CN100366637C - Human interleukin-8 antagonistic protein and preparation method thereof - Google Patents

Abstract

The present invention discloses a recombination mutation human cell medium element 8 protein and a preparation method thereof. In the method, active site ELR mutations in natural human IL-8 genes are used as AAR for constructing antagonism protein genes rmhIL-8 which can be combined with receptors being competitive with natural IL-8; the antagonism protein genes rmhIL-8 are expressed with high efficiency in escherichia coli and are purified for obtaining rmhIL-8 proteins; the chemotaxis activity of IL-8 can be inhibited through the competitive combination of mutants to receptors for reaching the function for treating inflammation diseases. The human cell medium element 8 antagonist protein of the present invention possibly provides a new protein medicine for the treatment of acute inflammation and chronic inflammation.

Description

Human interleukin-8 antagonistic protein and preparation method thereof

technical field

The invention belongs to the field of medical biotechnology, gene cloning, gene recombination, expression of exogenous gene in prokaryotic cells, purification of target protein, inhibition of natural human interleukin-8 chemotactic activity by human interleukin-8 antagonistic protein in vitro The invention relates to an anti-inflammatory drug protein and a preparation method thereof, in particular to a mutant protein capable of competing with natural human interleukin 8 for binding receptors and a preparation method thereof.

Background technique

1. Related research on IL-8

Interleukin 8 (Interleukin-8, IL-8) is a chemokine that can activate neutrophils. It was first discovered in the supernatant of activated monocytes. Factor meeting was named IL-8. IL-8 belongs to the C-X-C class chemokine of the chemokine family, which is produced by mononuclear macrophages and can induce a variety of leukocyte chemotactic movements.

1.1 The structure and characteristics of IL-8

Interleukin-8 (IL-8) is a chemokine capable of activating neutrophils and a base-heparin binding agent with endogenous leukocyte chemotaxis and activation protein. IL-8 was originally found in the supernatant of activated monocytes as a substance with neutrophil chemotactic activity, and was named IL-8 at a world cytokine conference in 1989. IL-8 belongs to the C-X-C class chemokine of the chemokine family, which is produced by mononuclear macrophages and can induce a variety of leukocyte chemotactic movements. IL-8 has different isoforms, which have different numbers of amino acids reduced at the N-terminus. Cells first synthesize IL-8 precursor protein, that is, type I IL-8 (99 peptides, 20 amino acid residues at the N-terminal form a highly hydrophobic signal peptide), and then directly enzymolyze IL-8 precursor N by different specific proteases. Different parts of the end produce IL-8 with different molecular weights. There are four mature forms of IL-8, consisting of 72 (70%), 77 (17%), 70 (8%) and 69 (5%) amino acid residues, respectively. Among them, type III IL-8 is 72 peptides with the strongest activity, which is usually referred to as mature IL-8. Its molecular weight is only 8Kd (SDS-PAGE), contains two disulfide bonds (7-34, 9-50) necessary for activity, has no glycosylation site, and its isoelectric point (PI) is 8-8.5. IL-8 is sensitive to trypsin and chymotrypsin, and is resistant to heat and acid. It cannot be inactivated by treatment at 70°C for 60 minutes, and it remains stable between pH 2.4 and 9.0. Nuclear Magnetic Resonance and X-ray crystallography prove that IL-8 in solution is a non-covalently linked homodimer, the interface of the dimer is dominated by β sheets, and the hydrogen bonds formed between Leu25, Val27, and Glu29 Involved in the stability of the dimer, the intersection of the α-helical structure and the β-sheet is also involved in dimer formation. The ELR (Glu4-Leu5-Arg6) structure of IL-8 is related to its activation of neutrophils and its pro-angiogenic effect, and C-X-C chemokines such as PF-4 and IP-10 without ELR cannot activate neutrophils and have anti-angiogenic effects. Angiogenesis, however, GRO with ELR structure inhibits angiogenesis. The C-terminus of IL-8 (Lys54~C-terminus) is the site of polyglucosamine on the surface of vein endothelial cells after capillary binding. This combination can promote the adhesion of leukocytes rolling on the surface of blood vessels to endothelial cells and further pass through blood vessels.

1.2 Production and regulation of IL-8

Many cells can produce IL-8 when properly stimulated, including monocyte-macrophages, lymphocytes, neutrophils, eosinophils, myeloid cell precursors, synoviocytes, smooth muscle cells, endothelial cells, epithelial cells cells, keratinocytes, chondrocytes, mesothelial cells, amnion cells, astrocytes, glomerular cells and hepatocytes. Some tumor cell lines can also produce IL-8, for example: melanoma cells, fibrosarcoma cells, large cell carcinoma cells of the lung, osteosarcoma cells, etc.

Many different factors induce IL-8 production. The first inducer that can induce monocytes to produce IL-8 is LPS, and later found that IL-1β and TNF-α can also induce some cells to produce IL-8, but LPS has the strongest effect on monocytes. In most cell types, IL-1β and TNF-α have the strongest effect on inducing IL-8 expression. PHA can induce T lymphocytes to produce IL-8. Alveolar macrophages and endothelial cells can also express IL-8 after LPS stimulation. Interleukin-17 induces epithelial cells, endothelial cells and fibroblasts to produce IL-8. In conclusion, different types of cells respond differently to different inducers.

