CN112521514A

CN112521514A - Protein compound and preparation method and application thereof

Info

Publication number: CN112521514A
Application number: CN202011521081.1A
Authority: CN
Inventors: 刘凯; 张洪杰
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2021-03-19
Also published as: WO2022135231A1

Abstract

The invention relates to the technical field of molecular biology, in particular to a recombinant protein, a protein compound containing the recombinant protein and application of the protein compound. The recombinant protein consists of humanized interleukin 1 receptor antagonist protein and protein containing VPGKG repeating units, can be used for the adjuvant treatment of rheumatoid arthritis, acute gouty arthritis and other rheumatic diseases with interleukin 1(IL-1) increase, hereditary diseases and tumors, and has obvious curative effect.

Description

Protein compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a protein compound and a preparation method and application thereof.

Background

Rheumatoid arthritis is a human immune disease, and the research on the etiology of the disease is still in the early stage. In addition to the genetic factors of the patient, infection, immune system abnormalities, environmental factors (e.g., exposure to silica, infectious agents, vitamin D deficiency in the body), poor lifestyle habits (e.g., smoking, obesity), changes in the microbiota of the body, and many other factors contribute to the increased risk of the disease. Patients with rheumatoid arthritis have 18.6%, 6.0%, 9.9% and 4.4% morbidity for hypertension, diabetes, hyperlipidemia and obesity, respectively, with these secondary cardiovascular diseases being common causes of premature death in the patient. Because the condition of the patient is not greatly influenced in the early stage of the disease, and the compliance of the patient after repeated administration is poor, most patients are easy to actively suspend or terminate the treatment process, so the condition of the patient is further deepened. In the middle and later period of disease, obvious arthritis appears at joints of four limbs, elbows, shoulders, cervical vertebrae, hips, knees and the like of a patient, dropsy in joint cavities extrudes into the back side of the joint to form popliteal cyst, so that the acromelic is weak and even difficult to return to a normal position, and finally, the joint and the acromelic deformity appear. The advanced rheumatoid arthritis cannot be improved by drug treatment, and the joint function of a patient can be adjusted only by joint replacement surgery therapy, so that the life quality is improved; however, the people receiving treatment are mostly concentrated in the middle-aged and the elderly, the operation risk is high, and the treatment cost is high. The pathogenesis of rheumatoid arthritis is not clear, and the inducement is complex. Therefore, the first-line clinical medicines mainly relieve the discomfort of patients by mainly diminishing inflammation, relieving pain and relieving fever, and mainly comprise nonsteroidal anti-inflammatory drugs and glucocorticoids. The medicine has low price, can cause side effects such as liver and kidney, gastrointestinal tract injury and the like after long-term use, and can reduce the curative effect after repeated use. In addition, the disease modification rheumatoid drug can obviously relieve the arthritis and the acute arthritis, but has slow effect, and relatively higher metabolic pressure and psychological burden on patients.

The onset of gout is similar to rheumatoid arthritis. Due to the deficiency of purine metabolizing enzymes in primates, with increased high purine food intake and abnormal excretion, uric acid increases in the body and accumulates at the joints to form mono-natriuretic salt crystals (tophus), causing inflammation. The gout attack process can be divided into an asymptomatic hyperuricemia period, an acute period, an intermittent period and a chronic period. After the patient shifts from asymptomatic hyperuricemia to acute phase, reactions such as fatigue, general discomfort, joint stabbing pain and the like begin to appear; the onset of gout can lead to the patient awakening from late night due to severe pain in the joints, and the patient develops laceration-like, knife-cut-like or bite-like pain after 12 hours, which is difficult to tolerate. Redness, swelling, heat, pain and limited function of the affected joint and its surrounding tissues can occur. In the intermission period, the gout can be relieved automatically for a plurality of days or weeks after the gout attack, and generally has no obvious sequelae; after recurrence for months or years, however, the frequency of the disease gradually increases, the duration of the disease continues to be prolonged, and the range of the affected joints is expanded to the finger, wrist, elbow, hip, sacroiliac, sternoclavicular or spinal joints. Gout attacks enter the pathological stage of chronic tophus for about 10 years. At the moment, a large amount of mono-natrium urate crystals are deposited on the subcutaneous tissues, the synovial membranes of joints, the cartilages, the sclerotin and the soft tissues around the joints of the patients, and when the joints move, the urate crystals rub the synovial membranes of the joints, the cartilages and other tissues of the joints, so that the tissues of the joints are abraded, inflamed, the articular sclerotin of the joints of the patients is damaged, the joints of the patients are continuously swollen and painful, even have malformation and dysfunction, and the living activities and the life quality. The current first-line medicines for treating gout comprise allopurinol, febuxostat and a uric acid excretion promoting medicine benzbromarone; the second line of application comprises colchicine, etc. The medicines have no obvious treatment effect in part of people, are easy to relapse and have serious hepatotoxicity and hepatotoxicity.

During the attack of rheumatoid arthritis and gout, the content of interleukin 1(IL-1) in joints is obviously increased, and further local inflammatory reaction is triggered, so that tissue swelling, inflammatory cell infiltration and cartilage damage are caused. The interleukin-1 receptor antagonist (IL-1RA) is used for inhibiting the combination of the IL-1 receptor and the IL-1, so that the inflammatory reaction caused by the IL-1 can be quickly inhibited, and the disease development can be effectively slowed down. At present, human interleukin 1 receptor antagonist (anakinra) has been successfully applied to the treatment of rheumatoid arthritis and acute gouty arthritis, but because of the short half-life period in vivo, patients need to repeatedly administer the drug to achieve the treatment effect, and the price is high.

