CN101612387A - Macrophage is regulated the application of albumen MIRF in the preparation anti-inflammatory drug - Google Patents

Abstract

The invention discloses the medical usage of MIRF in the preparation anti-inflammatory drug.A kind of anti-inflammatory drug is provided simultaneously, and it contains the MIRF of effective dose.Experiment shows, MIRF can suppress the expression of the inductive and inflammatory factor that autoimmune disease causes such as LPS, PGN, therefore can be used as some inflammation,, have potential clinical value as effective inhibitor of immunological diseases such as asthma, rheumatic arthritis.In addition, by technique for gene engineering, can obtain a large amount of, stable, activated reorganization MIRF albumen, cost is very low, is suitable for applying.

Description

Macrophage is regulated the application of albumen MIRF in the preparation anti-inflammatory drug

Technical field

The present invention relates to biomedicine field, specifically MIRF (the MacrophageInflammation Related Factor) purposes of albumen in medicine.

Background technology

Macrophage is that human body is cytophagous a kind of, is distributed in the tissue, the immunologic information transmission is arranged, work in coordination with and engulf processing antigen effect.Mononuclear cell oozes out blood vessel, enter tissue and organ after, further differentiation and development becomes macrophage, becomes the strongest cell of phagocytic activity in the body.Macrophage can be fixed, also can move with the mode of ameboid locomotion.Macrophage fixing and migration is homocellular different phase, and both can change, and its form also changes with the position at functional status and place.The title difference of macrophage in different tissues: in lung, claim " pulmonary macrophage "; In nervous system, be called " microgliacyte "; In bone, then be called " osteoclast ".

Mononuclear cell and macrophage can both eliminate the antibacterial of invading body, engulf foreign particles, eliminate old and feeble in the body, the cell of damage and intercellular substance, the killing tumor cell of degeneration, and participate in immunoreation.

The regulation and control of macrophage activity are vital for keeping anticusp and pressing down scorching balance in immune system.Behind the macrophage overactivity, can cause inflammation, even produce the macrophage activation syndrome.Studies show that inflammatory reaction and some cytokine TNF-α, IL-1, IL-6, IL-8 are closely related.These inflammatory cytokines are mainly by being secreted by Monocytes, neutrophilic granulocyte, lymphocyte, epithelial cell or endotheliocyte.Therefore, be expected to alleviate by the expression that suppresses cytokine TNF-α, IL-1, IL-6, IL-8 and the generation of inflammation-inhibiting.

MIRF albumen, the NCBI note name is CHID1 (Homo sapiens chitinasedomain containing a 1) albumen, be that inventor place laboratory is by the bioinformatics means, utilize in 25 kinds of human secretory proteins that PSORT and HMM model discrimination go out one (GenBank ID, NM_023947).This gene length 1182bp, 393 aminoacid of encoding are positioned No. 11 chromosomes (11p15.5), its Unknown Function.The result who compares in PDB (protein data bank) data base shows that MIRF albumen is the albumen of a structure the unknown.Protein sequence comparison finds that this albumen contains the conserved domain of a glycohydrolases 18 (Glyco_18), but with the proteic sequence homology of family less than 20%, belong to the unknown fully albumen of a 26S Proteasome Structure and Function.

The inventor further studies this albumen, finds that this albumen can suppress the expression of inflammatory cytokine, has therefore proposed the application.

Summary of the invention

The object of the present invention is to provide the new purposes of MIRF albumen in the preparation anti-inflammatory drug.

The present invention is by Se-SAD method (Brodersen, de La Fortelle et al.2000) resolved the three-dimensional crystalline structure of MIRF, find that MIRF has the typical TIM barrel-like structure of Glyco-18 chitinase family protein, but because the conserved domain (motif) that MIRF hypoproteinosis chitinase is lived, so it does not have the activity of chitinase.Be that with the most tangible difference of other protein structure of Glyco-18 chitinase family there is the zone that flexibility is very big in the proteic N of MIRF end, be likely bonded zones such as it and receptor protein.In addition, the MIRF structure has shown possible and the bonded crack of sugar (cleft), compares with other albumen, and this crack is long and flat.Because the albumen of other similar chitinase can be specifically in conjunction with different saccharides, find that by the analysis of surface energy resonance transfer MIRF has obvious the combination with monosaccharide derivatives such as glucamine, acetylglucosamine and galactose, already have certain combining with mannose and ribose, what is more important, MIRF can combine with the special oligosaccharide of pathogen such as lipopolysaccharide, Peptidoglycan and chitin, and shows concentration dependent.From seen at the MIRF protein surface long and wide in conjunction with the fissured structure characteristics can explain MIRF can in conjunction with polysaccharide (LPS, PGN) equimolecular quantity can be bigger saccharide.