After being properly stimulated, cells can quickly express IL-8mRNA. Using isotope-labeled oligonucleotide probes or IL-8cDNA probes for Northern hybridization, it was found that specific IL-8mRNA could be detected within 1 hour after stimulation. If endothelial cells were treated with actinomycin ketone before stimulation, the expression of IL-8 mRNA could be super-induced. It shows that the expression of IL-8 gene does not depend on the synthesis of some intracellular proteins, but is under the control of a repressor protein. One hour after laminar flow and low shear stress (posterior venule level) applied to cultured human umbilical vein endothelial cells, the expression of IL-8mRNA was significantly increased and accompanied by intracellular gene-binding nuclear factor (Nuclear-factor-genebinding, NF-кB) The degree of activation is related to the duration of action. It reached the peak at 2h and began to decrease at 3h. After 4h, the expression of IL-8mRNA continued to be low, showing a biphasic change rule of IL-8mRNA with the time of shear stress. Low shear stress can induce endothelial cells to express IL-8, which may be involved in the occurrence, development and prognosis of inflammatory diseases and cardiovascular and cerebrovascular diseases.

1.3L-8 receptor

IL-8 acts through receptors. There are IL-8 receptors on almost all cells. There was no cross-reactivity between IL-8 and other neutrophil chemotactic substances. There are two high-affinity receptors (Kd=0.2～1.2nmol/L): CXCR1 (IL-8Rα, IL-8R I or IL-8A) and CXCR2 (IL-8Rβ, IL-8RII or IL-8B), two Their amino acid sequences have 77% homology, and they all belong to the GTP-binding protein (G protein)-related receptor superfamily with 7 transmembrane regions. CXCR1 contains 350 amino acids, and its extracellular part has 5 glycosylation sites, a disulfide bond and two free Cys sulfhydryl groups, with a molecular weight of about 40kD and 55-69kD after glycosylation. CXCR2 contains 355 amino acids, only 5 glycosylation sites, and a molecular weight of about 59kD. CXCR1 specifically binds to IL-8, and has low affinity to other C-X-C chemokines, while CXCR2 can also bind to NAP-2, GRO-α, GRO-β, etc. with high affinity, and the heterogeneity of IL-8 receptor binding ligands Depending on the N-terminus of the receptor molecule, the region of the receptor that binds IL-8 has two free thiols in conformational proximity that are required for binding activity. Two IL-8R genes and one IL-8R pseudogene are located on human chromosome 2q34-35. There is also a 39kD IL-8 binding protein or Duffy antigen receptor for chemokines (DARC) on the red blood cell membrane, which is also a Duffy blood group antigen and a molecule that mediates the entry of Plasmodium vivax into red blood cells. Other C-C and C-X-C chemokines also bind to this receptor and compete with IL-8. DARC was expressed in the postcapillary vein endothelial cells of the brain, kidney and spleen.

1.4 Biological activity of IL-8

IL-8 selectively induces chemotaxis of neutrophils, T lymphocytes and basophils, but has no chemotaxis effect on eosinophils and monocytes.

IL-8 can enhance the function of neutrophils: induce neutrophil degranulation, release β-glucuronidase, elastase, myeloperoxidase, collagenase B, vitamin _B12 binding protein and milk in granules Ferritin; stimulates rapid increase of intracytoplasmic calcium ions; induces respiratory burst to produce superoxide ions and hydrogen peroxide. In the presence of exogenous arachidonic acid, IL-8, like C5a, FMLP, and PAF, can activate arachidonic acid-5-lipoxygenase in neutrophils and release LTB ₄ (leukotriene B ₄ ). IL-8 can stimulate neutrophils to phagocytize opsonized granules, and indirectly enhance the growth inhibitory effect of neutrophils on Candida albicans. IL-8 can inhibit the adhesion of neutrophils to cytokine-activated endothelial cell monolayer and prevent neutrophils from injuring endothelial cells; high-dose IL-8 can enhance the adhesion of neutrophils to unactivated endothelium cell capacity. IL-8 induces neutrophils to express L-selectin, CD11a/CD18 and CD11b/CD18 molecules, promoting cell adhesion to endothelial cell monolayers and passage through vessel walls. The response of neutrophils to IL-8 is enhanced after binding heparin or heparan sulfate.

The chemotactic effect of IL-8 on T cells has been reported differently: 10 times stronger than neutrophils, equal or unable to chemoattract T cells; T cells that respond to IL-8 are activated T cells or small T cells. IL-8 can selectively inhibit IL-4-induced IgE production by B cells.

IL-8 can induce the chemotaxis of basophils treated with GM-CSF or IL-3, and the chemotactic response of peripheral basophils in patients with atopic pylitis and allergic asthma to IL-8 is enhanced.

IL-8 can induce IL-2-activated NK cell chemotaxis, induce melanoma cell contact migration, and promote endothelial cell chemotaxis. IL-8 induces keratinocyte and melanocyte proliferation and chemotaxis. IL-8 can stimulate functional hematopoietic cells from the bone marrow to the periphery, but inhibit the proliferation of myeloid hematopoietic precursor cells. IL-8 can also induce angiogenesis.

In vivo, local injection of IL-8 can increase the permeability of blood vessels and induce the infiltration of neutrophils and lymphocytes. Intradermal injection of IL-8 leads to plasma exudation and local accumulation of neutrophils. Both reactions occur quickly and last for a short time. Anti-leukocyte integrin antibodies inhibit these effects. IL-8-induced histological damage includes neutrophil accumulation in blood vessels, clot formation, and vessel wall damage.