Disclosure of Invention

The compound comprises humanized IL-1RA protein and protein containing VPGKG repetitive sequence, can be used for the adjuvant treatment of rheumatoid arthritis, acute gouty arthritis and other rheumatic diseases, hereditary diseases and tumors which are characterized by the abnormal increase of interleukin 1(IL-1), and has obvious curative effect.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a recombinant protein, which comprises human IL-1RA protein and protein containing VPGKG repetitive sequence.

In some embodiments, the human IL-RA sequence is from human IL-1RA protein splicer 1(SEQ ID NO1), human IL-1RA protein splicer 2(SEQ ID NO 2), human IL-1RA protein splicer 3(SEQ ID NO 3), or human IL-1RA protein splicer 4(SEQ ID NO 4). In some preferred embodiments, the human IL-RA sequence is from human IL-1RA protein spliceosome 3. Specifically, the human IL-1RA protein has any one amino acid sequence shown in SEQ ID NO. 1-SEQ ID NO. 4; or has an amino acid sequence with the same or similar functions with the sequences shown in SEQ ID NO. 1-SEQ ID NO. 4.

In some embodiments, the VPGKG repeat sequence-containing protein consists of n repeat units (VPGKG) and m repeat units (VPGXG), wherein X is any one 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 combined at will. In some embodiments, the repeat protein unit comprising the (VPGKG) sequence has (VPGXG)_m(VPGKG)_nA repeating unit structure of a feature. In some embodiments, X is valine, alanine, or glycine. In some preferred embodiments, the repeat protein unit has [ (VPGAG)₂(VPGKG)₈]₉A characteristic structure; in some preferred embodiments, the repeating protein unit has [ (VPGGG)₁(VPGKG)₃(VPGAG)₂(VPGKG)₅(VPGVG)₂(VPGKG)₉]₅A characteristic structure; in some preferred embodiments, the repeat protein unit has [ VPGVG (VPGKG) ]₉]₆A characteristic structure; in other preferred embodiments, the repeat protein unit has [ VPGVG (VPGKG) ]₉]₈And (4) characteristic structure.

In some embodiments, the IL-1RA protein sequence and the sequence of repeat protein units comprising the (VPGKG) sequence are linked by a peptide bond or a linker peptide. In some preferred embodiments, the IL-1RA sequence and the sequence of the repeating protein unit comprising the (VPGKG) sequence are linked by a linker peptide. The connecting peptide is a plurality of continuous arbitrary amino acids. In some embodiments, the linker peptide has an amino acid number of 10% or less of the total length of the recombinant protein. In some preferred embodiments, the plurality is 13, 14 or 18. In some embodiments, the amino acid sequence of the linker peptide is as shown in any one of SEQ ID No.5 to SEQ ID No. 7.

In some embodiments, the recombinant proteins of the invention further comprise conservatively modified variants of the protein. The conservatively modified variants include variants in which 10% of the amino acids, preferably 5% of the amino acids, more preferably 2% of the amino acids, and most preferably 1 amino acid, of the recombinant protein of the invention are replaced, either entirely or in part, by conserved amino acids that are functionally similar. Such conservative substitutions are well known in the art and include the following 6 sets of amino acids:

alanine (a), serine (S), threonine (T);

aspartic acid (D), glutamic acid (E);

asparagine (N), glutamine (Q);

arginine (R), lysine (K);

isoleucine (I), leucine (L), methionine (M), valine (V);

phenylalanine (F), tyrosine (Y), tryptophan (W).

The total net charge and its molecular charge distribution of the recombinant protein variants thus obtained remain essentially the same as before the substitution.

In some embodiments, the recombinant protein of the invention has any one of the amino acid sequences shown in SEQ ID No.13 to SEQ ID No.72, or an amino acid sequence having at least 80% identity to any one of the sequences shown in SEQ ID No.13 to SEQ ID No.72, or an amino acid sequence functionally identical or similar to any one of the sequences shown in SEQ ID No.13 to SEQ ID No. 72.

In some embodiments, the recombinant protein has an amino acid sequence that is at least 85%, at least 90%, at least 95%, preferably at least 98%, more preferably at least 99% sequence identity to, and functionally identical or similar to, any one of SEQ ID No.13 to SEQ ID No. 72. Wherein, the same or similar functions are the same or similar with the affinity activity of interleukin 1 beta receptor.

In some more preferred embodiments, the recombinant protein has an amino acid sequence set forth in any one of 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.

In a second aspect, the invention provides a nucleic acid encoding a recombinant protein of the invention.

In some embodiments, the nucleic acid has:

any one nucleotide sequence shown in SEQ ID NO. 73-SEQ ID NO. 132; or

A nucleotide sequence which has more than 80 percent of identity with the sequence shown by SEQ ID NO. 73-SEQ ID NO. 132; or

The nucleotide sequence which is the same as the sequence shown in SEQ ID NO. 73-SEQ ID NO.132 in protein coding and is different from the sequence shown in SEQ ID NO. 73-SEQ ID NO.132 due to the degeneracy of the genetic code, or the nucleotide sequence which has more than 85 percent of identity with the sequence coding protein shown in SEQ ID NO. 73-SEQ ID NO. 132.

In some embodiments, the nucleic acid has a nucleotide sequence that 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.

