/min离心15min。收取上清为待测IL-1β活性的样品。
(2)IL-1β含量测定：用R&D公司的小鼠IL-1β酶联免疫检测试剂盒检测，按照试剂盒说明书操作如下：分别将所测样品和不同浓度标准品，用封板胶纸封住反应孔，37℃孵育90min；洗板四次；加入生物素化抗体工作液(100μL/孔)，用封板胶纸封住反应孔，37℃孵育60min；洗板四次；加入酶结合物工作液(100μL/孔)，用封板胶纸封住反应孔，37℃孵育30min；洗板四次；加入显色剂(100μL/孔)，避光37℃孵育10-20min，加入终止液(100μL/孔)，混匀后即可测量OD450值。实验结果见表11。
表11 蛋白一、蛋白二对小鼠腹腔巨噬细胞产生IL-1β的影响
Figure PCTCN2018077968-appb-000021
*P<0.05，**P<0.01vs control.
实验结果表明，融合蛋白一与蛋白二均能明显抑制小鼠腹腔巨噬细胞产生IL-1β。
实施例6
融合蛋白一、蛋白二对大鼠棉球肉芽肿亚急性炎症的影响
用分析天平精确称取脱脂棉40份，每份30mg，团成形状和大小基本相同的球形。1.5kpa高压灭菌30min，50℃烘干，备用。
取SD大鼠40只，雄性，将其随机分为4组，每组10只。分别为模型组，***阳性组(10mg/kg)，融合蛋白一、蛋白二为实验组有效剂量定位64mg/kg。于给药前将大鼠用戊巴比妥钠(40mg/kg)腹腔注射麻醉，剪去腹部被毛，无菌条件下剪开下腹部正中皮肤，切口长约1cm，血管钳扩充皮下组织，向一侧腹股沟皮下植入无菌干燥棉球，缝合切口，在切口处撒适量阿莫西林防止感染。术后当天起按组别每5天注射给药1次。在第七天给药后第24h颈椎脱臼处死大鼠，切开腹股沟皮肤，将棉球同周围肉芽组织一起取出，剔除周围组织。置 60℃烘箱中连续干燥48h后，精密称取重量。并计算肉芽肿重：肉芽肿重(mg/100g体重)＝肉芽净重(mg)/大鼠自身体重(100g)。实验结果见表12。
表12 蛋白一、蛋白二对大鼠棉球肉芽肿亚急性炎症的影响
Figure PCTCN2018077968-appb-000022
*P<0.05，**P<0.01vs control.
实验结果表明，与空白模型组比较，融合蛋白一与蛋白二在有效剂量64mg/kg下均能明显抑制大鼠棉球肉芽肿。阳性药虽然抑制率较高，但大鼠体重下降明显，毒副作用较大，相比较融合蛋白相对安全。
实施例7
融合蛋白一、蛋白二对小鼠腹腔毛细血管通透性的影响
取昆明小白鼠80只，随机将其分为8组，每组10只，分别为空表模型组，***阳性组(10mg/kg)，融合蛋白一和蛋白二高、中、低剂量(128、32、8mg/kg)为实验组。每5日注射给药1次，空白模型组给予同体积生理盐水，连续5d，正常饲喂。于给药后第5天，尾静脉注射5g/L伊文思蓝生理盐水溶液10kg/mL，随即腹腔注射10kg/mL HAc溶液(6mL/L)致炎。20min后颈椎脱臼处死小鼠，腹腔注射5mL生理盐水，轻揉腹部2min，剪开腹腔，收集腹腔洗液，4000rpm离心10min，取上清液1mL，加入3mL生理盐水得到4mL的稀释液，用紫外分光光度计于590nm波长测定稀释液的吸收度OD值，以OD590nm值表示色素渗出量，考察小白鼠腹腔毛细血管通透性。实验结果见表13。
表13 蛋白一、蛋白二对小鼠腹腔毛细血管通透性的影响
Figure PCTCN2018077968-appb-000023
Figure PCTCN2018077968-appb-000024
*P<0.05，**P<0.01vs control.