Intravenous administration of IL-8 to animals caused an immediate leukopenia followed by a longer period of neutropenia with various stages of release of neutrophils from the bone marrow. IL-8-induced neutropenia may play an important role in non-specific protection against infection in vivo. Adhesion of neutrophils to endothelial cells also causes systemic vascular effects: neutrophil accumulation, clot formation, and vessel wall damage.

The rat toe pressure test proved that IL-8 is one of the mediators of sympathetic pain. In addition to pro-inflammatory effects, IL-8 also has growth-regulating effects: it can promote angiogenesis in rat cornea, induce new blood vessels in rabbit corneal capsule model, initiate endothelial cell proliferation and increase neuron survival period, etc.

1.5 The mechanism of action of IL-8

After IL-8 binds to its receptor, the ligand-receptor complex is endocytosed, then the receptor recycles, and quickly reappears on the cell membrane surface within 60 minutes. Blocking the recycling of its receptor can cause IL-8-mediated Attenuation of induced chemotactic responses. IL-8 binds to receptors and activates pertussis toxin-sensitive receptor-coupled G proteins, especially G _αi proteins. After the G protein is converted into a GTP-bound form, it dissociates into two subunits, _Gα and _Gβγ . Among them, _Gβγ is located close to the receptor complex, recruits and activates phosphatidylinositol 3-kinase γ (PI3K-γ), which produces phosphatidylinositol 3,4,5-triphosphate (PIP ₃ ), PIP ₃ activates protein kinase K (Akt) and GTPases (GTPases), leading to direct cell migration. In the absence of phospholipase Cβ ₂₊₃ , a key enzyme that produces inositol 1,4,5-trisphosphate and diglyceride, IL-8-induced extracellular calcium ions, phosphorylation of protein kinase C, translocation of p47 The effect of the position and the generation of superoxide anion will disappear, but the chemotaxis mediated by it can be enhanced. In PI3K-γ knockout mice, IL-8-mediated neutrophil migration, _PIP3 production, Akt phosphorylation, and respiratory burst (a rapid increase in extracellular calcium concentration) were all attenuated . These results indicated that IL-8-mediated cell chemotaxis was carried out through PI3K-γ-dependent and independent pathways.

1.6 The role of IL-8 in the occurrence and development of diseases

More than 50 chemokines and more than 20 chemokine receptors have been found so far. However, IL-8 has been a relatively active research field among chemotactic cytokines since its discovery. According to incomplete statistics, between 1987 and 2000, there were more than 150,000 articles related to chemotactic cytokines published. Among them, there are as many as 1/5 of the literature related to IL-8, which is about 2 to 10 times that of other chemotactic cytokines. This phenomenon at least reflects a problem, that is, IL-8 is very frequently involved in the pathological process of various non-specific inflammations, and the IL-8 gene is easily activated by external stimuli.

1.6.1 The role of IL-8 in rheumatoid arthritis.

There is a large amount of IL-8 in the synovial tissue and synovial fluid of patients with rheumatoid arthritis, and the protein and mRNA levels of IL-8 in the affected joints are significantly higher than those in the uninvolved joints, suggesting that IL-8 -8 plays an important role in the occurrence and development of rheumatoid arthritis. Direct injection of IL-8 into the rabbit joint cavity can observe the initial pathological changes of arthritis in a dose- and time-dependent manner, including the accumulation of neutrophils in the joint cavity, which fully demonstrates the role of IL-8 in rheumatoid arthritis. play an important role in the pathological changes. Other studies have also confirmed that IL-8 plays a leading role in the movement of monocytes to synovial tissue.

1.6.2 The role of IL-8 in ischemia-reperfusion injury.

Reperfusion after brief ischemia can cause more severe injury than ischemia. A large number of oxygen free radicals are produced in ischemia-reperfusion injury, and the pathology is characterized by a large number of neutrophil infiltration. There is some evidence that oxygen free radicals can regulate the production of IL-8. During myocardial reperfusion, IL-8 gene expression precedes neutrophil infiltration and its mediated tissue damage. In pulmonary ischemia-reperfusion injury, ischemia can induce the local expression of IL-8, a large number of neutrophil infiltration, pulmonary edema, and finally lead to severe damage to the lung structure. In cerebral ischemia-reperfusion injury, the concentration of IL-8 in the homogenate of brain tissue increased significantly 6 hours after the start of reperfusion, while the level of IL-8 in plasma did not change significantly. In liver ischemia-reperfusion injury, anti-IL-8 monoclonal antibody can significantly block the infiltration of neutrophils into the liver during reperfusion. Although the liver cells still swell, the cells are in normal regular arrangement. The integrity of the cell wall structure indicated that IL-8 was a key factor for chemotactic neutrophil infiltration during liver reperfusion injury.