In some embodiments, the nucleic acid may be replaced by a codon encoding different consecutive three bases of the same amino acid. Such homo-amino acid base substitutions are well known in the art and comprise the following 20 codon substitutions:

1) phenylalanine (F): TTT, TTC;

2) leucine (L): TTA, TTG, CTT, CTC, CTA, CTG;

3) isoleucine (I): ATT, ATC, ATA;

4) valine (V): GTT, GTC, GTA, GTG;

5) serine (S): TCT, TCC, TCA, TCG;

6) proline (P): CCT, CCC, CCA, CCG;

7) threonine (T): ACT, ACC, ACA, ACG;

8) alanine (a): GCT, GCC, GCA, GCG;

9) tyrosine (Y): TAT and TAC;

10) histidine (H): CAT, CAC;

11) glutamine (Q): CAA, CAG;

12) asparagine (N): AAT, AAC;

13) lysine (K): AAA, AAG;

14) aspartic acid (D): GAT, GAC;

15) glutamic acid (E): GAA, GAG;

16) cysteine (C): TGT, TGC;

17) arginine (R): CGT, CGC, CGA, CGG, AGA, AGG;

18) serine (S): AGT, AGC;

19) glycine (G): GGT, GGC, GGA, GGG;

20) stop codon: TAA, TAG, TGA.

The nucleic acid variant thus obtained encodes the same amino acid sequence as the original nucleic acid.

In a third aspect, the invention provides an expression vector comprising a nucleic acid according to the second aspect of the invention.

In some embodiments, the backbone vector of the expression vector is pET25 b.

In a fourth aspect, the invention provides a host cell transformed or transfected with the expression vector of the third aspect of the invention.

In a fifth aspect, the invention provides a method of producing a recombinant protein according to the first aspect of the invention.

In some embodiments, the method of making comprises: culturing the host cell of the fourth aspect of the invention and inducing expression of the recombinant protein.

In some embodiments, the method of preparation specifically comprises the steps of:

1) artificially synthesizing a nucleic acid sequence for encoding the recombinant protein of the invention, and carrying the nucleic acid sequence on a prokaryotic expression vector to obtain an expression plasmid;

2) transferring the expression plasmid into prokaryotic expression host cell;

3) culturing and inducing prokaryotic expression cells to express recombinant proteins;

4) separating and purifying the obtained recombinant protein.

In some embodiments, the prokaryotic expression vector employed in step 1) of the method is pET25 b.

In some embodiments, the prokaryotic expression vector cell in step 2) of the method is an E.coli cell.

In some embodiments, the separation and purification process in step 4) of the method comprises centrifugation, salting-out, cation exchange chromatography, molecular sieve chromatography, and ultrafiltration. In some specific embodiments, 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. In some specific embodiments, the cation exchange chromatography elution buffer has a NaCl content of 200 to 500 mM; in some preferred embodiments, the cation exchange chromatography elution buffer has a NaCl content of 300 to 500 mM; in some more preferred embodiments, the cation exchange chromatography elution buffer has a NaCl content of 400 mM.

In a sixth aspect, the invention provides a protein complex formed by combining the recombinant protein of the first aspect of the invention with polyethylene glycol (PEG), and a method for preparing the same.

In some embodiments, the recombinant protein is combined with PEG by covalent or non-covalent association to form a protein complex. In some preferred embodiments, the molar ratio of recombinant protein to PEG is 1:0.25 n-1: 5 n; in some more preferred embodiments, the molar ratio of recombinant protein to PEG is 1: 0.5 n-1: 2 n; in some more preferred embodiments, the molar ratio of recombinant protein to PEG is 1: n is the same as the formula (I).

In some preferred embodiments, the PEG has an average molecular weight of 1000 to 40000; in some preferred embodiments, PEG has an average molecular weight of 1000 to 10000; in some more preferred embodiments, the PEG has an average molecular weight of 5000.

In some preferred embodiments, PEG is a carboxylated modified polyethylene glycol molecule that is mixed with a recombinant protein in a buffer solution to obtain a protein complex. The pH range of the buffer solution is preferably 6.0-8.0, more preferably 7.0-7.5, especially 7.2-7.4; the reaction time range is 0-24 h.

In some embodiments, the recombinant protein and PEG form a protein complex by covalent attachment. In some preferred embodiments, the PEG terminus is modified by maleimide, followed by mixing with the recombinant protein in a buffer solution to obtain a protein complex. The pH range of the buffer solution is preferably 6.0-8.5, more preferably 7.0-8.5, especially 7.5-8.0; the reaction time range is 0-72 h, the preferable time range is 0-24 h, and the more preferable time range is 12-24 h.

In some embodiments, the unsuccessfully attached PEG is removed by purification methods when preparing the protein complex. In some specific embodiments, the means of purification includes 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 method for producing the same.

In some embodiments, the recombinant protein is combined with CS by non-covalent association to form a protein complex. In some preferred embodiments, the molar ratio of recombinant protein to CS is 1:0.25 n-1: 5 n; in some more preferred embodiments, the molar ratio of recombinant protein to CS is 1: 0.5 n-1: 2 n; in some more preferred embodiments, the molar ratio of recombinant protein to CS is 1: n is the same as the formula (I).

In some preferred embodiments, CS is a protein complex obtained by mixing with a recombinant protein in a buffer solution. The pH range of the buffer solution is preferably 6.0-8.0, more preferably 7.0-7.5, especially 7.2-7.4; the reaction time range is 0-1 h.

