WO2022135231A1 - 一种蛋白复合物及其制备方法和应用 - Google Patents

一种蛋白复合物及其制备方法和应用 Download PDF

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WO2022135231A1
WO2022135231A1 PCT/CN2021/138269 CN2021138269W WO2022135231A1 WO 2022135231 A1 WO2022135231 A1 WO 2022135231A1 CN 2021138269 W CN2021138269 W CN 2021138269W WO 2022135231 A1 WO2022135231 A1 WO 2022135231A1
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protein
seq
recombinant protein
protein complex
disease
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French (fr)
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刘凯
张洪杰
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清华大学
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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the technical field of biosynthesis, in particular to a protein complex and a preparation method and application thereof.
  • Rheumatoid arthritis is a human immune disease, and the exploration of the etiology of the disease is still in its infancy.
  • the patient's own genetic factors, infection, abnormal immune system, environmental factors (such as exposure to silica, infectious factors, vitamin D deficiency in the body), unhealthy living habits (such as smoking, obesity), changes in the body's microbiota, etc. increase the risk of the disease.
  • the incidence of hypertension, diabetes, hyperlipidemia and obesity were 18.6%, 6.0%, 9.9% and 4.4%, respectively. Common cause of premature death in patients.
  • the first-line drugs used in clinical use are mainly anti-inflammatory, analgesic, and antipyretic to relieve the discomfort of patients, mainly non-steroidal anti-inflammatory drugs and glucocorticoids.
  • Such drugs are cheap, long-term use will cause side effects such as liver and kidney damage, and gastrointestinal tract damage, and the efficacy of repeated use will be reduced.
  • disease-modifying anti-rheumatoid drugs can significantly relieve joint inflammation and acute, but the onset is slow, and the metabolic stress and psychological burden they bring to patients are relatively high.
  • gout The symptoms of gout are similar to those of rheumatoid. Due to the deficiency of purine metabolizing enzymes in primates, with the increase of high-purine food intake and abnormal excretion, uric acid in the body increases and aggregates in the joints to form monosodium urate crystals (tophi), which causes inflammation.
  • the pathogenesis of gout can be divided into asymptomatic hyperuricemia phase, acute phase, intermittent phase, and chronic phase. After the patient transitioned from asymptomatic hyperuricemia to the acute phase, he began to experience fatigue, general malaise, and joint tingling. When a gout attack occurs, the patient wakes up from late at night due to severe joint pain, and develops into a tear 12 hours later.
  • the pain is unbearable, like a knife cut or a bite.
  • the joint and surrounding tissue may experience redness, swelling, heat, pain, and limited function.
  • gout can be relieved spontaneously in a few days or weeks after the attack, generally without obvious sequelae; but after a few months or years of recurrence, the frequency of the attack gradually increases, the duration continues to prolong, and the affected joints expand to the fingers and wrists , elbow or even hip, sacroiliac, sternoclavicular or spinal joints. About 10 years after the onset of gout, it enters the stage of chronic tophi.
  • first-line drugs for gout treatment include allopurinols and febuxostat, as well as the uricosuric drug benzbromarone; second-line drugs include colchicine, etc. These drugs have no obvious therapeutic effect in some people, and are prone to relapse and have severe liver and kidney toxicity.
  • interleukin-1 receptor antagonist IL-1RA
  • human interleukin-1 receptor antagonist anakinra
  • anakinra has been successfully used in the treatment of rheumatoid arthritis and acute gouty arthritis, but due to its short half-life in vivo, patients need repeated administration to achieve therapeutic effect and expensive.
  • the present invention provides a recombinant protein and its preparation method and application.
  • the complex includes human IL-1RA protein and VPGKG repeat-containing protein, which can be used for rheumatoid arthritis, acute gouty joints Inflammation and other adjuvant therapy of rheumatic immune diseases, hereditary diseases and tumors characterized by abnormal increase of interleukin-1 (IL-1), the curative effect is remarkable.
  • IL-1 interleukin-1
  • the first aspect of the present invention provides a recombinant protein, including human IL-1RA protein and VPGKG repeating sequence-containing protein.
  • the human IL-RA sequence is from human IL-1RA protein spliceosome 1 (SEQ ID NO 1), human IL-1RA protein spliceosome 2 (SEQ ID NO 2), human IL -1RA protein spliceosome 3 (SEQ ID NO 3) or human IL-1RA protein spliceosome 4 (SEQ ID NO 4).
  • the human IL-RA sequence is from human IL-1RA protein spliceosome 3.
  • the human IL-1RA protein has any one of the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.4; or has a sequence similar to that shown in SEQ ID NO.1 to SEQ ID NO.4 Functionally identical or similar amino acid sequences.
  • the VPGKG repeat-containing protein consists of n repeat units (VPGKG) and m repeat units (VPGXG), wherein X is any natural amino acid, and m and n are integers greater than zero ; Preferably, m is an integer between 0 and 20, and n is an integer between 1 and 150; the repeating units (VPGKG) and (VPGXG) are arbitrarily combined.
  • the repeating protein unit containing the (VPGKG) sequence has a repeating unit structure characteristic of (VPGXG) m (VPGKG) n .
  • X is valine, alanine, or glycine.
  • the repeat protein unit has the characteristic structure [(VPGAG) 2 (VPGKG) 8 ] 9 ; in some preferred embodiments, the repeat protein unit has [(VPPGG) 1 (VPGKG) 3 (VPGAG) 2 (VPGKG) 5 (VPGVG) 2 (VPGKG) 9 ] 5 characteristic structure; in some preferred embodiments, the repeating protein unit has [VPGVG (VPGKG) 9 ] 6 characteristic structure; in other preferred embodiments, The repeating protein unit has the characteristic structure of [VPGVG(VPGKG) 9 ] 8 .
  • the IL-1RA protein sequence and the repeat protein unit sequence containing the (VPGKG) sequence are linked by peptide bonds or linking peptides.
  • the IL-1RA sequence and the repeat protein unit sequence containing the (VPGKG) sequence are linked by a linker peptide.
  • the connecting peptide is a plurality of consecutive arbitrary amino acids.
  • the linker peptide has less than 10% of the amino acid number of the full length of the recombinant protein.
  • the plurality is 13, 14 or 18.
  • the amino acid sequence of the connecting peptide is shown in any one of SEQ ID NO.5 to SEQ ID NO.7.
  • the recombinant proteins of the present invention further comprise conservatively modified variants of the proteins.
  • Said conservatively modified variant comprises 10% of amino acids in the recombinant protein of the present invention, preferably 5% of amino acids, more preferably 2% of amino acids, most preferably 1 amino acid is replaced by all or part of its functionally similar conservative amino acids The resulting variant is replaced.
  • the conservative substitutions are well known in the art and include the following 6 groups of amino acids:
  • I Isoleucine
  • L Leucine
  • M Methionine
  • V Valine
  • the recombinant protein of the present invention has any one of the amino acid sequences shown in SEQ ID NO.13 to SEQ ID NO.72, or has any one of the amino acid sequences shown in SEQ ID NO.13 to SEQ ID NO.72 An amino acid sequence with at least 80% sequence identity, or an amino acid sequence that is functionally identical or similar to any of the sequences shown in SEQ ID NO.13 to SEQ ID NO.72.
  • the recombinant protein has at least 85%, at least 90%, at least 95%, preferably at least 98%, more preferably at least 99% sequence with any one of SEQ ID NO. 13 to SEQ ID NO. 72 An amino acid sequence that is identical and functionally identical or similar. Wherein, the same or similar function is specifically the same or similar to the affinity activity of the interleukin 1 ⁇ receptor.
  • the recombinant protein has SEQ ID NO.13, SEQ ID NO.21, SEQ ID NO.27, SEQ ID NO.33, SEQ ID NO.40, SEQ ID NO.45, The amino acid sequence shown in any one of SEQ ID NO.52, SEQ ID NO.56, SEQ ID NO.60, and SEQ ID NO.72.
  • a second aspect of the present invention provides nucleic acids encoding the recombinant proteins of the present invention.
  • the nucleic acid has:
  • the nucleic acid has at least 85%, at least 90%, at least 95%, preferably at least 98%, more preferably at least 99% sequence identity to any one of SEQ ID NO. 73 to SEQ ID NO. 132 sexual nucleotide sequence.
  • the nucleic acid may be replaced by a different three consecutive base codons encoding the same amino acid.
  • the same amino acid base substitutions are known in the art and include the following 20 codon substitutions:
  • the nucleic acid variant thus obtained encodes the same amino acid sequence as the original nucleic acid.
  • a third aspect of the present invention provides an expression vector comprising the nucleic acid of the second aspect of the present invention.
  • the backbone vector of the expression vector is pET25b.
  • the fourth aspect of the present invention provides host cells transformed or transfected with the expression vector of the third aspect of the present invention.
  • the fifth aspect of the present invention provides a preparation method for preparing the recombinant protein of the first aspect of the present invention.
  • the preparation method comprises: culturing the host cell of the fourth aspect of the present invention, and inducing the expression of the recombinant protein.