1.6.3 The role of IL-8 in tumors

IL-8 functions as a growth factor in melanoma, liver and pancreatic and colon cancer cells. Studies have found that IL-8 is an angiogenic factor, and recombinant IL-8 can mediate chemotactic and proliferative activities of endothelial cells in vitro, and mediate angiogenic activities in vivo ^[24.25] . The activity of IL-8 in inducing angiogenesis is comparable to that of TNF-a, aFGF, bFGF, angiogenin, angiotropin and VEGF. Human tumor cell lines can directly express IL-8, and the level of IL-8 in freshly isolated human non-small cell lung cancer is significantly increased, which is related to the angiogenic activity in 42-80% of human non-small cell lung cancer tumor tissues, which is It shows that tumor cells secrete IL-8, an angiogenic factor, which may be very important for the formation of tumor angiogenesis, and the latter is a necessary condition for tumorigenesis and development. Clinical studies have shown that the mRNA level of IL-8 in human breast cancer tissue is significantly higher than that in normal human breast tissue ^[28] , and the invasiveness of breast cancer cell lines with high expression of IL-8 is significantly enhanced. In melanoma cell lines, the expression level of IL-8 is closely related to their growth and invasion ^[29] .

1.6.4 Pathological role of IL-8 in lung diseases

IL-8 plays an important role in lung diseases. In bacterial or viral infectious lung diseases, the products of bacteria or viruses can induce the expression of IL-8, thereby causing neutrophils to chemotaxis to lung tissue, and finally causing inflammation in respiratory distress syndrome (ARDS) In patients, the content of IL-8 in bronchoalveolar lavage fluid was significantly increased, which suggested that IL-8 played an important role in the occurrence and development of ARDS. In addition, there are high levels of IL-8 in the alveolar lavage fluid of patients with atopic pulmonary fibrosis, sarcoidosis, metal fume fever, asbestosis, etc., and in the bronchial epithelial cells of patients with chronic asthma and cystic fibrosis. The production of IL-8 also increased, suggesting that IL-8 is an important mediator of pathological changes in lung tissue.

2.1 The therapeutic effect of blocking IL-8 activity on diseases

2.2.1 Application of anti-IL-8 monoclonal antibody in inflammatory diseases

Acute Respiratory Distress Syndrome (ARDS) is a clinically serious lung disease with a mortality rate of up to 50%. Mukaida et al. injected bacterial endotoxin (LPS) into rabbits intravenously to induce the symptoms of ARDS very similar to clinical symptoms in rabbits, thus making an animal model of ARDS. The use of anti-IL-8 monoclonal antibody can prevent the occurrence of ARDS: the activity of myeloperoxidase in bronchoalveolar lavage fluid (represents the aggregation of neutrophils), the protein concentration (represents the amount of protein leaked into the alveoli ) and lung wet/dry ratio (representing the degree of pulmonary edema) after receiving LPS were almost completely suppressed by anti-IL-8 monoclonal antibody. Likewise, anti-IL-8 mAb significantly increased the survival rate of rabbits: only 30% of rabbits in the anti-IL-8 mAb-treated group died within 6-8 hours, while 70% of rabbits in the control group died. This experiment shows that in LPS-induced ARDS animal model, IL-8 plays an important pathological role, and anti-IL-8 monoclonal antibody can effectively treat ARDS.

In the arthritis model induced by LPS and IL-1, anti-IL-8 monoclonal antibody can significantly reduce the infiltration of neutrophils into the joint cavity, the reduction rate is 93% and 89% at 4 hours and 8 hours, respectively, At the same time, it can protect the synovial membrane of the joint cavity from being damaged. In the animal model of pleurisy caused by endotoxin, it can reduce the infiltration of neutrophils into the pleural cavity, and the reduction rate reaches 77%.

A fully humanized anti-IL-8 monoclonal antibody (IgG ₂ ) called ABX-IL8 has entered clinical trials for the treatment of chronic obstructive pulmonary disease (COPD). The human body is safe, the human body tolerates it well, and no obvious toxic and side effects are found. COPD patients are injected with ABX-IL8 every other month for a total of three months, which can improve the symptoms of dyspnea in patients. Symptoms of dyspnea decreased from two weeks after the first injection, and the suppression lasted for two months. The results of this trial provide preliminary support for further research on the drug.

In our country, the emulsion containing anti-IL-8 monoclonal antibody has entered clinical application, and achieved good curative effect in the treatment of psoriasis.

2.2.2 Application of anti-IL-8 monoclonal antibody in tumor

Since the role of IL-8 in tumor growth and metastasis has attracted increasing attention in recent years, the use of anti-IL-8 monoclonal antibodies to treat tumors has become one of the research hotspots in tumor therapy.

Using anti-IL-8 monoclonal antibody combined with epidermal growth factor receptor (EGFR) antibody to treat breast cancer mouse xenograft tumor model, although anti-IL-8 monoclonal antibody alone has no obvious effect on inhibiting breast cancer metastasis, However, the combined application with EGFR antibody can significantly strengthen the latter's anti-tumor effect and improve the survival rate of tumor-bearing mice ^[45] . This effect of anti-IL-8 monoclonal antibody may be through neutralizing IL-8 produced by tumor cells, thereby reducing IL-8-induced metal matrix protease (MMP) production and inhibiting tumor metastasis.

The human melanoma cell lines A375SM (high expression of IL-8) and TXM-13 (moderate expression of IL-8) were subcutaneously inoculated into nude mice respectively, and then treated with anti-IL-8 monoclonal antibody (intraperitoneal injection, a total of 1 mg per week) , divided into three injections, and treated for 3 weeks). Compared with the control group, the tumor growth of the treatment group was significantly inhibited. At the same time, the anti-IL-8 monoclonal antibody can also significantly inhibit the tumor metastasis caused by intravenous injection of melanoma cells. Both in vitro and in vivo experiments have shown that anti-IL-8 monoclonal antibody can reduce the invasion of tumor cells by blocking IL-8, inhibiting the expression of MMP-2. In addition, in vitro tube formation test confirmed that anti-IL-8 monoclonal antibody can directly interfere with the angiogenesis test of umbilical cord vein endothelial cells ^[46] .