In some embodiments, the unsuccessfully attached CS is removed by purification methods when preparing the protein complex. In some specific embodiments, the means of purification includes molecular sieve chromatography and dialysis.

The eighth aspect of the invention provides a medicament comprising the recombinant protein of the first aspect of the invention and the protein complex of the sixth aspect of the invention, and other pharmaceutically active ingredients, pharmaceutically acceptable carriers, pharmaceutic adjuvants or excipients; or

The recombinant protein of the first aspect and the protein compound of the seventh aspect of the invention are medicaments consisting of other pharmaceutical active ingredients, pharmaceutically acceptable carriers, pharmaceutic adjuvants or excipients;

in some embodiments, the pharmaceutically active ingredient comprises one or more of the following: etanercept (Rilonacept), Infliximab (INFLECTRA), thalidomide, steroids (such as cortisone, prednisolone, prednisone, methylprednisolone, dexamethasone, betamethasone, triamcinolone acetonide, beclomethasone, fluticasone), anakinra (anakinra), colchicine, IL-18 binding protein (IL-18BP) or derivatives, IL-18 antibodies, IL-18 receptor (IL-18R1) antibodies, IL-18 receptor accessory protein (IL-18Racp) antibodies, aspirin, methotrexate, cyclosporin a, caspase-1, IKK1/2, cytotoxic T-cell antigen 4(CTLA-4Ig), IL-6 antibodies, and IL-6RA antibodies.

In a ninth aspect, the present invention provides the use of a recombinant protein according to the first aspect of the present invention, a protein complex according to the sixth aspect of the present invention, a protein complex according to the seventh aspect of the present invention or a medicament according to the eighth aspect of the present invention for the prophylactic and/or therapeutic manufacture of a medicament for an animal. The treatment is preferably recovery, further preferably complete recovery; the mammal is preferably a primate, more preferably a human; the disease is preferably a rheumatic immune disease, and more preferably a human rheumatoid immune disease with the characteristic of abnormally increased interleukin 1.

In some embodiments, the disease is characterized by abnormally elevated interleukin 1, and specifically includes rheumatoid immune diseases, hereditary diseases, and tumors. Specifically, the "disease forms with increased interleukin 1" include, but are not limited to, rheumatoid arthritis, erosive osteoarthritis, pyoderma gangrenosum, acne conglobata and aseptic arthritis, gout and pseudogout, systemic idiopathic juvenile arthritis, atherosclerosis, amyloid a amyloidosis, adult still's disease, asthma, behcet's disease, brussel's syndrome, crystalline deposit of calcium pyrophosphate dihydrate disease, castleman's disease, coldness-imidacloprid-associated periodic syndrome, interleukin-1 receptor antagonist deficiency, dermatomyositis, edham-chester's disease, familial mediterranean fever, psoriasis, graft-versus-host disease, hidradenitis suppurativa, hyper IgD syndrome, congenital cold urticaria, inclusion body myositis, inflammatory bowel disease, ischemic injury, macrophage activation syndrome, Magazine syndrome, mevalonate kinase deficiency, myeloid and other leukemias, osteoporosis, neutrophilic panniculitis, periodic fever with aphthous stomatitis, pharyngitis and lymphadenitis, polymyositis, recurrent congenital pericarditis, recurrent polychondritis, reperfusion injury, Schniemann's syndrome, myeloma, synovitis acne pustulosis osteoproliferation osteomyelitis, tumor necrosis factor receptor-related periodic syndrome, type 1 diabetes, type 2 diabetes, urticaria vasculitis, uveitis, neonatal onset multisystem inflammatory disorder, Munich syndrome, familial coldness autoinflammatory syndrome, adjuvant therapy of tumors, therapy of complications of tumor radiotherapy, radiation injury such as radiation oral mucositis, radiation pneumonitis, radiation esophagitis, radiation gastroenteritis, radiation cystitis, Radiation vaginitis, and radiation dermatitis.

In some embodiments, the recombinant proteins, protein complexes and drugs are preferably administered by subcutaneous or intravenous or intramuscular route, more preferably by subcutaneous route.

The tenth aspect of the present invention provides the use of a recombinant protein of the first aspect of the present invention or a protein complex of the sixth aspect or a protein complex of the seventh aspect of the present invention in the manufacture of a medicament for the prevention and/or treatment of inflammation mediated by an inflammatory factor in a primate. Wherein the inflammatory factor is interleukin 1.

An eleventh aspect of the present invention provides a therapeutic method for adjuvant therapy of rheumatic immune diseases, genetic diseases, and tumors, which are characterized by abnormally elevated interleukin 1, comprising administering to a subject a recombinant protein of the first aspect of the present invention, a protein complex of the sixth aspect of the present invention, a protein complex of the seventh aspect of the present invention, or a medicament of the eighth aspect of the present invention.

In some embodiments, the subject is a mammal, preferably a primate, more preferably a human.

In some embodiments, the recombinant protein, protein complex, codrug is preferably administered by the subcutaneous or intravenous or intramuscular route, more preferably by the subcutaneous route.

The recombinant protein consists of human interleukin 1 receptor antagonist protein and protein containing (VPGKG) n repeating units. The recombinant protein of the invention obviously prolongs the half-life period of IL-1RA in plasma concentration of rats, has obvious curative effect on acute gouty arthritis and rheumatic arthritis of rats, and has obvious difference (p is less than 0.001) compared with a single human interleukin 1 receptor antagonist. The recombinant protein can be used for the adjuvant treatment of rheumatoid arthritis, acute gouty arthritis and other rheumatic diseases, hereditary diseases and tumors which have the obvious characteristic of abnormally increased interleukin 1, and has more obvious curative effect.