  • the preparation method specifically includes the following steps:
  • the prokaryotic expression vector employed in step 1) of the method is pET25b.
  • the prokaryotic expression vector cells in step 2) of the method are E. coli cells.
  • the separation and purification process in step 4) of the method includes centrifugation, salting out, cation exchange chromatography, molecular sieve chromatography, and ultrafiltration.
  • the pH of the cation exchange chromatography buffer is in the range of 8.0 to 9.5, and in some more preferred embodiments, the pH of the cation exchange chromatography buffer is in the range of 8.8 to 9.0.
  • the NaCl content in the cation exchange chromatography elution buffer is 200-500 mM; in some preferred embodiments, the NaCl content in the cation exchange chromatography elution buffer is 300-500 mM; in some In a more preferred embodiment, the NaCl content in the cation exchange chromatography elution buffer is 400 mM.
  • the sixth aspect of the present invention provides a protein complex formed by combining the recombinant protein of the first aspect of the present invention with polyethylene glycol (PEG) and a preparation method thereof.
  • PEG polyethylene glycol
  • the recombinant protein is combined with PEG to form a protein complex by covalent or non-covalent binding.
  • the molar ratio of recombinant protein to PEG is 1:0.25n ⁇ 1:5n; in some more preferred embodiments, the molar ratio of recombinant protein to PEG is 1:0.5n ⁇ 1:2n; In some more preferred embodiments, the molar ratio of recombinant protein to PEG is 1:n.
  • the average molecular weight of PEG is 1,000-40,000; in some preferred embodiments, the average molecular weight of PEG is 1,000-10,000; in some more preferred embodiments, the average molecular weight of PEG is 5,000.
  • PEG is a carboxylated modified polyethylene glycol molecule, which is mixed with recombinant protein in a buffer solution to obtain a protein complex.
  • the pH range of the buffer solution is preferably 6.0-8.0, and the more preferable pH range is 7.0-7.5, especially 7.2-7.4; the reaction time range is 0-24h.
  • the recombinant protein forms a protein complex with PEG by covalent bonding.
  • the protein complex is obtained by mixing with the recombinant protein in a buffer solution.
  • the pH range of the buffer solution is preferably 6.0-8.5, and the more preferred pH range is 7.0-8.5, especially 7.5-8.0; the reaction time range is 0-72h, and the preferred time range is 0h-24h, more preferably The time range is 12 to 24 hours.
  • PEG that was not successfully attached during the preparation of the protein complex is removed by purification.
  • means of purification include molecular sieve chromatography and dialysis.
  • the seventh aspect of the present invention provides a protein complex formed by combining the recombinant protein of the first aspect of the present invention with chondroitin sulfate (CS) and a preparation method thereof.
  • CS chondroitin sulfate
  • the recombinant protein is combined with CS to form a protein complex by non-covalent association.
  • the molar ratio of recombinant protein to CS is 1:0.25n ⁇ 1:5n; in some more preferred embodiments, the molar ratio of recombinant protein to CS is 1:0.5n ⁇ 1:2n; In some more preferred embodiments, the molar ratio of recombinant protein to CS is 1:n.
  • CS is obtained by mixing with recombinant protein in a buffered solution to obtain a protein complex.
  • the pH range of the buffer solution is preferably 6.0-8.0, and the more preferable pH range is 7.0-7.5, especially 7.2-7.4; the reaction time range is 0-1 h.
  • the CS that was not successfully attached during the preparation of the protein complex is removed by purification.
  • means of purification include molecular sieve chromatography and dialysis.
  • the eighth aspect of the present invention provides a drug composed of the recombinant protein of the first aspect of the present invention and the protein complex of the sixth aspect of the present invention and other active pharmaceutical ingredients, pharmaceutically acceptable carriers, pharmaceutical excipients or excipients; or
  • the active pharmaceutical ingredient includes one or more of the following: etanercept (Rilonacept), infliximab (INFLECTRA), thalidomide, steroids (eg, cortisone, prednisone) Dragon, prednisone, methylprednisone, dexamethasone, betamethasone, triamcinolone acetonide, beclomethasone, fluticasone), anakinra, colchicine, IL-18 binding protein ( IL-18BP) or derivatives, IL-18 antibody, IL-18 receptor (IL-18R1) antibody, IL-18 receptor accessory protein (IL-18Racp) antibody, aspirin, methotrexate, cyclosporine A. Caspase-1, IKK1/2, cytotoxic T cell antigen 4 (CTLA-4Ig), IL-6 antibody and IL-6RA antibody.
  • CTL-4Ig cytotoxic T cell antigen 4
  • IL-6 antibody IL-6RA antibody
  • the ninth aspect of the present invention provides the recombinant protein of the first aspect of the present invention, the protein complex of the sixth aspect of the present invention, the protein complex of the seventh aspect of the present invention, or the medicine of the eighth aspect of the present invention for prevention and/or treatment Use in the preparation of medicaments for preventing and/or treating diseases in animals.
  • the treatment is preferably recovery, further preferably complete recovery;
  • the mammal is preferably a primate, more preferably a human being;
  • the disease is preferably a rheumatic immune disease, more preferably a rheumatism characterized by abnormally increased human interleukin-1. immune diseases.
  • the disease is a disease characterized by abnormally elevated interleukin-1, specifically including rheumatic immune diseases, genetic diseases and tumors.
  • the "disease forms with increased interleukin 1" include, but are not limited to, rheumatoid arthritis, erosive osteoarthritis, pyoderma gangrenosum, acne congregate and aseptic arthritis, gout and pseudoarthritis Gout, systemic idiopathic juvenile arthritis, atherosclerosis, amyloid A amyloidosis, adult Still's disease, asthma, Behcet's disease, Blau syndrome, calcium pyrophosphate dihydrate crystals deposition disease, Castleman disease, cold pyridine-related periodic syndrome, interleukin-1 receptor antagonist deficiency, dermatomyositis, Edheim-Chester disease, familial Mediterranean fever, psoriasis, Graft-versus-host disease, hidradenitis suppurativa, hyper-IgD syndrome
  • the recombinant proteins, protein complexes and medicaments are preferably administered by the subcutaneous or intravenous or intramuscular route, more preferably by the subcutaneous route.
  • the tenth aspect of the present invention provides the recombinant protein of the first aspect of the present invention or the protein complex of the sixth aspect or the protein complex of the seventh aspect of the present invention in the preparation of preventing and/or treating inflammation mediated by inflammatory factors in primates application in medicines.
  • the inflammatory factor is interleukin-1.
  • the eleventh aspect of the present invention provides a treatment method for adjuvant therapy of rheumatic immune diseases, genetic diseases, and tumors characterized by abnormally increased interleukin-1, comprising administering the first aspect of the present invention to a subject
  • the recombinant protein of the present invention, the protein complex of the sixth aspect of the present invention, the protein complex of the seventh aspect of the present invention, or the medicine of the eighth aspect of the present invention comprising administering the first aspect of the present invention to a subject.
  • the subject is a mammal, preferably a primate, more preferably a human.
  • the recombinant protein, protein complex, and co-drug are preferably administered by subcutaneous or intravenous or intramuscular route, more preferably by subcutaneous route.
  • the recombinant protein of the present invention is composed of a human interleukin-1 receptor antagonist protein and a protein containing (VPGKG) n repeating units.
  • the recombinant protein of the invention significantly prolongs the blood concentration half-life of IL-1RA in rats, and has a significant curative effect on acute gouty arthritis and rheumatoid arthritis in rats. Compared with single human interleukin-1 receptor antagonists The difference was significant (p ⁇ 0.001).
  • the recombinant protein can be used for the adjuvant therapy of rheumatoid arthritis, acute gouty arthritis and other rheumatic immune diseases, hereditary diseases and tumors which are characterized by abnormal increase of interleukin-1, and the curative effect is more significant.
  • Figure 1 shows the SDS-PAGE electrophoresis of recombinant protein
  • Fig. 2 shows the electron microscope photograph of the recombinant protein complex
  • Figure 3 shows the average particle size of protein complexes
  • Figure 4 shows the plasma concentration-time curve of the protein complex and IL-1RA protein in rats
  • Figure 5 shows the changes of ankle joint circumference of rats in each group after protein complex and IL-1RA protein injection
  • Figure 6 shows the changes in the thickness of the footpads of rats in each group after protein complex and IL-1RA protein injection.
  • the present invention provides a recombinant protein, a protein complex containing the recombinant protein and applications.
  • Those skilled in the art can achieve by completely repeating, or appropriately modifying the process parameters based on the content herein. It should be particularly pointed out that all similar substitutions and modifications apparent to those skilled in the art are deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.
  • the reagents and materials used in the present invention are all common commercial products and can be purchased in the market.
  • the inventors designed the following 10 recombinant protein sequences based on the (VPGKG) repeating polypeptide sequence and the human interleukin-1 receptor antagonist (IL-1RA) sequence, and the specific sequence information is shown in Table 2:
  • the corresponding nucleotide sequences of the above 10 recombinant proteins were synthesized by Sangon Bio (Shanghai) Company, and constructed on the pET25b expression plasmid vector for transformation into E. coli prokaryotic expression competent BLR (DE3).