实验结果表明，融合蛋白一与蛋白二均能对冰醋酸所致小鼠腹腔毛细血管通透性的增加有明显抑制作用，剂量越高，作用越强。
实施例8
融合蛋白一、蛋白二对二甲苯所致小白鼠耳肿胀的影响
取昆明小鼠80只，分为8组，每组10只，编号。以生理盐水组为空白对照组，以阿司匹林组(200mg/kg)为阳性对照组，融合蛋白一和蛋白二的高、中、低剂量(128、32、8mg/kg)为实验组。小鼠每5天注射给药1次。空白对照组给予等体积生理盐水。给药后第五天，于小鼠右耳两面涂二甲苯0.05mL致炎，左耳不涂为正常耳。2h后脱臼处死小鼠，沿耳廓剪下两耳，用打孔器取耳片、称重、计算肿胀度及肿胀率。肿胀度＝右耳片重-左耳片重，肿胀率＝(肿胀度/左耳片重)×100％。实验结果见表14。
表14 蛋白一、蛋白二对二甲苯所致小白鼠耳肿胀的影响
Figure PCTCN2018077968-appb-000025
*P<0.05，**P<0.01vs control.
实验结果表明，融合蛋白一与蛋白二中高剂量均能对二甲苯致小鼠耳肿胀有明显抑制作用，抑制作用随剂量的增大能增强。
实施例9
融合蛋白一、蛋白二对角叉菜胶诱导大鼠足趾肿胀急性炎症的影响
取SD大白鼠80只，将其随机分为8组，每组10只，分别为空白模型组，***阳性组(5mg/kg)和融合蛋白一与蛋白二的高、中、低剂量组(128、32、8mg/kg)为实验组。每5日注射给药1次，模型组给予同体积生理盐水，连续3d，正常饲喂。给药后第三天，于大鼠右后足跖皮下注射1％角叉菜胶0.1mL致炎，分别于致炎后1h，3h，5h，7h测量足容积。按照下列公式计算足肿胀度：足肿胀度(mL)＝致炎后足容积—致炎前容积。记录溢出液体的毫升数(方法：右关节的突出点处用圆珠笔划圈作为测量标志，依次将各鼠右后足放入容积测量器内，使后肢暴露在筒外，浸入的深度以划圈处与液面重合为度。该足进入液体后，液面升高，溢出液体的体积即为该鼠的右后足的体积，依次测定各鼠右后足的正常体积)。实验结果见表15。
表15 蛋白一、蛋白二对角叉菜胶诱导大鼠足趾肿胀急性炎症的影响
Figure PCTCN2018077968-appb-000026
*P<0.05，**P<0.01vs control.