Drawings

FIG. 1 shows a SDS-PAGE electrophoresis of recombinant proteins;

FIG. 2 shows an electron micrograph of a recombinant protein complex;

FIG. 3 shows the average particle size of the protein complexes;

FIG. 4 shows plasma concentration-time curves of protein complexes and IL-1RA protein in rats;

FIG. 5 shows ankle joint circumference changes in groups of rats after injection of protein complex and IL-1RA protein;

FIG. 6 shows the variation in thickness of footpads of rats in each group after injection of protein complexes and IL-1RA protein.

Detailed Description

The invention provides a recombinant protein, a protein compound containing the recombinant protein and application. Those skilled in the art can accomplish this by repeating it entirely, or by modifying the process parameters appropriately based on the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The reagents and materials adopted by the invention are all common commercial products and can be purchased in the market.

The sequences of the primers involved in the examples of the present invention are shown in Table 1.

TABLE 1 primer sequences

The invention is further illustrated by the following examples:

EXAMPLE 1 preparation of recombinant proteins

In this example, the inventors designed 10 recombinant protein sequences including the following based on the (VPGKG) repeat polypeptide sequence and the human interleukin 1 receptor antagonist (IL-1RA) sequence, and the specific sequence information is shown in table 2:

TABLE 2 sequence characterization of recombinant proteins

1.1 obtaining strains expressing recombinant proteins

The nucleotide sequences corresponding to the above 10 recombinant proteins were synthesized by biologies (Shanghai) and constructed on pET25b expression plasmid vector for transformation into Escherichia coli prokaryotic expression competent BLR (DE 3). Specific embodiments are as follows: 100ng of pET25b-A1 (or A2-A10) plasmid was added to 100. mu.L of BLR E.coli competence (available from Novagen) in an ice bath and maintained in the ice bath for 30 min; the mixture was then transferred to a 42 ℃ water bath with heat shock for 45s followed by ice bath for 2 min. To the mixture was added 600. mu.L of LB medium and treated at 37 ℃ for 1 hour with shaking at 180 rpm. The mixture was uniformly spread on LB solid medium containing ampicillin sodium (15. mu.g/mL) antibiotic and cultured at 37 ℃ for 18 hours to obtain a strain capable of stably expressing the recombinant protein. The grown colonies were picked up and inoculated in 10mL LB medium, cultured at 37 ℃ with shaking at 220rpm, and induced with isopropyl thiogalactoside (IPTG, final concentration 0.5mmol/L) when the OD600 value of the culture mixture reached 0.6, and cultured overnight at 16 ℃ with shaking at 220 rpm. After induction is finished, centrifuging the bacterial liquid at 4000rpm for 30min, and removing a supernatant; and (3) resuspending the bacterial pellet by PBS, centrifuging at 4000rpm for 30min again, and discarding the supernatant to obtain the bacterial pellet containing the target protein. Strains that correctly express the recombinant protein were identified by SDS-PAGE electrophoresis and immunoblotting experiments.

1.2 expression of recombinant proteins

Transferring the strain capable of correctly expressing the recombinant protein into 50mL LB culture medium containing ampicillin sodium (15 mug/mL) antibiotics, and culturing for 4-6h at 37 ℃ in a shaking environment of 220 rpm; when OD600 reaches 0.6, the bacterial liquid is transferred into 1L of fresh LB culture medium containing ampicillin sodium (15 mu g/mL) antibiotics, cultured in a shaking environment at 37 ℃ and 220rpm until OD600 reaches 0.6, then IPTG with the final concentration of 0.5mmol/L is added to induce the bacterial body to express recombinant protein, and the environmental temperature is adjusted to 16 ℃. After the induction culture is carried out for 4-18h, the bacterial liquid is centrifuged for 30min at 4000rpm, and the supernatant is discarded; and (3) resuspending the bacterial pellet by PBS, centrifuging at 4000rpm for 30min again, and discarding the supernatant to obtain the bacterial pellet containing the target protein.

1.3 purification of recombinant proteins

The cells were resuspended at pH 7.2-7.4 in 4 ℃ precooled 50mM Phosphate Buffer (PB) and the cell mixture was broken up with a high pressure homogenizer. The mixture was collected and centrifuged at 16000rpm at 4 ℃ for 30min, and the supernatant was collected. And (3) dropwise adding a4 ℃ precooled saturated ammonium sulfate aqueous solution into the supernatant until the final concentration of the saturated ammonium sulfate is 20%, centrifuging the mixture for 30min at 16000rpm and 4 ℃, and collecting the supernatant. And continuously dropwise adding a4 ℃ precooled saturated ammonium sulfate aqueous solution into the supernatant until the final concentration of the saturated ammonium sulfate is 50%, centrifuging the mixture for 30min at 16000rpm and 4 ℃, and collecting the precipitate. Slowly adding 50mMPB buffer solution into the precipitate at 4 ℃, adjusting the pH to 9.0, and slowly stirring until the precipitate is completely dissolved. The protein solution was further purified by SP cation exchange chromatography. After loading the protein solution on a10 mL SP cation exchange chromatography column (purchased from GE), the protein was eluted successively with 50mL of 50mM PB buffer containing 50mM, 100mM, 200mM, 400mM NaCl, and the fractions of the eluate containing 400mM NaCl were collected. The eluted fractions were subjected to molecular sieve chromatography in Superdex 100 (from GE). Loading the protein containing the eluent component of 400mM NaCl into a Superdex 100 molecular sieve chromatographic column, eluting with 50mM phosphate buffer solution (PB) with the pH of 7.2-7.4, and collecting the target protein component to obtain a purified product. The purified protein was identified by SDS-PAGE gel electrophoresis, as shown in FIG. 1.