  • the specific embodiment is as follows: 100 ng of pET25b-A1 (or A2-A10) plasmid was added to 100 ⁇ L of BLR E. coli competent (purchased from Novagen) in an ice bath environment, and maintained in an ice bath environment for 30 min; then The mixture was transferred to a 42°C water bath for 45 s heat shock followed by an ice bath for 2 min.
  • the bacterial solution was centrifuged at 4000 rpm for 30 min, and the supernatant was discarded; the bacterial cell pellet was resuspended in PBS, centrifuged again at 4000 rpm for 30 min, and the supernatant was discarded to obtain the bacterial cell precipitate containing the target protein.
  • the strains that correctly expressed the recombinant proteins were identified by SDS-PAGE electrophoresis and immunoblotting experiments.
  • the strains that can correctly express the recombinant protein were transferred into 50 mL of LB medium containing ampicillin sodium (15 ⁇ g/mL) antibiotics, and cultured at 37 ° C and 220 rpm shaking environment for 4-6 h; when the OD600 reached 0.6, the bacterial liquid was transferred into In 1L of fresh LB medium containing ampicillin sodium (15 ⁇ g/mL) antibiotics, culture at 37°C and 220rpm shaking environment until OD600 reaches 0.6, then add IPTG with a final concentration of 0.5mmol/L to induce bacterial expression of recombinant protein , and adjust the ambient temperature to 16°C.
  • the bacterial liquid was centrifuged at 4000 rpm for 30 min, and the supernatant was discarded; the bacterial pellet was resuspended in PBS, centrifuged again at 4000 rpm for 30 min, and the supernatant was discarded to obtain the bacterial pellet containing the target protein.
  • the cells were resuspended in 50 mM phosphate buffer (PB) pre-cooled at 4°C, the pH of which was 7.2-7.4, and the cell mixture was disrupted with a high-pressure homogenizer. The mixture was collected and centrifuged at 16,000 rpm and 4°C for 30 min, and the supernatant was collected. 4°C pre-cooled saturated aqueous ammonium sulfate solution was added dropwise to the supernatant until the final concentration of saturated ammonium sulfate was 20%, the mixture was centrifuged at 16000 rpm and 4°C for 30 min, and the supernatant was collected.
  • PB phosphate buffer
  • the protein solution was loaded on a 10 mL SP cation exchange chromatography column (purchased from GE)
  • the protein was eluted with 50 mL of 50 mM PB buffer containing 50 mM, 100 mM, 200 mM, and 400 mM NaCl successively, and the eluate containing 400 mM NaCl was collected. components.
  • the eluted fractions were subjected to molecular sieve chromatography in Superdex 100 (purchased from GE).
  • the protein containing the eluate fraction of 400 mM NaCl was loaded on a Superdex 100 molecular sieve chromatography column, and eluted with 50 mM phosphate buffer (PB) with a pH of 7.2-7.4, and the target protein fraction was collected to obtain a purified product .
  • the purified protein was identified by SDS-PAGE gel electrophoresis, as shown in Figure 1.
  • Protein complex 1 The purified recombinant protein A1 was mixed with carboxylated PEG in a molar ratio of 1:18, and stirred at 4°C for 1 h. The mixture was loaded on a desalting chromatography column (purchased from GE), eluted with 50 mM PB buffer at pH 7.2-7.4, and the protein complex fractions were collected.
  • Protein complex 2 The purified recombinant protein A2 was mixed with carboxylated PEG in a molar ratio of 1:72, and stirred at 4°C for 1 h. The mixture was loaded on a desalting chromatography column (purchased from GE), eluted with 50 mM PB buffer at pH 7.2-7.4, and the protein complex fractions were collected.
  • Protein complex 3 The purified recombinant protein A3 was mixed with carboxylated PEG according to a molar ratio of 1:288, and stirred at 20-25° C. for 4 hours. The mixture was loaded on a desalting chromatography column (purchased from GE), eluted with 50 mM PB buffer at pH 7.2-7.4, and the protein complex fractions were collected.
  • Protein complex 4 The purified recombinant protein A4 and carboxylated PEG were mixed in a molar ratio of 1:9, and stirred at 20-25° C. for 4 hours. The mixture was loaded on a desalting chromatography column (purchased from GE), eluted with 50 mM PB buffer at pH 7.2-7.4, and the protein complex fractions were collected.
  • Protein complex 5 The purified recombinant protein A5 was mixed with carboxylated PEG according to a molar ratio of 1:54, and stirred at 2-8° C. for 24 hours. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 2-8°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; Change the dialysis replacement fluid every 8 hours. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 6 The purified recombinant protein A6 was mixed with carboxylated PEG in a molar ratio of 1:162, and stirred at 20-25° C. for 2 hours. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 2-8°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; Change the dialysis replacement fluid every 8 hours. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 7 The purified recombinant protein A7 was mixed with maleimide-modified PEG in a molar ratio of 1:720, and stirred in a water bath environment at 37° C. for 30 minutes. The mixture was loaded on a desalting chromatography column (purchased from GE), eluted with 50 mM PB buffer at pH 7.2-7.4, and the protein complex fractions were collected.
  • Protein complex 8 The purified recombinant protein A8 was mixed with carboxylated PEG in a molar ratio of 1:72, and stirred at 20-25° C. for 1 h. The mixture was loaded on a desalting chromatography column (purchased from GE), eluted with 50 mM PB buffer at pH 7.2-7.4, and the protein complex fractions were collected.
  • Protein complex 9 The purified recombinant protein A9 and the maleimide-modified PEG were mixed in a molar ratio of 1:108, and stirred for 30 min in a 37° C. water bath environment. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 2-8°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; Change the dialysis replacement fluid every 8 hours. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 10 The purified recombinant protein A10 was mixed with maleimide-modified PEG in a molar ratio of 1:144, and stirred for 30 min in a water bath environment at 20-25°C. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 2-8°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; Change the dialysis replacement fluid every 8 hours. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 11 The purified recombinant protein A1 and CS were mixed in a molar ratio of 1:162, and stirred at 20-25° C. for 10 min. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 2-8°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; Change the dialysis replacement fluid every 8 hours. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 12 The purified recombinant protein A2 and CS were mixed in a molar ratio of 1:72, and stirred at 20-25° C. for 30 min. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 2-8°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; Change the dialysis replacement fluid every 8 hours. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 13 The purified recombinant protein A3 and CS were mixed in a molar ratio of 1:720, and stirred at 2-8° C. for 1 h. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 2-8°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; Change the dialysis replacement fluid every 8 hours. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 14 The purified recombinant protein A4 and CS were mixed in a molar ratio of 1:90, and stirred at 2-8° C. for 1 h. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 2-8°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; Change the dialysis replacement fluid every 8 hours. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 15 The purified recombinant protein A5 and CS were mixed in a molar ratio of 1:180, and stirred at 37° C. for 10 min. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 2-8°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; Change the dialysis replacement fluid every 8 hours. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 16 The purified recombinant protein A6 and CS were mixed in a molar ratio of 1:54, and stirred at 37° C. for 10 min. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 2-8°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; Change the dialysis replacement fluid every 8 hours. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 17 The purified recombinant protein A7 and CS were mixed in a molar ratio of 1:432, and stirred at 37° C. for 10 min. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 2-8°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; Change the dialysis replacement fluid every 8 hours. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 18 The purified recombinant protein A8 and CS were mixed in a molar ratio of 1:72, and stirred at 20-25° C. for 30 min. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 25°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; replaced every 8h A dialysis replacement fluid. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 19 The purified recombinant protein A9 and CS were mixed in a molar ratio of 1:324, and stirred at 20-25° C. for 30 min. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 25°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; replaced every 8h A dialysis replacement fluid. Protein complex fractions were collected after 24 h of dialysis.
  • Protein complex 20 The purified recombinant protein A10 and CS were mixed in a molar ratio of 1:288, and stirred at 20-25° C. for 30 min. The mixture was transferred into a dialysis tube, and the dialysis reaction was carried out at 25°C.
  • the dialysis replacement fluid was 50mM PB buffer with a pH of 7.2-7.4, and the volume of the dialysis replacement fluid was at least 10 times the volume of the protein complex solution; replaced every 8h A dialysis replacement fluid. Protein complex fractions were collected after 24 h of dialysis.
  • Example 2 The protein complexes purified in Example 2 were detected by transmission electron microscopy, and the average particle size of the proteins was analyzed. The results are shown in Figures 2-3, and Figure 2 shows the electron microscope picture of the protein complex. Fig. 3 shows the results of the analysis of the average particle size of the protein.