实验结果表明，各组大鼠造模后足趾部迅速肿胀，大约在3～5h达到肿胀的高峰，7h开始消退。融合蛋白一与蛋白二的高剂量均能对角叉菜胶诱导的大鼠足趾肿胀均有明显的抑制作用，低剂量抑制作用不明显。
实施例10
融合蛋白一、蛋白二对大鼠佐剂型关节炎慢性炎症的影响
模型建立：
取SPF级SD大鼠80只，随机分成8组，各组大鼠用***浅麻醉，随后在大鼠左后足趾皮下注射含灭活的结核分枝杆菌完全弗氏佐剂0.1mL，大鼠的左后足即出现原发性关节炎，造模13d左右会在右后足出现继发性关节炎。空白对照组注射等体积的生理盐水。造模13d后开始给药，其中甲氨蝶呤组每5天注射给药一次，给药15天，一共4次；融合蛋白一与蛋白二的高、中、低三个剂量(128mg/kg、32mg/kg、8mg/kg)每5天注射给药一次，给药15天。
疗效评价：
1.原发性及继发性足趾肿胀度
采用足容积测定方法，在每只大鼠左右后足踝关节处用脂溶性记号笔作一标志，将动物左右后足分别浸入容积测定装置内。浸入深度以标记处为界，以该装置的刻度吸管处读取数值即为动物左右后足的初始体积。
造模当天算作第0天，记为d0，从造模第一天d1开始测量左后足(造模足)的体积，每隔2天一次，直到对侧非致炎足(右后足)出现肿胀(即出现了继发性关节炎)时开始给药，并每隔2天测量一次左右后足的体积，求出原发性和继发性足趾肿胀度。计算公式如下：
原发性足趾肿胀度(mL)＝测量当天的左后足体积-左后足的初始体积
继发性足趾肿胀度(mL)＝测量当天的右后足体积-右后足的初始体积
2.临床评分
全身评分：从继发性炎症出现后，每隔2天进行全身评分。
后足：无肿胀＝0分，一个后足肿胀＝1分，两个后足肿胀＝2分；
前足：无肿胀＝0分，一个前足肿胀＝1分，两个前足肿胀＝2分；
耳朵：无发红症状和结节＝0分，一个耳朵出现发红症状或者结节＝1分，两个耳朵出现发红症状和结节＝2分；
鼻：无肿胀＝0分，明显的肿胀＝1分；
尾巴：无结节＝0分，有结节＝1分；满分8分。
关节炎指数评分：从继发性炎症出现后，每隔2天进行关节炎指数评分。
正常＝0分；踝关节出现红斑和轻度肿胀＝1分；踝关节到跖关节或掌关节出现红斑和轻度肿胀＝2分；踝关节到跖趾关节或掌关节出现红斑和中度肿胀＝3分；踝关节到跖趾关节或掌关节出现红斑和重度肿胀＝4分；每只足满分4分，每只大鼠最多记16分。
3.体重增加值
造模前称量各组大鼠的初始体重，自造模d1天开始，每隔2天测一次体重，减去初始体重， 即为各组大鼠的体重增加值。实验结果见表16。
表16 蛋白一、蛋白二对大鼠佐剂型关节炎慢性炎症的影响
Figure PCTCN2018077968-appb-000027
*P<0.05,**P<0.01vs control.
实验结果表明，各组大鼠造模后，左后足迅速肿胀(原发性炎症)，第13d左右后足(非对侧致炎足)开始红肿(即出现了继发性炎症)，关节炎指数和全身评分开始升高，第19d达到最高值，随着给药各组肿胀度及评分逐渐下降。以原发性足趾肿胀度来反映各治疗组对原发性关节炎的治疗作用，各给药组的高、中两个剂量与模型组比较均能一定程度的治疗原发性关节炎，阳性药甲氨蝶呤的效果最好，融合蛋白一与蛋白二以高剂量组的效果好，具有极显著性差异(**P<0.01)；以继发性足趾肿胀度来反映各治疗组对继发性关节炎的治疗作用。
实施例11
融合蛋白一、蛋白二对人视网膜血管内皮细胞(HRCEC)的增殖抑制作用
采用MTT法检测整合素阻断剂多肽抑制人视网膜血管内皮细胞增殖的活性。HRCEC细胞在37℃、5％CO2的培养箱中培养至密度90％以上时用胰蛋白酶消化收集，用培养液重悬细胞并在显微镜下计数，将细胞浓度调整为3.0×10 4个/mL，将细胞悬液接种到96孔板中，每孔100μL，并于37℃，5％CO 2培养箱中培养过夜。将多肽I、多肽II、多肽III、Avastin用培养液稀释到各个预定浓度。待细胞完全贴壁后，将各个稀释液分别加入96孔板中，每孔100μL。以加入整合素阻断剂多肽作为给药组，Avastin作为阳性对照组，以不加任何药物的培养液作为空白对照组，在37℃，5％CO 2培养箱孵育48小时。向96孔板中每孔加入20μL 5mg/mL的MTT，继续培养4小时。吸去培养基，每孔加入100μL DMSO溶解。用酶标仪在570nm下检测，参比波长为630nm处测定吸光值，并计算生长抑制率(proliferation inhibition，PI)，公式如下：
Figure PCTCN2018077968-appb-000028
其中N test为测试组的OD值，N control为空白对照组的OD值。
数据统计：
试验独立重复5次，试验得到的结果计算mean±SD，并进行统计t-test检验，P<0.05为显著性差异，P<0.01为极显著性差异。实验结果见表17。
表17 蛋白一、蛋白二对人视网膜血管内皮细胞(HRCEC)增殖抑制作用
Figure PCTCN2018077968-appb-000029
*P<0.05,**P<0.01vs control.