Anti-IL-8 monoclonal antibody had no effect on the growth of bladder cancer cells in vitro, but if bladder cancer cells were subcutaneously inoculated into nude mice, after four weeks of treatment (100 μg intraperitoneal injection per week), anti-IL-8 monoclonal antibody It can significantly inhibit the growth of tumors, and can significantly inhibit the expression, activity and transcription of MMP-2 and MMP-9 by blocking IL-8. This effect is achieved by regulating the expression and transcription of the transcription factor NF-кB.

2.3 Research progress of IL-8 receptor antagonists

In addition to monoclonal antibodies, inhibiting the binding of IL-8 to its receptor is another effective way to reduce its activity. Since the ELR sequence at the N-terminal of the IL-8 molecule is necessary for binding and activating the receptor, it has been reported that the ELR motif mutated into AAR by chemical synthesis can bind to the IL-8 receptor, but cannot activate the receptor . So far, there is no report on the expression of IL-8 receptor antagonist protein by genetic engineering.

Contents of the invention

The object of the present invention is to provide an IL-8 receptor antagonistic protein (rmhIL-8) capable of competing with natural IL-8 molecules for binding to receptors, thereby inhibiting its biological activity and its preparation method. The human interleukin-8 antagonistic protein of the present invention may provide a new protein drug for the treatment of acute and chronic inflammation.

In order to achieve the above object, the technical scheme adopted by the present invention is to use PCR method to mutate the active site of natural IL-8, namely the N-terminal ELR motif, into AAR, so that it retains the ability to bind to the receptor and loses the ability to activate the receptor. The ability of the body, thereby constructing an IL-8 receptor antagonist protein. Its characteristic is that it can competitively inhibit the activity of natural IL-8, and play a therapeutic role in inflammatory diseases. The IL-8 mutant gene constructed by the PCR method can be highly expressed in Escherichia coli, and the rmhIL-8 protein can be obtained after purification.

The expression product is purified by lysing, heating, and SP ion chromatography column, and the protein purity can reach more than 95%. Purified rmhIL-8 can inhibit IL-8-induced neutrophil chemotaxis in vitro. In rats, rmhIL-8 can inhibit glycogen-induced neutrophil migration to the peritoneal cavity.

The IL-8 receptor antagonist protein of the present invention is prepared by mutating the natural IL-8 gene by PCR to construct the rmhIL-8 gene; constructing a prokaryotic expression vector containing the target gene rmhIL-8 and recombining the protein Expression; Identification of expression products; Purification of recombinant proteins; Inhibition of natural IL-8 chemotaxis by rmhIL-8 in vitro and IL-8-induced neutrophil chemotaxis in rats Research.

The IL-8 receptor antagonist protein (rmhIL-8) of the present invention competitively inhibits the chemotaxis of natural IL-8 by binding to the IL-8 receptor, thus it is possible to treat acute and chronic inflammation A novel protein drug is provided.

Description of drawings

Fig. 1 is the result of the sequenced gene sequence of pET-22b(+)-rmhIL-8;

Figure 2 is the agarose electrophoresis diagram of the PCR product, in which 1 represents the DNAmarker; 2 represents the target gene;

Figure 3 is a diagram of the enzyme digestion identification results of pET-22b(+)-rmhIL-8 recombinant plasmid; in the figure 1 represents DNAmarker; 2 represents pET-22b(+)-rmhIL-8;

Figure 4 is the SDS-PAGE of the rmhIL-8 expression product; 41 in the figure is a peptide marker; 42 is before induction; 43 after induction; 44 lysate precipitation; 45 lysate supernatant;

Figure 5 is the supernatant after heating the lysate product expressing the bacterium; the number 41 in the figure is a peptide marker; 52 is the supernatant of the lysate before heating; 53 is the supernatant heated at 50°C for 10 minutes; 54 is the supernatant heated at 60°C for 10 minutes; 55 The supernatant was heated at 70°C for 10 minutes; the supernatant was heated at 80°C for 10 minutes at 56;

Figure 6 is the SDS-PAGE of the purified product of rmhIL-8; where Figure (A) is a picture of ArmhIL-8 after purification, and Figure (B) is a picture of BWestern-blot, the number 61 in the figure, peptide marker, 62, upper after heating Qing, 63, purified rmhIL-8 (non-reduced), 64, purified rmhIL-8 (reduced); 65, purified rmhIL-8 (non-reduced), 66 purified rmhIL-8 (reduced);

Figure 7 shows the inhibitory effect of rmhIL-8 on the chemotactic activity of natural IL-8 in vitro; symbols in the figure ◆ rmhIL-8, ▲ anti-TNF irrelevant antibody, ■ anti-IL-8 antibody.

Figure 8 is a histogram of the inhibitory effect on IL-8-induced neutrophil chemotaxis in vivo.

In order to understand the present invention more clearly, the present invention will be further described in detail below in conjunction with the embodiments completed by the inventor.