  • IL-1RA protein 6.95 protein complex 11 14.45 protein complex 1 14.17 protein complex 12 20.95 protein complex 2 25.31 protein complex 13 34.57 protein complex 3 30.22 protein complex 14 18.48 protein complex 4 16.42 protein complex 15 25.23 protein complex 5 22.49 protein complex 16 19.00 protein complex 6 22.67 protein complex 17 28.99 protein complex 7 31.94 protein complex 18 28.97 protein complex 8 27.16 protein complex 19 21.85 protein complex 9 23.94 protein complex 20 34.28 protein complex 10 33.08
  • Example 5 Therapeutic effect of protein complex in rat acute gouty arthritis model
  • Rats were then modeled for gouty arthritis disease: 50 ⁇ L of a 60 mg/mL sodium urate crystal suspension was injected into the ankle joint cavity of all rats. All rat ankle joint circumferences were measured.
  • Example 6 Therapeutic effect of protein complex in rat rheumatoid arthritis model
  • Rats 60 8-week-old female SD rats were selected, the rats were marked, and the thickness of the footpads of all rats was measured. Rats were then modeled for rheumatoid arthritis: 2 mg/mL type II collagen PBS solution was mixed with an equal volume of complete Freund's adjuvant, and mixed into a homogeneous water-in-oil emulsion under the action of ultrasound. Obtain immunogen 1. Immunogen 1 was then injected subcutaneously into the footpad and tail base of all rats. After 14 days, all rats received a booster immunization.
  • the 2 mg/mL collagen type II PBS solution was mixed with an equal volume of incomplete Freund's adjuvant, and mixed into a homogeneous water-in-oil emulsion under the action of ultrasound to obtain immunogen 2.
  • Immunogen 2 was then injected subcutaneously into the footpad and base of the tail of all rats.

Abstract

提供了一种重组蛋白、含该重组蛋白的蛋白复合物和应用。该重组蛋白由人源白介素1受体拮抗剂蛋白与含有VPGKG重复单元的蛋白组成，可用于包括类风湿性关节炎、急性痛风性关节炎及其它存在白介素1（interleukin，IL-1）增多的风湿免疫病、遗传性疾病及肿瘤的辅助治疗，疗效显著。

Description

一种蛋白复合物及其制备方法和应用
本申请要求于2020年12月21日提交中国专利局、申请号为202011521081.1、发明名称为“一种蛋白复合物及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物合成技术领域，尤其涉及一种蛋白复合物及其制备方法和应用。
背景技术
类风湿性关节炎是一种人类免疫性疾病，目前对于该疾病病因的探索还处于初级阶段。除患者本身遗传因素，感染、免疫系统异常、环境因素(如接触二氧化硅、感染因子、体内维生素D缺乏)、不良生活习惯(如吸烟、肥胖)、体内微生物群改变等多中因素均会导致该疾病风险升高。患有类风湿性关节炎的患者，其罹患高血压、糖尿病、高脂血症和肥胖症的发病率分别为18.6％、6.0％、9.9％和4.4％，这些继发的心血管疾病是导致患者过早死亡的常见原因。由于在发病早期病情对患者影响不大，且反复给药患者依从性差，多数患者易主动暂停或终止治疗过程，使得病情进一步加深。在发病中后期，患者四肢关节、肘、肩、颈椎、髋、膝等关节处出现明显的关节炎症，关节腔积液挤入关节后侧形成腘窝囊肿、导致肢端无力甚至难以回复到正常位置，最终出现关节、肢端畸形。晚期类风湿性关节炎症状无法通过药物治疗改善，只能通过关节置换外科手术疗法调整患者关节功能，改善生活质量；但此时接受治疗的人群多集中于中老年群体，手术风险高，治疗花销高昂。由于类风湿性关节炎发病机制尚不明确，诱因复杂。所以临床用一线药物主要通过消炎、止痛、解热为主，从而缓解患者的不适感，主要为非甾类抗炎药和糖皮质激素类。此类药物价格低廉，长期使用会导致肝肾、胃肠道损伤等副作用，且反复用药疗效会降低。另外，疾病修饰类抗类风湿药可明显缓解关节炎症和急性，但起效缓慢，其对患者带来的代谢压力和心理负担相对较高。
痛风的发病症状与类风湿类似。由于灵长类动物存在嘌呤代谢酶缺陷，随着高嘌呤食物摄入增多和排泄异常，体内尿酸增多并在关节处聚集形成单钠尿酸盐晶体(痛风石)，从而引发炎症。痛风发病过程可分为无症状高尿酸血症期、急性期、间歇期、慢性期。患者从无症状的高尿酸血症转入急性期后，开始出现疲乏、全身不适和关节刺痛等反应；痛风发作时会导致患者因关节剧烈疼痛从深夜惊醒，在12小时后发展为撕裂样、刀割样或咬噬样痛感，难以忍受。发作关节及其周围组织会出现红、肿、热、痛和功能受限。在间歇期，痛风发作后数天或数周可以自行缓解，一般无明显后遗症；但在数月或数年复发后，发病频率逐渐升高、持续时间持续延长，受累关节范 围扩大至指、腕、肘甚至、髋、骶髂、胸锁或脊柱关节。痛风发作10年左右进入慢性痛风石病变期。此时患者皮下、关节滑膜、软骨、骨质及关节周围软组织处沉积有大量单钠尿酸盐晶体，在关节活动时，这些尿酸盐结晶摩擦关节滑膜、软骨等组织，导致关节处组织磨损、发炎、关节骨质出现破坏，患者关节处持续肿痛、甚至出现畸形和功能障碍，严重影响患者的生存活动和生活质量。当前痛风治疗一线用药包括别嘌醇类和非布司他，以及促尿酸排泄药物苯溴马隆；二线用药包括秋水仙碱等。这些药物在部分人群中无明显治疗效果，且容易复发、存在严重肝肾毒性。
类风湿性关节炎和痛风发作过程中，关节处的白介素1(IL-1)含量明显升高，并进一步引发局部的炎症反应，导致组织肿胀、炎症细胞浸润以及软骨损伤。通过白介素1受体拮抗剂(IL-1RA)抑制IL-1受体与IL-1的结合，可快速抑制IL-1导致的炎症反应，有效减缓病情发展。目前，人源白介素1受体拮抗剂(阿那白滞素)已成功应用于类风湿性关节炎和急性痛风性关节炎的治疗中，但由于其在体内半衰期短，患者需要反复给药达到治疗效果，而且价格昂贵。
发明内容
有鉴于此，本发明提供了一种重组蛋白及其制备方法和应用，该复合物包括人源IL-1RA蛋白和含VPGKG重复序列的蛋白，可用于包括类风湿性关节炎、急性痛风性关节炎及其它以白介素1(interleukin-1，IL-1)异常增高为显著特点的风湿免疫病、遗传性疾病及肿瘤的辅助治疗，疗效显著。
为了实现上述发明目的，本发明提供以下技术方案：
本发明第一方面提供了一种重组蛋白，包括人源IL-1RA蛋白和含VPGKG重复序列的蛋白。
在一些实施方案中，所述人源IL-RA序列来自人源IL-1RA蛋白剪接体1(SEQ ID NO 1)、人源IL-1RA蛋白剪接体2(SEQ ID NO 2)、人源IL-1RA蛋白剪接体3(SEQ ID NO 3)或人源IL-1RA蛋白剪接体4(SEQ ID NO 4)。在一些优选的实施方案中，所述人源IL-RA序列来自人源IL-1RA蛋白剪接体3。具体地，所述人源IL-1RA蛋白具有如SEQ ID NO.1～SEQ ID NO.4所示的任意一种氨基酸序列；或具有与SEQ ID NO.1～SEQ ID NO.4所示序列功能相同或相似的氨基酸序列。