结果显示，融合蛋白一、蛋白二能显著抑制人视网膜血管内皮细胞(HRCEC)增殖抑制作用，呈现剂量依赖关系，在64μg/mL浓度下，抑制率达到50％以上。
实施例12
鸡胚尿囊绒膜(CAM)分析融合蛋白一、蛋白二的体内抑制血管生成活性作用
本研究采用CAM试验探讨融合蛋白一、蛋白二体内抑制血管生成的活性。研究表明在鸡胚发育的第8天到第11天，胶原蛋白的生物合成速率达到最大，此时正是血管生成的最旺盛阶段，而机体免疫***尚未完全建立，因此选择发育到第8天的鸡胚开始给药。考虑到载药纸片上的多肽会在鸡胚***上有一定的弥散范围限制，因此试验中只计数距离纸片边缘5mm半径的范围内新生血管数量。采用如下操作步骤：
(1)将第6天的白杭鸡胚在60％-70％湿度的37℃培养箱培养两天。
(2)在鸡胚气室上方钻1.0cm×1.0cm的窗口，用镊子将内膜撕去，暴露出***。以直径为5mm的擦镜纸片为加样载体，放入鸡胚气室尿囊绒膜上。滤纸片加PBS为空白组，给药组分别加不同剂量的融合蛋白，阳性对照为Avastin。
(3)将鸡胚气室用无菌透明胶带封上，于37℃培养72小时后，打开鸡胚气室，加入固定液(甲醛：丙酮＝1：1)固定15min。取出粘附有擦镜纸片的***，观察其新生血管分布状况，对新生血管进行计数并拍照。每组剂量设5个重复，试验结果进行统计分析。
鸡胚尿囊绒膜(CAM)分析融合蛋白的体内抑制血管生成活性结果：阴性对照采用PBS处理，阳性对照Avastin的剂量为10μg，融合蛋白一、蛋白二设置高、中、低三个剂量处理鸡胚，分别是128μg、32μg、8μg。结果见表18。
表18 蛋白一、蛋白二鸡胚尿囊绒膜(CAM)新生血管的抑制作用
Figure PCTCN2018077968-appb-000030
*P<0.05,**P<0.01vs control.