Detailed ways

According to the technical scheme of the present invention, the IL-8 receptor antagonist protein selects the active site of the natural IL-8 molecule, that is, the N-terminal ELR motif is mutated into an AAR motif to form an IL-8 molecular mutant, which has the same -8 receptor binding ability, but unable to activate IL-8 receptor. The rmhIL-8 gene was cloned by PCR method, the prokaryotic expression vector was constructed, and highly expressed in Escherichia coli. The expression product is heated and purified by SP ion chromatography column, and the protein purity can reach more than 95%. Purified rmhIL-8 can inhibit IL-8-induced neutrophil chemotaxis in vitro. In rats, rmhIL-8 can inhibit glycogen-induced neutrophil migration to the peritoneal cavity.

Realize the receptor antagonist protein (rmhIL-8) of IL-8 of the present invention, its preparation method is carried out according to the following steps: 3.1 The cloning of rmhIL-8 gene and the construction of prokaryotic expression vector

The PCR reaction primers were designed as follows, the NdeI restriction site was introduced into the 5' end primer, and the SalI restriction site was introduced into the 3' end primer:

P1 (5' end primer 29nt): 5'AGCATATGGCCGCGCGTTGTCAGTGCATA3'

P2 (3' end primer 29nt): 5'AGGTCGACTTATGAGTTTCTCAGCCCCTT3'

Perform PCR amplification on the pBV220-IL-8 plasmid according to the following conditions.

PCR amplification reaction tube composition:

PBV220-IL-8 (template DNA, 200ng/ml) 0.5μl

2.5 mMdNTPsl μl

25mMMgCl24μl

P1 (20 μM) 0.5 μl

P2 (20 μM) 0.5 μl

Taq enzyme (5.0U/μl) 0.5μl

10×PCRBuffer5μl

H ₂ O 38μl

50 μl total

The preparation of the reaction tubes was carried out in an ice bath. During the preparation process, water, template DNA and other reaction components were first added, and reacted at 95°C for 5 minutes, then Taq enzyme was added, and then PCR reaction was carried out. The cycle parameters of PCR amplification were: 94 ℃, 60s (denaturation); 55℃, 60s (annealing); 72℃, 60s (amplification), 30 cycles were performed, and then extended at 72℃ for 15min. The PCR product was subjected to 2% agarose gel electrophoresis, and a DNA fragment of the expected size could be seen (Fig. 2).

The prokaryotic expression vector pET-22b(+) was digested with NdeI and SalI to recover a large fragment, and the recovered fragment was ligated with the recovered product of the above-mentioned PCR product after the same double digestion. The ligation product was transformed into competent cells BL21(DE3), and a single clone was picked and cultured overnight, the plasmid was extracted, and the insert fragment of the expected size was obtained by enzyme digestion (Figure 3), and sequenced (Figure 1). The correctly sequenced plasmid was named pET-22b(+)-rmhIL-8. The engineered bacteria were named pET-22b(+)-rmhIL-8/BL21(DE3).

Its gene sequence is:

CATATGGCCGCGCGTTGTCAGTGCATAAAGACATACTCCAAAACCTTTCCACCCCAAATTTATCAAAGAACTGAGAG

TGATTGAGAGTGGACCACACTGCGCCAACACAGAAATTATTGTAAAACTTTCTGATGGAAGAGAGCTCTGTCTGGA

CCCCAAGGAAAACTGGGTGCAGAGGGTTGTGGAGAAGTTTTTTGAAGAGGGCTGAGAACTCATAAGTCGAC (see Figure 1).

3.2 Expression of recombinant protein

Pick a single colony of engineered bacteria and inoculate it in 5 mL of LB culture solution (containing Amp100mg/L), culture it on a shaker at 37°C overnight, transfer it to the LB culture solution containing Amp at a ratio of 1:100 the next day, and inoculate it on a shaker at 37°C. After culturing for 3 hours to the mid-logarithmic growth phase (OD ₆₀₀ is 0.4-0.6), add IPTG at a final concentration of 1 mmol/L, and continue culturing for 4 hours. Bacteria were collected by centrifugation.

3.3 Identification of expression products

The expression product and expression form were analyzed by Trcine-SDS-PAGE (Figure 4), and the immunogenicity of the expression product was identified by Western-Blot method.

3.3.1Tricine-SDS-PAGE

3.3.1.1 The preparation of electrophoresis buffer is shown in the table below

Buffer TrisTricinepHSDS(mol/L)(mol/L)(%) Anode buffer 0.2-8.9*-cathode buffer 0.10.18.25**0.1 Gel buffer 3.0-8.4*0.3

*Adjust pH with HCl, **pH is about 8.25.

3.3.1.2 The preparation of acrylamide stock solution is shown in the table below

Monopropylene-Dipropylene Mixture Percentage of Monopropylene Percentage of Dipropylene 49.5%T, 3%C481.549.5%T, 6%C46.53.0

Where T: total concentration of acrylamide; C: degree of crosslinking

3.3.1.3 Preparation of glue

Similar to general SDS-PAGE, prepare separating gel and stacking gel according to the following table

Component separation gel stacking gel 16%T, 6%C6%T, 3%C 49.5% T, 3% C acrylamide stock solution (ml)-0.48 49.5% T, 6% C acrylamide stock solution (ml) 3.3-

Gel buffer (ml) 3.31.00 urea (g)/glycerol (ml) 3.6/2.4-water (ml) 11.5010% ammonium persulfate (μl) 4025 TEMED (μl) 4.02.5 total volume (ml) 10.043.03

3.3.1.4 Electrophoresis process

Mix the sample with the sample buffer and boil for 2 minutes, fix the gel-filled glass plate on the electrophoresis device, seal the edge with 1% agarose, pour the cathode buffer, add the sample, put it into the electrophoresis tank, pour the anode buffer, and keep Electrophoresis at 70V for 5-7h, staining, decolorization, gel preservation.