在一些实施方案中，所述含VPGKG重复序列的蛋白由n个重复单元(VPGKG)和m个重复单元(VPGXG)组成，其中，X为任意一种天然氨基酸，m、n为大于零的整数；优选地，m为0～20之间的整数，n为1～150之间的整数；重复单元(VPGKG)和(VPGXG)随意组合。在一些实施方案中，含有(VPGKG)序列的重复蛋白单元具有(VPGXG) m(VPGKG) n特征的重复单元结构。在一些实施方案中，X为缬氨酸、丙氨酸或甘氨酸。在一些优选的实施方案中，重复蛋白单元具有[(VPGAG) 2(VPGKG) 8] 9特征结构；在一些优选的实施方案中，重复蛋白单元具有[(VPGGG) 1(VPGKG) 3(VPGAG) 2(VPGKG) 5(VPGVG) 2(VPGKG) 9] 5特征结构；在一些优选的实施方案中，重复蛋白单元具有[VPGVG(VPGKG) 9] 6特征结构；在另一些优选的 实施方案中，重复蛋白单元具有[VPGVG(VPGKG) 9] 8特征结构。
在一些实施方案中，IL-1RA蛋白序列和含有(VPGKG)序列的重复蛋白单元序列间通过肽键或者连接肽连接。在一些优选的实施方案中，IL-1RA序列和含有(VPGKG)序列的重复蛋白单元序列由连接肽连接。所述连接肽为多个连续的任意氨基酸。一些实施方案种，所述连接肽具有重组蛋白全长的10％以下的氨基酸个数。一些优选实施方案中，所述多个为13个、14个或18个。一些具体实施例中，所述连接肽的氨基酸序列如SEQ ID NO.5～SEQ ID NO.7中任一项所示。
在一些实施方案中，本发明重组蛋白还包含该蛋白的保守性修饰变体。所述保守性修饰变体包括本发明重组蛋白中的10％的氨基酸，优选5％的氨基酸，更优选2％的氨基酸，最优选1个氨基酸被其功能上相似的保守性氨基酸全部、或者部分置换所得的变体。所述保守性置换是本领域公知的，包括以下6组氨基酸：
丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
天冬氨酸(D)、谷氨酸(E)；
天冬酰胺(N)、谷氨酰胺(Q)；
精氨酸(R)、赖氨酸(K)；
异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；
苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
由此获得的重组蛋白变体的总净电荷和其分子上电荷分布与置换前基本上保持相同。
在一些实施方案中，本发明重组蛋白具有SEQ ID NO.13～SEQ ID NO.72所示的任意一种氨基酸序列，或具有与SEQ ID NO.13～SEQ ID NO.72所示的任一序列至少80％同一性的氨基酸序列，或具有与SEQ ID NO.13～SEQ ID NO.72所示的任一序列功能相同或相似的氨基酸序列。
在一些实施方案中，所述重组蛋白具有与SEQ ID NO.13～SEQ ID NO.72中任一个至少85％、至少90％、至少95％，优选至少98％、更优选至少99％的序列同一性、且与其功能相同或相似的氨基酸序列。其中，功能相同或相似具体为与白介素1β受体的亲和活性相同或相似。
在一些更优选的实施方案中，所述重组蛋白具有SEQ ID NO.13、SEQ ID NO.21、SEQ ID NO.27、SEQ ID NO.33、SEQ ID NO.40、SEQ ID NO.45、SEQ ID NO.52、SEQ ID NO.56、SEQ ID NO.60、SEQ ID NO.72中任一个所示的氨基酸序列。
本发明的第二方面提供了编码本发明重组蛋白的核酸。
在一些实施方案中，所述核酸具有：
SEQ ID NO.73～SEQ ID NO.132所示的任意一种核苷酸序列；或
与SEQ ID NO.73～SEQ ID NO.132所示序列80％以上同一性核苷酸序列；或
与SEQ ID NO.73～SEQ ID NO.132所示序列编码相同蛋白，但因遗传密码的简并性而与SEQ ID NO.73～SEQ ID NO.132所示序列不同的核苷酸序列，或与SEQ ID NO.73～SEQ ID NO.132所示序列编码蛋白具有85％以上同一性的核苷酸序列。
在一些实施方案中，所述核酸具有与SEQ ID NO.73～SEQ ID NO.132中任一个至少85％、至少90％、至少95％，优选至少98％、更优选至少99％的序列同一性的核苷酸序列。
在一些实施方案中，所述核酸可由编码相同氨基酸的不同的连续三个碱基的密码子所替换。所述同氨基酸碱基替换是本领域所公知的，包含以下20种密码子替换：
1)苯丙氨酸(F)：TTT、TTC；
2)亮氨酸(L)：TTA、TTG、CTT、CTC、CTA、CTG；
3)异亮氨酸(I)：ATT、ATC、ATA；
4)缬氨酸(V)：GTT、GTC、GTA、GTG；
5)丝氨酸(S)：TCT、TCC、TCA、TCG；
6)脯氨酸(P)：CCT、CCC、CCA、CCG；
7)苏氨酸(T)：ACT、ACC、ACA、ACG；
8)丙氨酸(A)：GCT、GCC、GCA、GCG；
9)酪氨酸(Y)：TAT、TAC；
10)组氨酸(H)：CAT、CAC；
11)谷氨酰胺(Q)：CAA、CAG；
12)天冬酰胺(N)：AAT、AAC；
13)赖氨酸(K)：AAA、AAG；
14)天冬氨酸(D)：GAT，GAC；
15)谷氨酸(E)：GAA、GAG；
16)半胱氨酸(C)：TGT、TGC；
17)精氨酸(R)：CGT、CGC、CGA、CGG、AGA、AGG；
18)丝氨酸(S)：AGT、AGC；
19)甘氨酸(G)：GGT、GGC、GGA、GGG；
20)终止密码子：TAA、TAG、TGA。
由此获得的核酸变体编码与原核酸相同的氨基酸序列。
本发明第三方面提供了包含本发明第二方面的核酸的表达载体。
在一些实施方案中，所述表达载体的骨架载体为pET25b。
本发明第四方面提供了转化或转染本发明第三方面的表达载体的宿主细胞。
本发明的第五方面提供了制备本发明第一方面的重组蛋白的制备方法。
在一些实施方案中，该制备方法包括：培养本发明第四方面的宿主细胞，诱导所述重组蛋白的表达。
在一些实施方案中，所述制备方法具体包括以下步骤：
1)人工合成编码本发明重组蛋白的核酸序列，并搭载于原核表达载体上，获得表达质粒；
2)将表达质粒转入原核表达宿主细胞中；
3)培养和诱导原核表达细胞表达重组蛋白；
4)分离纯化后获得的重组蛋白。
在一些实施方案中，所述方法的步骤1)中采用的原核表达载体是pET25b。
在一些实施方案中，所述方法步骤2)中的原核表达载体细胞是大肠杆菌细胞。
在一些实施方案中，所述方法的步骤4)中的分离和纯化过程包括离心、盐析盐溶、阳离子交换层析、分子筛层析和超滤。在一些具体的实施方案中，阳离子交换层析缓冲液pH范围为8.0～9.5，在一些更优选的实施方案中，阳离子交换层析缓冲液pH范围为8.8～9.0。在一些具体的实施方案中，阳离子交换层析洗脱缓冲液中NaCl含量为200～500mM；在一些优选的实施方案中，阳离子交换层析洗脱缓冲液中NaCl含量为300～500mM；在一些更优选的实施方案中，阳离子交换层析洗脱缓冲液中NaCl含量为400mM。
本发明的第六方面提供了本发明第一方面的重组蛋白与聚乙二醇(PEG)组合形成的蛋白复合物及其制备方法。
在一些实施方案中，所述重组蛋白与PEG通过共价结合或非共价结合的方式组合形成蛋白复合物。在一些优选的实施方案中，重组蛋白与PEG的摩尔比例1：0.25n～1：5n；在一些更优选的实施方案中，重组蛋白与PEG的摩尔比例为1：0.5n～1：2n；在一些更优选的实施方案中，重组蛋白与PEG的摩尔比例为1：n。
在一些优选的实施方案中，PEG的平均分子量1000～40000；在一些优选的实施方案中，PEG平均分子量为1000～10000；在一些更优选的实施方案中，PEG平均分子量为5000。
在一些优选的实施方案中，PEG为经过羧基化修饰的聚乙二醇分子，该分子通过与重组蛋白在缓冲溶液中混合后得到蛋白复合物。所述缓冲液pH范围优选是6.0～8.0，更优选的pH范围为7.0～7.5，尤其是7.2～7.4；所述反应时间范围是0～24h。
在一些实施方案中，所述重组蛋白与PEG通过共价结合的方式形成蛋白复合物。在一些优选的实施方案中，PEG末端通过马来酰亚胺化修饰后，通过与重组蛋白在缓冲溶液中混合后得到蛋白复合物。所述缓冲液pH范围优选是6.0～8.5，更优选的pH范围为7.0～8.5，尤其是7.5～8.0；所述反应时间范围是0～72h，优选的时间范围是0h～24h，更优选的时间范围是12～24h。
在一些实施方案中，制备蛋白复合物时未成功连接的PEG通过纯化的方法除去。在一些具体的实施方案中，纯化的手段包括分子筛层析和透析。
本发明的第七方面提供了本发明第一方面的重组蛋白与硫酸软骨素(chondroitin sulfate，CS)组合形成的蛋白复合物及其制备方法。
在一些实施方案中，所述重组蛋白与CS通过非共价结合的方式组合形成蛋白复合物。在一些优选的实施方案中，重组蛋白与CS的摩尔比例1：0.25n～1：5n；在一些更优选的实施方案中，重组蛋白与CS的摩尔比例为1：0.5n～1：2n；在一些更优选的实施方案中，重组蛋白与CS的摩尔比例为1：n。