实验结果显示，融合蛋白一与蛋白二均能对CAM新生血管形成抑制，高剂量下有较强的抑制效果，接近50％。
实施例13
融合蛋白一、蛋白二对小鼠角膜新生血管的作用
(1)BALB/c小鼠碱烧伤诱导角膜新生血管模型的制备：
健康BALB/c小鼠15只，雄性，体重20-25g，裂隙灯显微镜下检查双眼眼前段及附属器，排除眼部病变。碱烧伤模型制备前1d给0.3％氧氟沙星眼药水点眼，每日2次。小鼠经腹腔注射1.8％Avertin麻醉后，用镊子夹住直径为2mm的单层滤纸片，浸于1mol/L氢氧化钠溶液中，使其达饱和状态，去除多余液体，将滤纸片置于BALB/c小鼠角膜中央40S，弃掉滤纸，立即用15mL的PBS充分冲洗烧伤区及结膜囊1min。棉签拭去过多水分，于手术显微镜下以角膜刮铲平行于角膜缘的方式旋转刮除角膜上皮，注意勿伤及上皮下基质层及角膜缘，术毕结膜囊内涂红霉素眼膏预防感染。
(2)实验动物分组及标本获取：
15只小鼠按随机分组，标记为融合蛋白一组、融合蛋白二组和对照组，每组5只，自碱烧伤后分别给予64μg融合蛋白一、64μg融合蛋白二和生理盐水玻璃体腔注射，每7日1次，碱烧伤后1d、7d、14d于裂隙灯显微镜下观察各组角膜的炎症反应及新生血管情况。碱烧伤后第14d于带眼前段照相的裂隙灯显微镜下拍照记录各组角膜新生血管形成情况，随即以颈椎脱臼法处死所有小鼠并摘除眼球，生理盐水冲洗血迹，4％多聚甲醛固定1.5h，含30％蔗糖的PBS中脱水过夜，OCT冰冻切片包埋剂包埋，-80℃冰箱中保存，8μm冰冻切片，免疫组织化学法检测CD31的表达。
(3)角膜组织微血管密度定量测定：
微血管密度(Microvessel density，MVD)是评价血管形成的指标。我们采用抗CD31抗体免疫组织化学法标记血管内皮细胞，计数单位面积中的微血管数目，由此来衡量新生血管生成的程度。统计微血管的标准：显微镜下观察角膜组织中与邻近组织分界清楚，并被染成棕黄色或褐色的内皮细胞或细胞团均计入新生血管。在10×20镜下计数整张切片新生血管数目，角膜组织片照相后以图像处理软件Image J计算出整个角膜组织片面积，求出该例整张切片的新生血管密度。结果见表19。
表19 蛋白一、蛋白二对小鼠角膜新生血管的作用MVD计数
Figure PCTCN2018077968-appb-000031
*P<0.05,**P<0.01vs control.
结果显示，CD31作为微血管标记物，主要表达于血管内皮细胞胞质中，染色阳性细胞为血管内皮细胞染成棕黄色或褐色，无背景染色。融合蛋白一、蛋白二实验组CD31阳性新生血管与对照组相比显著减少。融合蛋白一、蛋白二相比对照组具有显著性差异。实验结果表明融合蛋白一、蛋白二均能够抑制角膜新生血管的生长，能够作为治疗角膜新生血管性眼病的药物。
实施例14
融合蛋白一、蛋白二对家兔虹膜新生血管的作用
采用577nm氩离子激光凝阻家兔视网膜主分支静脉，经眼底荧光素血管造影(FFA)证实静脉阻塞成功。5-12天后眼虹膜荧光素血管造影(IFA)显示虹膜血管与正常对照组对比荧光素渗漏明显，证实虹膜新生血管化的动物模型(NVI)形成。