EXAMPLE 2 preparation of protein complexes

Protein complex 1: purified recombinant protein a1 was mixed with carboxylated PEG in a molar ratio of 1: 18 and stirring for 1h at the temperature of 4 ℃. The mixture was applied to a desalting column (available from GE) and eluted with 50mM PB buffer solution having a pH of 7.2 to 7.4 to collect protein complex fractions.

Protein complex 2: purified recombinant protein a2 was mixed with carboxylated PEG in a molar ratio of 1: 72 and stirring for 1h at 4 ℃. The mixture was applied to a desalting column (available from GE) and eluted with 50mM PB buffer solution having a pH of 7.2 to 7.4 to collect protein complex fractions.

Protein complex 3: purified recombinant protein a3 was mixed with carboxylated PEG in a molar ratio of 1: 288, and stirring at 20-25 ℃ for 4 h. The mixture was applied to a desalting column (available from GE) and eluted with 50mM PB buffer solution having a pH of 7.2 to 7.4 to collect protein complex fractions.

Protein complex 4: purified recombinant protein a4 was mixed with carboxylated PEG in a molar ratio of 1: 9, and stirring for 4 hours at the temperature of between 20 and 25 ℃. The mixture was applied to a desalting column (available from GE) and eluted with 50mM PB buffer solution having a pH of 7.2 to 7.4 to collect protein complex fractions.

Protein complex 5: purified recombinant protein a5 was mixed with carboxylated PEG in a molar ratio of 1: 54, and stirring for 24 hours at the temperature of between 2 and 8 ℃. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 2-8 deg.C with 50mM PB buffer solution having pH of 7.2-7.4 as dialysis displacement solution with a volume at least 10 times of that of protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 6: purified recombinant protein a6 was mixed with carboxylated PEG in a molar ratio of 1: 162, and stirring for 2 hours at the temperature of between 20 and 25 ℃. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 2-8 deg.C with 50mM PB buffer solution having pH of 7.2-7.4 as dialysis displacement solution with a volume at least 10 times of that of protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 7: purified recombinant protein a7 was mixed with maleimide-modified PEG at a molar ratio of 1: 720, and stirring for 30min in a water bath environment at 37 ℃. The mixture was applied to a desalting column (available from GE) and eluted with 50mM PB buffer solution having a pH of 7.2 to 7.4 to collect protein complex fractions.

Protein complex 8: purified recombinant protein A8 was mixed with carboxylated PEG in a molar ratio of 1: 72, and stirring for 1h at the temperature of 20-25 ℃. The mixture was applied to a desalting column (available from GE) and eluted with 50mM PB buffer solution having a pH of 7.2 to 7.4 to collect protein complex fractions.

Protein complex 9: purified recombinant protein a9 was mixed with maleimide-modified PEG at a molar ratio of 1: 108 and stirring for 30min in a water bath environment at 37 ℃. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 2-8 deg.C with 50mM PB buffer solution having pH of 7.2-7.4 as dialysis displacement solution with a volume at least 10 times of that of protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 10: purified recombinant protein a10 was mixed with maleimide-modified PEG at a molar ratio of 1: 144, and stirring for 30min in a water bath environment at the temperature of between 20 and 25 ℃. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 2-8 deg.C with 50mM PB buffer solution having pH of 7.2-7.4 as dialysis displacement solution with a volume at least 10 times of that of protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 11: mixing the purified recombinant protein A1 with CS according to a molar ratio of 1: 162, and stirring at 20-25 ℃ for 10 min. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 2-8 deg.C with 50mM PB buffer solution having pH of 7.2-7.4 as dialysis displacement solution with a volume at least 10 times of that of protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 12: mixing the purified recombinant protein A2 with CS according to a molar ratio of 1: 72, and stirring for 30min at 20-25 ℃. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 2-8 deg.C with 50mM PB buffer solution having pH of 7.2-7.4 as dialysis displacement solution with a volume at least 10 times of that of protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 13: mixing the purified recombinant protein A3 with CS according to a molar ratio of 1: 720, and stirring for 1h at the temperature of 2-8 ℃. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 2-8 deg.C with 50mM PB buffer solution having pH of 7.2-7.4 as dialysis displacement solution with a volume at least 10 times of that of protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 14: mixing the purified recombinant protein A4 with CS according to a molar ratio of 1: 90, and stirring for 1 hour at the temperature of 2-8 ℃. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 2-8 deg.C with 50mM PB buffer solution having pH of 7.2-7.4 as dialysis displacement solution with a volume at least 10 times of that of protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 15: mixing the purified recombinant protein A5 with CS according to a molar ratio of 1: 180, and stirring at 37 ℃ for 10 min. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 2-8 deg.C with 50mM PB buffer solution having pH of 7.2-7.4 as dialysis displacement solution with a volume at least 10 times of that of protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 16: mixing the purified recombinant protein A6 with CS according to a molar ratio of 1: 54 and stirring at 37 ℃ for 10 min. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 2-8 deg.C with 50mM PB buffer solution having pH of 7.2-7.4 as dialysis displacement solution with a volume at least 10 times of that of protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 17: mixing the purified recombinant protein A7 with CS according to a molar ratio of 1: 432, and stirring at 37 deg.C for 10 min. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 2-8 deg.C with 50mM PB buffer solution having pH of 7.2-7.4 as dialysis displacement solution with a volume at least 10 times of that of protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 18: mixing the purified recombinant protein A8 with CS according to a molar ratio of 1: 72, and stirring for 30min at the temperature of 20-25 ℃. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 25 deg.C with 50mM PB buffer solution with pH of 7.2-7.4 as dialysis displacement solution, wherein the volume of the dialysis displacement solution is at least 10 times of that of the protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 19: mixing the purified recombinant protein A9 with CS according to a molar ratio of 1: 324, and stirring for 30min at the temperature of 20-25 ℃. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 25 deg.C with 50mM PB buffer solution with pH of 7.2-7.4 as dialysis displacement solution, wherein the volume of the dialysis displacement solution is at least 10 times of that of the protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Protein complex 20: mixing the purified recombinant protein A10 with CS according to a molar ratio of 1: 288, and stirring for 30min at 20-25 ℃. Transferring the mixture into a dialysis tube, and performing dialysis reaction at 25 deg.C with 50mM PB buffer solution with pH of 7.2-7.4 as dialysis displacement solution, wherein the volume of the dialysis displacement solution is at least 10 times of that of the protein complex solution; the dialysis replacement fluid was replaced every 8 h. And collecting protein complex components after dialysis for 24 h.