在一些优选的实施方案中，CS通过与重组蛋白在缓冲溶液中混合后得到蛋白复合物。所述缓冲液pH范围优选是6.0～8.0，更优选的pH范围为7.0～7.5，尤其是7.2～7.4； 所述反应时间范围是0～1h。
在一些实施方案中，制备蛋白复合物时未成功连接的CS通过纯化的方法除去。在一些具体的实施方案中，纯化的手段包括分子筛层析和透析。
本发明第八方面提供了本发明第一方面的重组蛋白和第六方面的蛋白复合物与其它药物活性成分、药学上可接受的载体、药用辅料或赋形剂组成的药物；或
发明第一方面的重组蛋白和第七方面的蛋白复合物与其它药物活性成分、药学上可接受的载体、药用辅料或赋形剂组成的药物；
在一些实施方案中，药物活性成分包括一种或多种以下成分：依那西普(Rilonacept)，英夫利昔单抗(INFLECTRA)，沙立度胺、类固醇(如可的松、泼尼松龙、泼尼松、甲基强的松、地塞米松、倍他米松、曲安奈德、倍氯米松、氟替卡松)、阿那白滞素(anakinra)、秋水仙碱、IL-18结合蛋白(IL-18BP)或衍生物、IL-18抗体、IL-18受体(IL-18R1)抗体、IL-18受体辅助蛋白(IL-18Racp)抗体、阿司匹林、甲氨蝶呤、环胞菌素A、胱天蛋白酶-1、IKK1/2、细胞毒T细胞抗原4(CTLA-4Ig)、IL-6抗体和IL-6RA抗体。
本发明第九方面提供了本发明第一方面的重组蛋白、本发明第六方面的蛋白复合物、本发明第七方面的蛋白复合物或本发明第八方面的药物用于预防和/或治疗制备用于动物预防和/或治疗疾病的药物中的应用。所述治疗优选恢复、进一步优选完全恢复；所述哺乳动物优选为灵长类动物，更优选为人类；所述疾病优选为风湿免疫病，更优选为人类的白介素1异常增高为显著特点的风湿免疫类疾病。
在一些实施方案中，所述疾病为以白介素1异常增高为显著特点的疾病，具体包括风湿免疫疾病、遗传性疾病及肿瘤。具体地，所述“存在白介素1增多的疾病形式”包括但不限于类风湿性关节炎、侵蚀性骨关节炎、坏疽性脓皮病、聚会性痤疮和无菌性关节炎、痛风和假性痛风、全身型特发性幼年型关节炎、动脉粥样硬化、淀粉样蛋白A淀粉样变性、成人期斯蒂尔病、哮喘、白塞氏病、布劳综合征、二水焦磷酸钙结晶沉积疾病、卡斯尔曼病、冷吡啉相关周期性综合征、白介素-1受体拮抗剂缺陷、皮肌炎、埃德海姆-切斯特病、家族性地中海热、银屑病、移植物抗宿主病、化脓性汗腺炎、高IgD综合征、先天性寒冷性荨麻疹、包涵体肌炎、炎症性肠病、缺血性损伤、巨噬细胞活化综合征、马吉德综合征、甲羟戊酸激酶缺乏症、骨髓性及其他白血病、骨质疏松症、嗜中性脂膜炎、伴有口疮性口炎、咽炎和淋巴结炎的周期性发热、多发性肌炎、复发性先天性心包炎、复发性多软骨炎、再灌注损伤、施尼茨勒综合征、骨髓瘤、滑膜炎痤疮脓疱病骨质增生性骨炎、肿瘤坏死因子受体相关周期性综合征、1型糖尿病、2型糖尿病、荨麻疹性血管炎、葡萄膜炎、新生儿期起病的多系统炎性病症、穆-韦综合征、家族性寒冷型自身炎症性综合征、肿瘤的辅助治疗、肿瘤放疗并发症的治疗、放射性损伤如放射性口腔黏膜炎、放射性肺炎、放射性食道炎、放射性胃肠炎、放射性膀胱炎、放射性阴道炎、放射性皮肤炎症。
在一些实施方案中，所述重组蛋白、蛋白复合物及药物优选通过皮下或静脉或肌肉途径进行给药，更优选通过皮下途径进行给药。
本发明的第十方面提供了本发明第一方面的重组蛋白或第六方面的蛋白复合物或第七方面的蛋白复合物在制备预防和/或治疗灵长类动物由炎症因子介导的炎症的药物中的应用。其中，所述炎症因子为白介素1。
本发明的第十一方面提供了一种治疗以白介素1异常增高为显著特点的风湿免疫类疾病、遗传类疾病、以及肿瘤的辅助治疗的治疗方法，包括向受试者施用本发明第一方面的重组蛋白、本发明第六方面的蛋白复合物、本发明第七方面的蛋白复合物或者本发明第八方面的药物。
在一些实施方案中，所述受试者为哺乳动物，优选为灵长类动物，更优选为人。
在一些实施方案中，所述重组蛋白、蛋白复合物、复合药物优选通过皮下或静脉或肌肉途径进行给药，更优选通过皮下途径进行给药。
本发明重组蛋白由人源白介素1受体拮抗剂蛋白与含有(VPGKG)n重复单元的蛋白组成。本发明重组蛋白显著地延长了IL-1RA在大鼠体内的血药浓度半衰期，对大鼠急性痛风性关节炎、风湿性关节炎具有显著疗效，与单独人源白介素1受体拮抗剂相比差异显著(p<0.001)。表明，该重组蛋白可用于包括类风湿性关节炎、急性痛风性关节炎及其它存在以白介素1异常增高为显著特点的风湿免疫病、遗传性疾病及肿瘤的辅助治疗，疗效更加显著。
附图说明
图1示重组蛋白SDS-PAGE电泳图；
图2示重组蛋白复合物的电镜照片；
图3示蛋白复合物的平均粒径；
图4示蛋白复合物和IL-1RA蛋白在大鼠体内血药浓度-时间曲线；
图5示蛋白复合物和IL-1RA蛋白注射后各组大鼠踝关节周长变化；
图6示蛋白复合物和IL-1RA蛋白注射后各组大鼠足垫厚度变化。
具体实施方式
本发明提供了一种重组蛋白、含该重组蛋白的蛋白复合物和应用。本领域技术人员可以本领域技术人员可以通过完全重复，或基于本文内容适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明内。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试剂与材料皆为普通市售品，皆可于市场购得。
本发明实施例中涉及的引物序列如表1所示。
表1引物序列
Figure PCTCN2021138269-appb-000001
Figure PCTCN2021138269-appb-000002
Figure PCTCN2021138269-appb-000003
Figure PCTCN2021138269-appb-000004
Figure PCTCN2021138269-appb-000005
Figure PCTCN2021138269-appb-000006
Figure PCTCN2021138269-appb-000007
Figure PCTCN2021138269-appb-000008
Figure PCTCN2021138269-appb-000009
Figure PCTCN2021138269-appb-000010
Figure PCTCN2021138269-appb-000011
Figure PCTCN2021138269-appb-000012
Figure PCTCN2021138269-appb-000013
Figure PCTCN2021138269-appb-000014
Figure PCTCN2021138269-appb-000015
Figure PCTCN2021138269-appb-000016
Figure PCTCN2021138269-appb-000017
Figure PCTCN2021138269-appb-000018
Figure PCTCN2021138269-appb-000019
Figure PCTCN2021138269-appb-000020
Figure PCTCN2021138269-appb-000021
Figure PCTCN2021138269-appb-000022
Figure PCTCN2021138269-appb-000023
Figure PCTCN2021138269-appb-000024
Figure PCTCN2021138269-appb-000025
Figure PCTCN2021138269-appb-000026
Figure PCTCN2021138269-appb-000027
Figure PCTCN2021138269-appb-000028
Figure PCTCN2021138269-appb-000029
Figure PCTCN2021138269-appb-000030
Figure PCTCN2021138269-appb-000031
Figure PCTCN2021138269-appb-000032
Figure PCTCN2021138269-appb-000033
Figure PCTCN2021138269-appb-000034
Figure PCTCN2021138269-appb-000035
Figure PCTCN2021138269-appb-000036
Figure PCTCN2021138269-appb-000037
Figure PCTCN2021138269-appb-000038
Figure PCTCN2021138269-appb-000039
Figure PCTCN2021138269-appb-000040
Figure PCTCN2021138269-appb-000041
Figure PCTCN2021138269-appb-000042
Figure PCTCN2021138269-appb-000043
Figure PCTCN2021138269-appb-000044
Figure PCTCN2021138269-appb-000045
下面结合实施例，进一步阐述本发明：