取造模成功的9只眼，随机分成3组，每组3只。分别标记为阴性对照组、融合蛋白一治疗组、融合蛋白二治疗组，分别以生理盐水、128μg融合蛋白一、128μg融合蛋白二玻璃体腔注射给药，每7日1次，持续2周。第3周用光学及电子显微镜观察。
结果：光学显微镜下可观察到虹膜前表面是主要由纤维组织组成的纤维血管膜残迹，仅有极少的开放血管腔。在虹膜基质内可看到血管残存物，为坏死细胞及细胞碎片。光镜下的对照眼的虹膜表面为有分枝的和潜在管腔的纤维血管膜。
治疗组虹膜的超微结构为一系列的退行性改变。虹膜基质中部的大血管的内皮细胞有正常的细胞核、细胞质和细胞连接。虹膜基质中及虹膜前表面有毛细血管残迹，周围有细胞碎片和巨噬细胞浸润。无潜在管腔的毛细血管及退变的壁细胞，表明新生血管消退。
通过虹膜新生血管化的动物模型实验，证明了融合蛋白一与融合蛋白二能够抑制新生血管形成并使已形成的血管退化。
实施例15
融合蛋白一、蛋白二对兔眼脉络膜血流的影响
取体重为2.5-3.0公斤的新西兰白兔，随机分为3组，分别标记为对照组、融合蛋白一组、 蛋白二组。每组的白兔用35mg/kg甲苯噻嗪混合后肌注麻醉，之后每小时用起始量的一半肌注维持麻醉。升高左眼眼压至40mmHg，该压力下可使眼血流降至正常值的1/3。经右颈动脉插管至左心室，用于注射微球(计算眼血流量)，股动脉插管用于采血。各组分别进行玻璃体腔注射生理盐水、128μg融合蛋白一、128μg融合蛋白二，给药后于0、30、60分钟用彩色微球技术测定高眼压兔眼的眼血流量。在每个时间点，注入0.2mL(约200万)微球，微球注入后立即经股动脉采血60秒整，并置于肝素化抗凝管，记录采血量。最后一次采血后，用100mg/kg的***静注处死动物，摘取眼球，分离视网膜、脉络膜、虹膜和睫状体，记录组织重量。每个时间点组织血流的计算用以下公式：Qm＝(Cm×Qr)/Cr。其中Qm代表组织血流，单位μL/min/mg；Cm是每毫克组织微球数；Qr是血流量，单位μL/min；Cr是作为参照的血液微球数。实验结果见表20。
表20 蛋白一、蛋白二对白兔眼脉络膜血流的影响
Figure PCTCN2018077968-appb-000032
结果显示，在所有观察时间点，融合蛋白一、蛋白二的治疗组脉络膜血流量都有显著增加。
实施例16
融合蛋白一、蛋白二在OIR小鼠中对视网膜血管的影响
OIR模型的建立：在C57/B16小鼠出生后第7天至第12天将小鼠幼畜及其母鼠暴露置于75％高氧环境中，可致其中央视网膜中毛细管迅速消失。在第12天返回到室内空气中，暴露与高氧中的视网膜血管迅速消失，这会引起广泛的异常新生血管形成，视网膜的中央部分长期在很大程度上保持无血管状态。在血管消失完全之后，于第13天向玻璃体内注射融合蛋白(给药组，融合蛋白一、蛋白二剂量均为64μg)或生理盐水(阴性组)，在第17天对视网膜 血管进行评价(为标记未闭合的血管，将50mL德克萨斯红标记的番茄凝集素注射入左心室并循环5分钟)。实验结果见表21。
表21 蛋白一、蛋白二对OIR小鼠中对视网膜血管的影响
Figure PCTCN2018077968-appb-000033
*P<0.05,**P<0.01vs control.