Example 3: protein complex particle size determination

The protein complex obtained by purification in example 2 was examined by transmission electron microscopy and analyzed for average particle size of protein. The results are shown in FIGS. 2-3, and FIG. 2 shows the electron microscope pictures of the protein complexes. FIG. 3 shows the results of the average particle size analysis of the protein.

Example 4: in vivo drug metabolism change detection of protein complexes in rats

36 male SD rats with the weight of 200 +/-20 g are collected in infraorbital veins after isoflurane anesthesia, centrifuged immediately at 1100g and 4 ℃ for 20min, and the supernatant is stored at-80 ℃. The rats were then randomly divided into 12 groups of 3 rats each. 0.1 mu mol of protein complexes A1-A10, IL-RA protein and physiological saline with the same volume are injected into the neck at three subcutaneous points respectively. After administration, blood was collected from infraorbital veins after isoflurane anesthesia at time points of 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h, 30h, 36h, 48h, 60h, 72h, 96h, 120h, 144h, and 168h in all rats, and immediately centrifuged at 1100g and 4 ℃ for 20min, and the supernatant was stored at-80 ℃. And (3) detecting the content of the IL-1RA in the blood by using a human source IL-1RA detection kit for the plasma sample, and calculating the half-life period of each group of medicaments in vivo. FIG. 4 shows the in vivo half-life curve of protein complex A8 and IL-1RA protein in rats; table 3 shows the calculated half-life of each histone complex/protein in rat plasma concentration.

TABLE 3 half-life of each histone complex/protein in rat plasma concentration

Example 5: therapeutic effect of protein complex in rat acute gouty arthritis model

65 male SD rats of 8 weeks of age were taken and marked and ankle circumferences of all rats were measured. Then, the rat is subjected to gouty arthritis disease molding: to the ankle cavity of all rats, 50. mu.L of a 60mg/mL suspension of sodium urate crystals was injected. All rats were tested for ankle circumference.

All rats successfully modeled were randomly divided into 13 groups, and injected with 0.1. mu. mol/100. mu.L/dose of protein complex 1-10, IL-RA protein, and physiological saline and indomethacin in equal volumes at three points under the neck. Ankle joint circumferences at 0h, and 6h, 12h, 24h, 48h, and 72h after dosing were measured for all rats, respectively. Figure 5 shows the change in ankle circumference in rats after administration. The result shows that the protein compound has obvious curative effect on acute gouty arthritis of rats, and the effect is obviously superior to IL-1RA protein and indometacin (P is less than 0.001).

EXAMPLE 6 therapeutic Effect of protein complexes in rat rheumatoid arthritis model

60 female SD rats of 8 weeks of age were taken and marked and all rat footpad thicknesses were measured. Then, the rats are subjected to rheumatoid arthritis disease molding: mixing a 2mg/mL type II collagen PBS solution with a complete Freund's adjuvant in the same volume, and mixing the mixture into a uniform water-in-oil emulsion under the action of ultrasound to obtain the immunogen 1. All rats were then injected subcutaneously with immunogen 1 at the footpad and tail root. After 14 days, all rats were boosted once. Mixing a 2mg/mL type II collagen PBS solution with an incomplete Freund's adjuvant in the same volume, and mixing the mixture into a uniform water-in-oil emulsion under the action of ultrasound to obtain the immunogen 2. All rats were then injected subcutaneously with immunogen 2 at the footpad and tail root.

On day 15, all successfully molded rats were randomly divided into 12 groups, and injected with 0.1. mu. mol/100. mu.L/dose of protein complex 11-20, IL-RA protein, and physiological saline of the same volume at three points under the neck skin. All rats were tested for footpad thickness on day 0, and on

days

1, 2, 4, 6, 9, 11, 14 post-dose, respectively. The results are shown in FIG. 5, and FIG. 6 shows the variation in thickness of footpads of rats in each group after injection of the protein complex and IL-1RA protein.