3.3.2 Western blot reaction

After the Tricine-SDS-PAGE is finished, according to the Bio-Rad product instructions, the gel is close to the cathode side, the nitrocellulose membrane (NC membrane) is close to the anode side, and the transfer buffer (25mmol/LTris, 192mmol/LGlycine, 20% methanol) , 100V constant voltage for 1h, electrotransfer the protein from the gel to the NC membrane, after the electrotransfer, take out the NC membrane, wash 3 times with the washing solution TBST (containing 0.02mol/LpH7.4TBS, 0.4% Tween20) at room temperature, and immerse in Blocking solution (TBST containing 2% BSA) at 37°C for 1 hour, washing solution (TBST) at room temperature for 3 times, adding mouse anti-human IL-8 mAb, incubating at 37°C for 1 hour, washing membrane with TBST at room temperature for 3 times, adding goat anti-mouse IgG - AP secondary antibody, incubated at 37°C for 1 h, washed the membrane 3 times with TBST at room temperature, and then washed 3 times with TBS, immersed the NC membrane in the color developing solution, and developed color at room temperature in the dark for 5 min, rinsed with distilled water to terminate the reaction.

3.4 Purification of recombinant protein

Suspend each gram of induced cells in 10ml of STE [50mmol/LTris.HCl (pH8.0), 1mmol/LEDTA, 100mmol/LNaCl], add lysozyme (0.8mg/g cells), stir for 20 minutes, then add Sodium deoxycholate (DOC), continue to stir until the suspension becomes viscous, then add 1mol/LMgCl ₂ 0.4ml to 1 gram of bacteria, after stirring evenly, add 1mg/ml DNAaseI 20μl to 1 gram of bacteria, stir evenly, then room temperature Leave for 30min for digestion. The lysate product was heated at 70° C. for 10 minutes, rapidly cooled to 0° C., centrifuged at 12,000 r/min for 15 minutes, and the supernatant was taken ( FIG. 5 ). Dialyze with 50 times the volume of solution A (20mmol/L citric acid buffer, pH6.0) for 12 hours, change the dialyzed external fluid once in the middle, pass the supernatant after dialysis through SP-Sepharose FF which is fully balanced with solution A, and elute B Liquid is 20mmol/L citric acid buffer solution pH6.0, 1mol/LNaCl, continuous gradient, elution 4-5 column bed volume, collect the elution peak at 0.5mol/LNaCl place, carry out SDS-PAGE electrophoresis detection (Fig. 6 ).

3.5 Study on Inhibition of IL-8 Chemotactic Activity in Vitro

Take fresh human venous blood and put it into a heparin anticoagulant tube, mix it with Hank's solution 1:1, carefully add it on the liquid surface of 2 parts of PMN cell separation solution, centrifuge at 1000-1500rpm for 20 minutes, collect the cells on the interface, put Put it into a test tube containing 4-5ml of Hank's solution, mix thoroughly, and centrifuge at 1500-2000rpm for 10 minutes. Aspirate the supernatant, wash the pellet twice in the same way to obtain the desired cells, suspend the cells in 1 ml of RPMI-1640 culture medium containing 5% BSA, and test the cell viability. Then use the same culture medium to adjust the cells to 3×106/ml for chemotaxis test immediately. 96-well chemotaxis plate with a membrane thickness of 10 μm and a pore size of 3 μm. The concentration of freshly isolated peripheral blood PMNs (neutrophils) was adjusted to 10 ⁶ /ml. Add 20 μl of cell suspension to the upper layer, and add 29 μl of sample to the lower layer (rmhIL-8 serially diluted with RPMI-1640 containing 5% BSA and 10ng/ml IL-8, the concentrations are 0, 0.01, 0.1, 1, 10 and 100 μg/ml) , an anti-IL-8 neutralizing monoclonal antibody was used as a positive control, and an anti-TNF irrelevant antibody was used as a negative control. After being placed in a CO ₂ incubator at 37°C for 2 hours, fix with 70% methanol and stain with Giemsa solution to observe the cells on the lower membrane surface, and use a 400× optical microscope to observe and count the number of cells in the lower layer. Chemotaxis activity was expressed by chemotaxis index (chemotatic index, CI), that is, the ratio of the number of cells chemoattracted to the lower surface of the membrane to the number of cells randomly migrating to the lower surface of the membrane (negative control group) (Figure 7).

3.6 Chemotaxis inhibition test in vivo

15 rats were randomly divided into 3 groups. The rats in the test group were intraperitoneally injected with 10 ml of PBS (pH 7.2) containing 0.1% (w/v) oyster glycogen (sigma) and 50 μg/ml rnhIL-8. Rats were intraperitoneally infused with 10ml of PBS containing 0.1% (w/v) oyster glycogen and 50μg/ml anti-IL-8 neutralizing monoclonal antibody, and rats in the negative control group were intraperitoneally infused with 10ml containing only 0.1% (w/v ) PBS of oyster glycogen. After 4 hours, the rats were sacrificed, the peritoneal effusion was collected, and the content of neutrophils was counted ( FIG. 8 ).