结果显示，对OIR小鼠施用融合蛋白一、蛋白二后，能够改善病理性新生血管形成。与阴性对照相比，用融合蛋白一、蛋白二处理的OIR小鼠视网膜中新生血管丛明显减少，所占的面积分别减少了52.97％和35.17％。
实施例17
融合蛋白一、蛋白二对早产视网膜病变大鼠模型新生血管的作用
采取波动氧诱导动物模型，将同一天自然分娩的新生鼠(12h内)随机分成3组：给氧模型组和给氧治疗组、正常对照组。给氧模型再分成三个亚组模型组和治疗组均置于有机玻璃制作的半封闭氧舱中，舱内接入医用氧气，测氧仪调整浓度至80％±2％，24h后往氧气舱内通入氮气，迅速气将氧浓度调整至10％±2％，并维持24h。如此反复，保持氧气舱内的氧气浓度每隔24h在80％和10％之间交替，持续7d后再转入空气中饲养。每天监测氧浓度8次，控制舱内环境温度在23℃±2℃，更换垫料、加食、换水、替换母鼠1次。正常对照组置于动物房饲养环境中。模型组与对照组比较，若视网膜铺片ADP酶染色示血管改变明显，突破视网膜内界膜长入玻璃体的血管内皮细胞核计数增多，差异有统计学意义，则造模成功。
给氧治疗组分成两个亚组，于造模第7天，玻璃体腔注射给药，分别给予融合蛋白一、蛋白二，剂量均为100μg；给氧模型组和对照组只给予生理盐水，连续给药1周。
第14天时，***麻醉处死后，摘除眼球，于40g/L多聚甲醛溶液中固定24h。梯度酒精脱水、二甲苯透明。浸蜡后连续切片，厚度4μm，尽量避开视盘周围。切片平行于角膜至视盘的矢状位平面。每只眼球随机取10张切片行苏木精伊红染色，计数突破视网膜内界膜的血管内皮细胞核数目(仅计数与内界膜有紧密联系的血管内皮细胞核)，统计平均每只眼球每张切片细胞数。
结果：对照组中未发现或仅极少数切片中偶有突破视网膜内界膜长入玻璃体的血管内皮 细胞核。模型组可见较多突破视网膜内界膜的血管内皮细胞核，有些单独出现，有些成簇出现，同时在一些切片上还可见这些血管内皮细胞核邻近深层视网膜血管，证实他们来源于视网膜而非玻璃体或眼部其他组织。治疗组切片中仅可见少数突破视网膜内界膜的血管内皮细胞核。实验结果见表22。
表22 各组视网膜血管内皮细胞核计数
Figure PCTCN2018077968-appb-000034
结果显示，融合蛋白一、蛋二治疗组视网膜血管内皮细胞核计数为6.693±2.109、7.333±1.263，与给氧模型组28.392±2.220比较，血管内皮细胞核数都显著减少，证明其能够一定程度上抑制氧诱导新生鼠视网膜病变模型新生血管的形成。
实施例19
融合蛋白一、蛋白二对糖尿病视网膜病变大鼠模型新生血管的作用
实验用糖尿病大鼠用链脲佐菌素STZ造模。将STZ溶于0.1mol/L，pH4.5的柠檬酸缓冲液中配制成2％的溶液。所有实验Wistar大鼠注射前禁食12h，每只大鼠按65mg/kg剂量腹腔注射2％STZ溶液。注射后单笼饲养，48h检测尿糖和血糖。尿糖在+++以上，血糖高于16.7mmol/L为成模标准。通过血糖、尿糖、尿量检测以及视网膜VEGF免疫组化检测，糖尿病视网膜病变模型建模成功。
取成模大鼠15只，随机分为三组，标记为对照组、融合蛋白一治疗组、融合蛋白二治疗组。玻璃体腔给药，对照组注射生理盐水(0.1mL)，融合蛋白一、融合蛋白二均给药100μg(0.1mL)，每7日1次，给药2周，第4周、第8周、第12周天观察。实验结果见表23。
表23 蛋白一、蛋白二对糖尿病视网膜病变大鼠模型新生血管的作用
Figure PCTCN2018077968-appb-000035
结果表明，光学显微镜下检测，每只眼球计数10张后极部视网膜神经节细胞数目，每只眼球测量10张后极部视网膜厚度。实验组大鼠视网膜组织较对照组大鼠视网膜组织各层厚度增加。实验组大鼠视网膜神经节细胞数目与对照组比较，治疗组视细胞数目和对照组视细胞数目对比增加。表明融合蛋白一、蛋白二在100μg剂量下均能够对糖尿病视网膜病变产生一定的治疗作用。