The result shows that the therapeutic effect of the protein compound of the invention on rheumatoid arthritis is obviously better than that of IL-1RA protein (p < 0.01).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. Recombinant proteins, including human IL-1RA proteins and proteins containing (VPGKG) repeat sequences.

2. The recombinant protein according to claim 1, wherein the human IL-1RA protein and the VPGKG repeat sequence-containing protein are linked directly by a peptide bond or via a linker peptide.

3. The recombinant protein according to claim 1, wherein the human IL-1RA protein has any one of the amino acid sequences shown in SEQ ID No. 1-SEQ ID No. 4; or has an amino acid sequence with the same or similar functions with the sequences shown in SEQ ID NO. 1-SEQ ID NO. 4.

4. The recombinant protein according to claim 1, wherein 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 more 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 combined at will; preferably, X is valine, alanine or glycine.

5. The recombinant protein according to claim 2, wherein said linker peptide consists of a plurality of consecutive amino acid amino groups of any of the amino acids.

6. The recombinant protein according to any one of claims 1 to 5, having any one of the amino acid sequences shown in SEQ ID No.13 to SEQ ID No.72, or having an amino acid sequence with at least 80% identity to any one of the sequences shown in SEQ ID No.13 to SEQ ID No.72, or having an amino acid sequence functionally identical or similar to any one of the sequences shown in SEQ ID No.13 to SEQ ID No. 72.

7. A nucleic acid encoding the recombinant protein of any one of claims 1 to 6, having:

any one nucleotide sequence shown in SEQ ID NO. 73-SEQ ID NO. 132; or

A nucleotide sequence with at least 80% of identity with the sequences shown in SEQ ID NO. 73-SEQ ID NO. 132; or

A nucleotide sequence which encodes the same protein as the sequence shown in SEQ ID NO. 73-SEQ ID NO.132 but is different from the sequence shown in SEQ ID NO. 73-SEQ ID NO.132 due to the degeneracy of the genetic code.

8. A method for producing a recombinant protein according to any one of claims 1 to 6, wherein:

(1) constructing an expression vector of the nucleic acid of claim 7;

(2) transforming or transfecting the nucleic acid expression vector of claim 7 into a host cell;

(3) culturing the host cell to induce expression of the recombinant protein of claim 7.

9. A protein complex comprising the recombinant protein of any one of claims 1 to 6 and polyethylene glycol.

10. The protein complex of claim 9, wherein the molar ratio of the recombinant protein to the polyethylene glycol is 1:0.25n to 1:5 n.

11. A protein complex comprising the recombinant protein according to any one of claims 1 to 6 and Chondroitin Sulfate (CS).

12. The protein complex of claim 11, wherein the molar ratio of the recombinant protein to the chondroitin sulfate is 1:0.25n to 1:5 n.

13. Use of the recombinant protein according to any one of claims 1 to 6 or the protein complex according to any one of claims 9 to 12 for the preparation of a medicament for the prophylaxis and/or treatment of a disease; the disease is characterized by significant interleukin 1 increase; the diseases include rheumatoid arthritis, erosive osteoarthritis, pyoderma gangrenosum, acne conglobata and aseptic arthritis, gout and pseudogout, systemic idiopathic juvenile arthritis, atherosclerosis, amyloid A amyloidosis, adult still's disease, asthma, Behcet's disease, Braun's syndrome, calcium pyrophosphate dihydrate crystal deposition disease, Cassman's disease, cold pyrroline-associated periodic syndrome, interleukin-1 receptor antagonist deficiency, dermatomyositis, Edheimer-Chester disease, familial mediterranean fever, psoriasis, graft-versus-host disease, hidradenitis suppurativa, Gaigd syndrome, congenital cold urticaria, inclusion body myositis, inflammatory bowel disease, ischemic injury, macrophage activation syndrome, Magidd syndrome, mevalonate kinase deficiency, myelogenous and other leukemias, Osteoporosis, neutrophilic panniculitis, periodic fever with aphthous stomatitis, pharyngitis and lymphadenitis, polymyositis, recurrent congenital pericarditis, recurrent polychondritis, reperfusion injury, Sinitsier syndrome, myeloma, synovitis acne pustulosis osteoproliferation osteomyelitis, tumor necrosis factor receptor-related periodic syndrome, type 1 diabetes, type 2 diabetes, urticaria vasculitis, uveitis, multiple system inflammatory disorders of neonatal origin, Mucuneir syndrome, familial cold autoinflammatory syndrome, adjuvant therapy of tumors, treatment of tumor complications by radiotherapy, radioactive injury such as radioactive oral mucositis, radiation pneumonitis, radioactive esophagitis, radioactive gastroenteritis, radiation cystitis, radiation vaginitis, and radiation dermatitis.

14. Medicament comprising a protein according to any one of claims 1 to 6 or a protein complex according to any one of claims 9 to 12 and one or more of the following pharmaceutically active ingredients:

etanercept (Rilonacept), Infliximab (INFLECTRA), thalidomide, such as cortisone, prednisolone, prednisone, methylprednisolone, dexamethasone, betamethasone, triamcinolone acetonide, beclomethasone, fluticasone, anakinra, colchicine, IL-18 binding protein (IL-18BP) or derivatives, IL-18 antibodies, IL-18 receptor (IL-18R1) antibodies, IL-18 receptor accessory protein (IL-18Racp) antibodies, aspirin, methotrexate, cyclosporin a, caspase-1, IKK1/2, cytotoxic T-cell antigen 4(CTLA-4Ig), IL-6 antibodies, and IL-6RA antibodies.