Claims (3)

1. human interleukin 8 antagonist protein, it is characterized in that, select the reactive site ELR in the natural human IL-8 gene to sport AAR, structure can be competed the antagonist protein gene rmhIL-8 of bind receptor with natural IL-8, and in intestinal bacteria, obtain to efficiently express, obtain rmhIL-8 albumen after purified, its gene order is:

CATATGGCCGCGCGTTGTCAGTGCATAAAGACATACTCCAAACCTTTCCACCCCAAATTTATCAAAGAACTGAGAGTGATTGAGAGTGGACCACACTGCGCCAACACAGAAATTATTGTAAAACTTTCTGATGGAAGAGAGCTCTGTCTGGACCCCAAGGAAAACTGGGTGCAGAGGGTTGTGGAGAAGTTTTTGAAGAGGGCTGAGAACTCATAAGTCGAC。

2. realize the preparation method of the described human interleukin 8 antagonist protein of claim 1, it is characterized in that, carry out according to the following steps:

1) structure of people rmhIL-8 expression vector

Obtain people rmhIL-8 gene by the PCR reaction, simultaneously the NdeI restriction enzyme site is introduced 5 ' end primer, the SalI restriction enzyme site is introduced 3 ' end primer, and the plasmid that contains the natural IL-8 gene of people is carried out pcr amplification, and design PCR primer is as follows:

Primere15’AGCATATGGCCGCGTGTCAGTGCATAAAG3’

Primere25’AGGTCGACTTATGAGTTCTCAGCCCTCTT3’

The preparation of pcr amplification reaction pipe is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive components, 95 ℃ of reaction 5min add the Taq enzyme again, carry out the PCR reaction then, and the loop parameter of pcr amplification is: 94 ℃, the 60s sex change; 55 ℃, 60s annealing; 72 ℃, 60s extends, and carries out 30 circulations, extends 15min in 72 ℃ again; The PCR product carries out 2% agarose gel electrophoresis, the dna fragmentation of visible expection size;

Plasmid pET-22b (+) reclaims big fragment behind NdeI and SalI double digestion, the fragment of recovery and above-mentioned PCR product carry out ligation through the recovery product of same double digestion; Connect product transformed competence colibacillus cell BL21, picking mono-clonal overnight incubation is extracted plasmid, cuts through enzyme and identifies the insertion fragment that obtains the expection size, checks order, and the result is consistent with expection; Plasmid called after pET-22b (+)-rmhIL-8 after the reorganization;

2) rmhIL-8 Recombinant Protein Expression

The e. coli bl21 that will contain recombinant plasmid pET-22b (+)-rmhIL-8 is inserted activation jolting overnight incubation in the LB substratum in 37 ℃, m seq, be inoculated in the LB substratum in 1: 100 ratio after, continue 37 ℃ of cultivation 3h to logarithmic growth later stage, OD _650nm=0.4 o'clock, add IPTG, 1mmol/L cultivates 4h, collects thalline;

3) evaluation of expression product

To the evaluation Western-blot of expression product, behind Tricine-SDS-PAGE separation expression product, change film, add mouse-anti people IL-8 monoclonal antibody respectively and resist as one, the sheep anti-mouse antibody of alkaline phosphatase AP mark is anti-as two, the substrate colour developing;

4) purifying of recombinant protein

Induce the back thalline to be suspended among the 10mlSTE every gram, the prescription of STE is: the Tris.HCl of 50mmol/LpH8.0,1mmol/LEDTA, 100mmol/LNaCl, add the N,O-Diacetylmuramidase of 0.8mg/g thalline, stir after 20 minutes, add Sodium desoxycholate DOC, continue to stir and become thickness until suspension, 1 gram thalline adding 1mol/LMgCl ₂0.4ml the back that stirs adds 1mg/mlDNAaseI20 μ l, the back room temperature that stirs is placed 30min and is digested; To split the bacterium product at 70 ℃ of heating 10min, and be cooled to 0 ℃ rapidly, the centrifugal 15min of 12000r/min gets supernatant;

With the 20mmol/L citrate buffer solution of 50 times of volumes, the A liquid of pH6.0, dialysis 12h, the centre is changed extracellular fluid dialysis one time, and the supernatant after the dialysis is crossed with the abundant equilibrated of A liquid and is crossed SP-SepharoseFF, wash-out B liquid is 20mmol/L citrate buffer solution pH6.0,1mol/LNaCl, continuous gradient, 4～5 column volumes of wash-out, the target protein peak elutes when 0.5mol/LNaCl, collect the purpose peak, carry out the Tricine-SDS-PAGE electrophoresis detection, promptly obtain rmhIL-8 albumen.

3. the preparation method of human interleukin 8 antagonist protein as claimed in claim 2 is characterized in that, described pcr amplification reaction pipe consists of:

200ng/ml pBV220-IL-8 0.5μl

2.5mM dNTPs 1μl

25mM MgCl ₂ 4μl

20μMP1 0.5μl

20μMP2 0.5μl

5.0U/ul Taq enzyme 0.5 μ l

10×PCR Buffer 5μl

H ₂O 38μl

Amount to 50 μ l.

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