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Claims (13)

  1. 一种融合蛋白，其特征在于：融合蛋白分子中含有血管抑制多肽HM-3序列、白介素4序列以及抗体IgG1的Fc序列。
  2. 根据权利要求1所述的一种融合蛋白，其特征在于：血管抑制多肽HM-3序列、白介素4序列以及抗体IgG1的Fc序列之间由柔性Linker链接。
  3. 根据权利要求2所述的一种融合蛋白，其特征在于：其氨基酸序列为SEQ ID NO.1。
  4. 根据权利要求1所述的一种融合蛋白，其特征在于：血管抑制多肽HM-3序列、白介素4序列以及抗体IgG1的Fc序列之间由刚性Linker链接。
  5. 根据权利要求4所述的一种融合蛋白，其特征在于：其氨基酸序列为SEQ ID NO.2。
  6. 编码权利要求3或5所述的融合蛋白的基因，其特征在于：编码序列SEQ ID NO.1、SEQ ID NO.2的核酸序列分别为SEQ ID NO.3、SEQ ID NO.4。
  7. 权利要求1中所述的融合蛋白在制备治疗炎症、抗肿瘤和治疗眼科疾病药物中的应用。
  8. 根据权利要求7所述的融合蛋白在制备治疗炎症、抗肿瘤和治疗眼科疾病药物中的应用，其特征在于：所述的炎症包括类风湿关节炎、骨关节炎、痛风性关节炎、强直性脊柱炎、银屑病关节炎、反应性关节炎、感染性关节炎和创伤性关节炎、***性红斑狼疮、银屑病。
  9. 根据权利要求7所述的融合蛋白在制备治疗炎症、抗肿瘤和治疗眼科疾病药物中的应用，其特征在于：所述的肿瘤包括胃癌、肺癌、肝癌、乳腺癌、结肠癌、胶质瘤、黑色素瘤和***以及起源于人的头颈部、脑部、甲状腺、食管、胰腺、肺脏、肝脏、胃、乳腺、肾脏、胆囊、结肠或直肠、卵巢、子宫颈、子宫、***、膀胱、睾丸的原发或继发的癌、黑色素瘤以及肉瘤。
  10. 根据权利要求7所述的融合蛋白在制备治疗炎症、抗肿瘤和治疗眼科疾病药物中的应用，其特征在于：所述的眼科疾病包括虹膜新生血管性眼病、脉络膜新生血管性眼病、视网膜新生血管性眼病或角膜新生血管性眼病。
  11. 根据权利要求10所述的融合蛋白在制备治疗炎症、抗肿瘤和治疗眼科疾病药物中的应用，其特征在于：所述的虹膜新生血管性眼病包括新生血管性青光眼、糖尿病视网膜病变或视网膜中央静脉栓塞引起的虹膜新生血管性眼病；所述的脉络膜新生血管性眼病包括年龄相关性黄斑变性、中心性渗出性视网膜脉络炎、眼组织胞浆菌病综合征或葡行性脉络膜病变脉络膜新生血管性眼病；所述的视网膜新生血管性眼病包括糖尿病、肿瘤、视网膜脱落、视网膜中央静脉阻塞、视网膜静脉周围炎、全身性红斑狼疮、Eales病或Coat病相关的视网膜新生血管性眼病；所述的角膜新生血管性眼病包括角膜接触镜所致角膜新生血管性疾病，以及碱及其他化学物质烧伤、角膜手术、细菌感染、衣原体感染、病毒感染或原虫感染引起的角膜新生血管性眼病。
  12. 根据权利要求7-11中任意一项所述的融合蛋白在制备治疗炎症、抗肿瘤和治疗眼科疾病药物中的应用，其特征在于：药物的剂型为胶囊、片剂、药丸、注射剂、鼻喷剂或气雾剂。
  13. 权利要求1中所述的融合蛋白的制备方法，其特征在于：利用真核细胞表达及纯化方法。
PCT/CN2018/077968 2017-03-20 2018-03-05 一种融合蛋白及其制备方法和其在制备治疗眼科疾病、抗炎、抗肿瘤药物中的应用 WO2018171411A1 (zh)

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