实施例1重组蛋白的制备
在本实施例中，发明人基于(VPGKG)重复多肽序列和人源白介素1受体拮抗剂(IL-1RA)序列设计了包括如下10种重组蛋白序列，具体序列信息参见表2：
表2重组蛋白序列特征
Figure PCTCN2021138269-appb-000046
1.1获得表达重组蛋白的菌株
以上所述10种重组蛋白对应核苷酸序列由生工生物(上海)公司合成，并构建于pET25b表达质粒载体上，用于转化入大肠杆菌原核表达感受态BLR(DE3)中。具体的实施方案如下：将100ng的pET25b-A1(或A2～A10)质粒在冰浴环境中加入100μL的BLR大肠杆菌感受态中(购自Novagen公司)，并在冰浴环境中维持30min；然后将混合物转入42℃水浴中热激45s，随后冰浴2min。向混合物中加入LB培养基600μL，于37℃、180rpm震荡环境中处理1h。将混合物均匀涂布在含有氨苄青霉素钠(15μg/mL)抗生素的LB固体培养基上，37℃培养18h，即可获得可以稳定表达重组蛋白的菌株。挑取生长的菌落接种在10mL LB培养基中，于37℃、220rpm震荡环境中培养，并在培养混合物OD600值达到0.6时，用异丙基硫代半乳糖苷(IPTG，终浓度0.5mmol/L)进行诱导培养，于16℃、220rpm震荡环境中培养过夜。诱导完成后，将菌液以4000rpm离心30min，弃去上清；用PBS重悬菌体沉淀，再次以4000rpm离心30min，弃去上清，得到含有目的蛋白的菌体沉淀。通过SDS-PAGE电泳和免疫印迹实验鉴定正确表达重组蛋白的菌株。
1.2重组蛋白的表达
将可以正确表达重组蛋白的菌株转入50mL含有氨苄青霉素钠(15μg/mL)抗生素的LB培养基中，于37℃、220rpm震荡环境中培养4-6h；当OD600达到0.6时将菌液转入1L新鲜的含有氨苄青霉素钠(15μg/mL)抗生素的LB培养基中，于37℃、220rpm震荡环境中培养至OD600达到0.6，然后加入终浓度为0.5mmol/L的IPTG诱导菌体表达重组蛋白，并调节环境温度至16℃。诱导培养4-18h后，将菌液以4000rpm离心30min，弃去上清；用PBS重悬菌体沉淀，再次以4000rpm离心30min，弃去上清，得到含有目的蛋白的菌体沉淀。
1.3重组蛋白的纯化
用4℃预冷的50mM磷酸盐缓冲液(PB)重悬菌体,其pH为7.2-7.4，并用高压均质机将菌体混合液破碎。收集混合物并于16000rpm、4℃条件下离心30min，收集上清。向上清中滴加4℃预冷的饱和硫酸铵水溶液至饱和硫酸铵终浓度为20％，将混合物于16000rpm、4℃条件下离心30min，收集上清。继续向上清中滴加4℃预冷的饱和硫酸铵水溶液至饱和硫酸铵终浓度为50％，将混合物于16000rpm、4℃条件下离心30min，收集沉淀。在4℃环境中向沉淀中缓慢滴加50mMPB缓冲液，并调节pH至9.0，缓慢搅拌至沉淀完全溶解。用SP阳离子交换层析对蛋白液进行进一步纯化。将蛋白液上样于10mL SP阳离子交换层析柱(购自GE公司)后，相继用50mL含有50mM、100mM、200mM、400mMNaCl的50mMPB缓冲液对蛋白进行洗脱，并收集含有400mMNaCl的洗脱液组分。将该洗脱组分于Superdex 100(购自GE公司)中进行分子筛层析。即将含有400mMNaCl的洗脱液组分的蛋白上样于Superdex 100分子筛层析柱中，并用pH为7.2-7.4的50mM磷酸盐缓冲液(PB)进行洗脱，收集目的蛋白组分，得到纯化产物。纯化蛋白经SDS-PAGE凝胶电泳进行鉴定，如图1。
实施例2蛋白复合物的制备
蛋白复合物1：将纯化的重组蛋白A1与羧基化的PEG按照摩尔比1：18的比例混合，在4℃环境搅拌1h。将混合物上样于脱盐层析柱(购自GE公司)中，用pH为7.2-7.4的50mM PB缓冲液进行洗脱，收集蛋白复合物组分。
蛋白复合物2：将纯化的重组蛋白A2与羧基化的PEG按照摩尔比1：72的比例混合，在4℃环境搅拌1h。将混合物上样于脱盐层析柱(购自GE公司)中，用pH为7.2-7.4的50mM PB缓冲液进行洗脱，收集蛋白复合物组分。
蛋白复合物3：将纯化的重组蛋白A3与羧基化的PEG按照摩尔比1：288的比例混合，在20-25℃环境搅拌4h。将混合物上样于脱盐层析柱(购自GE公司)中，用pH为7.2-7.4的50mM PB缓冲液进行洗脱，收集蛋白复合物组分。
蛋白复合物4：将纯化的重组蛋白A4与羧基化的PEG按照摩尔比1：9的比例混合，在20-25℃环境搅拌4h。将混合物上样于脱盐层析柱(购自GE公司)中，用pH为7.2-7.4的50mM PB缓冲液进行洗脱，收集蛋白复合物组分。
蛋白复合物5：将纯化的重组蛋白A5与羧基化的PEG按照摩尔比1：54的比例混合，在2-8℃环境搅拌24h。将混合物转入透析管中，于2-8℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物6：将纯化的重组蛋白A6与羧基化的PEG按照摩尔比1：162的比例混合，在20-25℃环境搅拌2h。将混合物转入透析管中，于2-8℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物7：将纯化的重组蛋白A7与马来酰亚胺修饰的PEG按照摩尔比1： 720的比例混合，在37℃水浴环境中搅拌30min。将混合物上样于脱盐层析柱(购自GE公司)中，用pH为7.2-7.4的50mM PB缓冲液进行洗脱，收集蛋白复合物组分。
蛋白复合物8：将纯化的重组蛋白A8与羧基化的PEG按照摩尔比1：72的比例混合，在20-25℃环境搅拌1h。将混合物上样于脱盐层析柱(购自GE公司)中，用pH为7.2-7.4的50mM PB缓冲液进行洗脱，收集蛋白复合物组分。
蛋白复合物9：将纯化的重组蛋白A9与马来酰亚胺修饰的PEG按照摩尔比1：108的比例混合，在37℃水浴环境中搅拌30min。将混合物转入透析管中，于2-8℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物10：将纯化的重组蛋白A10与马来酰亚胺修饰的PEG按照摩尔比1：144的比例混合，在20-25℃水浴环境中搅拌30min。将混合物转入透析管中，于2-8℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物11：将纯化的重组蛋白A1与CS按照摩尔比1：162的比例混合，在20-25℃环境搅拌10min。将混合物转入透析管中，于2-8℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物12：将纯化的重组蛋白A2与CS按照摩尔比1：72的比例混合，在20-25℃环境搅拌30min。将混合物转入透析管中，于2-8℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物13：将纯化的重组蛋白A3与CS按照摩尔比1：720的比例混合，在2-8℃环境搅拌1h。将混合物转入透析管中，于2-8℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物14：将纯化的重组蛋白A4与CS按照摩尔比1：90的比例混合，在2-8℃环境搅拌1h。将混合物转入透析管中，于2-8℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物15：将纯化的重组蛋白A5与CS按照摩尔比1：180的比例混合，在37℃环境搅拌10min。将混合物转入透析管中，于2-8℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物16：将纯化的重组蛋白A6与CS按照摩尔比1：54的比例混合，在37℃环境搅拌10min。将混合物转入透析管中，于2-8℃环境中进行透析反应，透析 置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物17：将纯化的重组蛋白A7与CS按照摩尔比1：432的比例混合，在37℃环境搅拌10min。将混合物转入透析管中，于2-8℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物18：将纯化的重组蛋白A8与CS按照摩尔比1：72的比例混合，在20～25℃环境搅拌30min。将混合物转入透析管中，于25℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物19：将纯化的重组蛋白A9与CS按照摩尔比1：324的比例混合，在20～25℃环境搅拌30min。将混合物转入透析管中，于25℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
蛋白复合物20：将纯化的重组蛋白A10与CS按照摩尔比1：288的比例混合，在20～25℃环境搅拌30min。将混合物转入透析管中，于25℃环境中进行透析反应，透析置换液为pH为7.2-7.4的50mM PB缓冲液，透析置换液体积为蛋白复合物溶液体积的至少10倍；每8h更换一次透析置换液。透析24h后收集蛋白复合物组分。