Further experimental result shows, MIRF is at PMA (phorbol 12-myristate13-acetate, phorbol ester) induces that expression raises in the THP-1 macrophage of differentiation, but its albumen that is secreted in the cell culture medium reduces, in order to study its reason, the present invention has detected the Subcellular Localization of MIRF in the macrophage of mononuclear cell and differentiation.Immunofluorescence analysis is the result show, MIRF albumen and PMA induce the macrophage surface combination of differentiation, and flow cytometry (FACS) detects and observes same result, but does not then observe this phenomenon in mononuclear cell THP-1.And MIRF strengthens with the prolongation that combines the time of handling along with PMA of macrophage.Therefore infer that MIRF can influence cell activity with combining of macrophage.

The present invention at first uses MIRF albumen (the 0 μ g/ml of variable concentrations purification, 0.01 μ g/ml, 0.1 μ g/ml, 1 μ g/ml and 10 μ g/ml) stimulation class macrophage, analyze cytokine expression such as TNF α and IL-6 by real-time RT-PCR, the result shows that mode that MIRF albumen relies on Concentraton gradient has really suppressed the expression of TNF α and IL-6, shows that MIRF albumen can suppress autoimmune disease, the cellular inflammation reaction that causes as lupus or rheumatism.

LPS is the main component of gram negative bacteria epicyte, causing many biological effects by gram positive bacterial infection all is to be produced by LPS as endotoxin shock, heating and deteriorated blood etc., LPS can activate the lymph and the macrophage of immunity of organism non-specificly, amplify activation NF κ B path by some signals, induce the generation of inflammation-related factor such as TNF α.The present invention uses LPS to stimulate the macrophage body to simulate the infection of gram negative bacteria, add MIRF albumen simultaneously, real-time RT-PCR detects the expression of inflammation-related factor, found that at PMA and induce in the macrophage of differentiation, LPS stimulates identical with the macrophage response of normal development, has induced inflammation-related factor such as TNF α, IL-6, IL-1 β and IL-8 to express equally; After adding MIRF albumen, the inductive anticusp factor of LPS (pro-inflammation factor) TNF α and IL-6 are obviously suppressed, and the expression of IL-1 β and IL-8 also is suppressed to a certain extent.The above results shows that MIRF is by the protein binding that shows with macrophage and then suppress the cellular inflammation reaction that LPS causes.

The main component Peptidoglycan PGN of gram-positive bacteria cell wall is made of through the recurring unit that 6-7 amino acid whose small peptide is connected to form acetylglucosamine and acetylmuramic acid, caused most of clinical symptoms that causes by gram positive bacteria such as infection of staphylococcus aureus, as heating, inflammation, deteriorated blood shock, leukocytosis, lethargy, abscess and arthritis etc.These clinical symptoms are mainly regulated proteic release by the inductive cytokine of PGN and other inflammation and are caused, and PGN is as the special pathogen-associated molecular pattern of antibacterial, it is the ideal part of pattern recognition receptor, above-mentioned sugar shows that in conjunction with experiment MIRF albumen can have intensive the combination with PGN, the present invention uses PGN to stimulate the macrophage body to simulate the infection of gram positive bacteria, add MIRF albumen simultaneously, real-time RT-PCR detects the expression of inflammation-related factor.Found that similar in the influence that LPS induces inflammation-related factor to express on changing with MIRF, MIRF albumen has suppressed the expression of the inductive pro-inflammatory cytokine TNF of PGN α, IL-6.

The regulation and control of macrophage activity are vital for keeping anticusp and pressing down scorching balance in immune system.The present invention finds that first MIRF is the novel secretion albumen that can suppress macrophage activity specifically: it can combine with macrophage, and can be specifically combines with the special oligosaccharide of pathogen such as LPS, PGN.Since pathogen infect the reaction that will cause inflammation of inductive proinflammatory cytokine expression, MIRF has macrophage (deactivation) but alive effect the supression explanation MIRF of TNF α and IL-6 expression, promptly shows as MIRF and has the effect that inflammation-inhibiting reacts.

Other albumen that contains the Glyco-8 domain of finding so far in the mammal relatively, we find that member's and inflammation disease generation is closely related in this family.Acid chitinase and chitotriose enzyme be high expressed in asthma and patient Guacher respectively, YKL-39 and YKL-40 then have important effect in the pathogeny of rheumatic arthritis, and high expressed is also arranged in the Atheromatosis people, and mice Ym1/2 brings into play regulating action in asthma or other inflammation disease.They are all expressed in macrophage, are subjected to the regulation and control of Th2 cytokine, participate in dendritic cell (DC cell) and cytophagous activation etc.MIRF albumen also shows tangible inherent immunity and regulates activity, and it can be as effective inhibitor of immunological diseases such as asthma, rheumatic arthritis.

MIRF albumen of the present invention can suppress the expression of the inductive and cellular inflammation factor that autoimmune disease (as rheumatism or lupus etc.) causes such as LPS, PGN, therefore can be as some inflammation, as effective inhibitor of immunological diseases such as asthma, rheumatic arthritis, has potential clinical value.In addition, by technique for gene engineering, can obtain a large amount of, stable, activated reorganization MIRF albumen, cost is very low, is suitable for applying.