实施例3：蛋白复合物粒径测定
将实施例2纯化获得的蛋白复合物进行透射电镜的检测，并对蛋白平均粒径进行分析。结果见图2～3，图2表示蛋白复合物电镜图片。图3表示蛋白的平均粒径分析结果。
实施例4：蛋白复合物在大鼠中的体内药物代谢变化检测
取体重为200±20g的雄性SD大鼠36只，经异氟烷麻醉后于眶下静脉采血，并立即于1100g、4℃环境下离心20min，取上清于-80℃中保存。然后将大鼠随机分为12组，每组3只大鼠。分别于颈部皮下三点注射0.1μmol的蛋白复合物A1～A10、IL-RA蛋白，以及同等体积的生理盐水。给药后，对所有大鼠在0.5h、1h、2h、4h、6h、8h、12h、18h、24h、30h、36h、48h、60h、72h、96h、120h、144h、168h时间点经异氟烷麻醉后于眶下静脉采血，并立即于1100g、4℃环境下离心20min，取上清于-80℃中保存。血浆样品用人源IL-1RA检测试剂盒检测血药中IL-1RA含量，并计算各组药物在体内半衰期。图4展示了蛋白复合物A8和IL-1RA蛋白在大鼠体内半衰期曲线；表3展示了各组蛋白复合物/蛋白在大鼠血药浓度半衰期的计算结果。
表3各组蛋白复合物/蛋白在大鼠血药浓度半衰期
蛋白/蛋白复合物 血药半衰期(小时) 蛋白/蛋白复合物 血药半衰期(小时)
IL-1RA蛋白 6.95 蛋白复合物11 14.45
蛋白复合物1 14.17 蛋白复合物12 20.95
蛋白复合物2 25.31 蛋白复合物13 34.57
蛋白复合物3 30.22 蛋白复合物14 18.48
蛋白复合物4 16.42 蛋白复合物15 25.23
蛋白复合物5 22.49 蛋白复合物16 19.00
蛋白复合物6 22.67 蛋白复合物17 28.99
蛋白复合物7 31.94 蛋白复合物18 28.97
蛋白复合物8 27.16 蛋白复合物19 21.85
蛋白复合物9 23.94 蛋白复合物20 34.28
蛋白复合物10 33.08    
实施例5：蛋白复合物在大鼠急性痛风性关节炎模型中的治疗效果
取8周龄的雄性SD大鼠65只，并对大鼠进行标记，并测量所有大鼠踝关节周长。然后对大鼠进行痛风性关节炎疾病造模：向所有大鼠的踝关节腔中注射50μL的60mg/mL尿酸钠晶体悬浊液。检测所有大鼠踝关节周长。
将所有造模型成功的大鼠随机分为13组，分别于颈部皮下三点注射0.1μmol/100μL/剂的蛋白复合物1～10、IL-RA蛋白，以及同等体积的生理盐水和吲哚美辛。分别检测所有大鼠在第0h，及给药后第6h、12h、24h、48h、72h时的踝关节周长。图5展示了给药后大鼠踝关节周长变化。结果表明，本发明蛋白复合物对大鼠急性痛风性关节炎具有显著疗效，效果显著优于IL-1RA蛋白和吲哚美辛(P<0.001)。
实施例6蛋白复合物在大鼠类风湿性关节炎模型中的治疗效果
取8周龄的雌性SD大鼠60只，并对大鼠进行标记，并测量所有大鼠足垫厚度。然后对大鼠进行类风湿性关节炎疾病造模：将2mg/mL的Ⅱ型胶原蛋白PBS溶液与等体积的完全弗氏佐剂混合，并于超声作用下混合成均匀的油包水乳剂，获得免疫原1。随后向所有大鼠的足垫和尾根部皮下注射免疫原1。14天后，对所有大鼠进行一次加强免疫。将2mg/mL的Ⅱ型胶原蛋白PBS溶液与等体积的不完全弗氏佐剂混合，并于超声作用下混合成均匀的油包水乳剂，获得免疫原2。随后向所有大鼠的足垫和尾根部皮下注射免疫原2。
在第15天对所有造模成功的大鼠随机分为12组，分别于颈部皮下三点注射0.1μmol/100μL/剂的蛋白复合物11～20、IL-RA蛋白，以及同等体积的生理盐水。分别检测所有大鼠在第0天，及给药后第1、2、4、6、9、11、14天足垫厚度。结果见图5，图6为蛋白复合物和IL-1RA蛋白注射后各组大鼠足垫厚度变化。
结果表明，本发明蛋白复合物对类风湿性关节炎的治疗效果显著优于IL-1RA蛋白的治疗效果(p<0.01)。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (14)

  1. 重组蛋白，包括人源IL-1RA蛋白和含(VPGKG)重复序列的蛋白。
  2. 根据权利要求1所述的重组蛋白其特征在于，所述人源IL-1RA蛋白和含VPGKG重复序列的蛋白通过肽键直接相连或通过连接肽相连。
  3. 根据权利要求1所述的重组蛋白，其特征在于，所述人源IL-1RA蛋白具有如SEQ ID NO.1～SEQ ID NO.4所示的任意一种氨基酸序列；或具有与SEQ ID NO.1～SEQ ID NO.4所示序列功能相同或相似的氨基酸序列。
  4. 根据权利要求1所述的重组蛋白，其特征在于，所述含(VPGKG)重复序列的蛋白由n个重复单元(VPGKG)和m个重复单元(VPGXG)组成；其中，X为任意一种天然氨基酸，m、n为大于零的整数；优选地，m为0～20之间的整数，n为1～150之间的整数；重复单元(VPGKG)和(VPGXG)随意组合；优选地，X为缬氨酸、丙氨酸或甘氨酸。
  5. 根据权利要求2所述的重组蛋白，其特征在于，所述连接肽由多个连续的任意氨基酸氨基组成。
  6. 根据权利要求1～5任一项所述的重组蛋白，其特征在于，具有SEQ ID NO.13～SEQ ID NO.72所示的任意一种氨基酸序列，或具有与SEQ ID NO.13～SEQ ID NO.72所示的任一序列至少80％同一性的氨基酸序列，或具有与SEQ ID NO.13～SEQ ID NO.72所示的任一序列功能相同或相似的氨基酸序列。
  7. 编码权利要求1～6任一项所述重组蛋白的核酸，其特征在于，具有：
    SEQ ID NO.73～SEQ ID NO.132所示的任意一种核苷酸序列；或
    与SEQ ID NO.73～SEQ ID NO.132所示序列至少有80％同一性的核苷酸序列；或
    与SEQ ID NO.73～SEQ ID NO.132所示序列编码相同蛋白，但因遗传密码的简并性而与SEQ ID NO.73～SEQ ID NO.132所示序列不同的核苷酸序列。
  8. 权利要求1～6任一项所述的重组蛋白的制备方法，其特征在于：
    (1)构建权利要求7所述核酸的表达载体；
    (2)将权利要求7所述核酸表达载体转化或转染入宿主细胞；
    (3)培养宿主细胞，诱导权利要求7所述重组蛋白的表达。
  9. 蛋白复合物，包含权利要求1～6任一项所述的重组蛋白和聚乙二醇。
  10. 根据权利要求9所述的蛋白复合物，其特征在于，所述重组蛋白与聚乙二醇的摩尔比为1:0.25n～1:5n。
  11. 蛋白复合物，包含权利要求1～6任一项所述的重组蛋白和硫酸软骨素(chondroitin sulfate，CS)。
  12. 根据权利要求11所述的蛋白复合物，其特征在于，所述重组蛋白与硫酸软骨素的摩尔比为1:0.25n～1:5n。
  13. 权利要求1～6任一项所述的重组蛋白、权利要求9～12任一项所述的蛋白复合物在制备预防和/或治疗疾病的药物中的应用；所述疾病具有白介素1增多的显著 特征；所述疾病包括类风湿性关节炎、侵蚀性骨关节炎、坏疽性脓皮病、聚会性痤疮和无菌性关节炎、痛风和假性痛风、全身型特发性幼年型关节炎、动脉粥样硬化、淀粉样蛋白A淀粉样变性、成人期斯蒂尔病、哮喘、白塞氏病、布劳综合征、二水焦磷酸钙结晶沉积疾病、卡斯尔曼病、冷吡啉相关周期性综合征、白介素-1受体拮抗剂缺陷、皮肌炎、埃德海姆-切斯特病、家族性地中海热、银屑病、移植物抗宿主病、化脓性汗腺炎、高IgD综合征、先天性寒冷性荨麻疹、包涵体肌炎、炎症性肠病、缺血性损伤、巨噬细胞活化综合征、马吉德综合征、甲羟戊酸激酶缺乏症、骨髓性及其他白血病、骨质疏松症、嗜中性脂膜炎、伴有口疮性口炎、咽炎和淋巴结炎的周期性发热、多发性肌炎、复发性先天性心包炎、复发性多软骨炎、再灌注损伤、施尼茨勒综合征、骨髓瘤、滑膜炎痤疮脓疱病骨质增生性骨炎、肿瘤坏死因子受体相关周期性综合征、1型糖尿病、2型糖尿病、荨麻疹性血管炎、葡萄膜炎、新生儿期起病的多系统炎性病症、穆-韦综合征、家族性寒冷型自身炎症性综合征、肿瘤的辅助治疗、肿瘤放疗并发症的治疗、放射性损伤如放射性口腔黏膜炎、放射性肺炎、放射性食道炎、放射性胃肠炎、放射性膀胱炎、放射性阴道炎、放射性皮肤炎症。
  14. 药物，其特征在于，包含权利要求1～6任一项所述蛋白或9～12任一项所述的蛋白复合物和以下药物活性成分中的一种或多种：
    依那西普(Rilonacept)，英夫利昔单抗(INFLECTRA)，沙立度胺、如可的松、泼尼松龙、泼尼松、甲基强的松、地塞米松、倍他米松、曲安奈德、倍氯米松、氟替卡松、阿那白滞素(anakinra)、秋水仙碱、IL-18结合蛋白(IL-18BP)或衍生物、IL-18抗体、IL-18受体(IL-18R1)抗体、IL-18受体辅助蛋白(IL-18Racp)抗体、阿司匹林、甲氨蝶呤、环胞菌素A、胱天蛋白酶-1、IKK1/2、细胞毒T细胞抗原4(CTLA-4Ig)、IL-6抗体和IL-6RA抗体。
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