Description of drawings

What Fig. 1 showed is the proteic overall structure of MIRF.(a) the silk ribbon figure of MIRF.Each secondary structure element has all been done labelling.All figure are all by the PyMOL program making.The zone of arrow indication is a new domain of the N end of MIRF.(b) comparison chart of MIRF (black, PDB accession no.3BXW) and YM 1 (Lycoperdon polymorphum Vitt, PDB accession no.1VF8) protein structure.

Fig. 2 shows is MIRF and the combining of part.(a) comparison chart of MIRF and HCgp39-N-acetyl glucosamine amine compound (1LG1) structure has shown the crack in conjunction with sugar.N-acetylglucosamine (NAG ₆) with bar-shaped expression.MIRF main chain (backbone) is represented with Dark grey.The HCgp39 complex is represented with light gray.On the fissured surface of MIRF, there are several aromatic amino acids (Tyr62, Trp66, Tyr239, Tyr241, Tyr280 and Trp358) in HCgp39 albumen, also to guard, these residues may participate in MIRF and sugared combining.(b) MIRF albumen and NAG ₆The molecular surface sketch map of butt joint.Combining between surface resonance energy transfer analysis checking different sugar (c) and lipopolysaccharide LPS (d) and the MIRF.(e) the bonded pull down of insoluble PGN and Chitin and MIRF analyzes.After MIRF albumen and PGN or Chitin suspension are hatched, centrifugal removal supernatant, after PBS washed five times, upward cleer and peaceful precipitation was not taken a sample and is carried out SDS-PAGE, changeed film, and anti-MIRF analyzes; Use GluNAc-argrose and Glu-argrose to carry out pull down simultaneously respectively, as the positive and negative control.P, precipitation; S, supernatant.

Fig. 3 illustrates that MIRF and PMA-induce the THP-1 cell surface of differentiation to combine, and suppresses the expression of inflammatory factor in this cell.(a) immunofluorescence detects the combining of THP-1 cell that MIRF and monokaryon THP-1 cell and PMA-induce differentiation.(b) facs analysis detects the combining of THP-1 cell that endogenous and reorganization MIRF albumen and monokaryon THP-1 cell and PMA-induce differentiation.The numeral that the upper right corner shows is the number in conjunction with cell.(c) MIRF induces the THP-1 cell of differentiation to combine with the time-dependent form with PMA-.PMA induces collecting cell after the different time, hatches with 80 μ g/ml MIRF albumen, and flow cytometry MIRF is proteic in conjunction with strong and weak behind the two anti-labellings of anti-MIRF and FITC labelling.Every pictures upper right corner shown respectively PMA induction time (on) and surface combination the proteic cell number percentage ratio of MIRF (descending) is arranged.

Fig. 4 illustrates that MIRF can suppress the expression of the cellular inflammation factor.(a) RT-PCR analyzes the influence (left side) of MIRF pair cell factor TNF α and IL-6 expression.That show among the figure is 1.5% agarose gel electrophoresis figure.This tests independent triplicate.Real time RT-PCR analyzes the influence (right side) of variable concentrations MIRF pair cell factor TNF alpha expression.Experimental result is the meansigma methods of 4 independent experiments, and standard deviation as shown in the figure.(b) MIRF suppresses the expression of inductive TNF α of LPS and IL-6 significantly in PMA-induces the THP-1 cell of differentiation.What the result showed is the meansigma methods of 4 independent experiments, and standard deviation is P＜0.01, ^*Expression is compared with independent LPS processing, and TNF α that MIRF+LPS produces and IL-6 express and change difference and remarkable.(c) PGN or PGN+MIRF handle the THP-1 cell of PMA differentiation, and ctrl. is as untreated contrast.Extract total RNA and carry out reverse transcription reaction, the expression of real time pcr analysis inflammation relevant cell factor.Block diagram has shown the meansigma methods of 4 independent experiments, and standard deviation as shown in the figure. ^*Expression is compared with independent PGN processing, and the TNF alpha expression that MIRF+PGN produces changes significant difference (P＜0.05); ^*Expression is compared with independent PGN processing, and the expression difference in change heteropole of the IL-6 that MIRF+PGN produces is (P＜0.01) significantly.

Fig. 5 explanation is compared with undifferentiated cell, induces the expression of MIRF in the THP-1 cell of differentiation obviously to improve at PMA-.(a) result of sxemiquantitative RT-PCR (left side) and real-time quantitative RT-PCR (right side).(b) Western blot result.Band 1, untreated monokaryon THP-1 cell; Band 2, PMA-are induced the THP-1 cell of differentiation.Actin is as the standard of last sample.(c) induce the proteic secretion of MIRF in the THP-1 cell of differentiation at monokaryon THP-1 cell and PMA-.The THP-1 cell having/cultivated 48 hours in the culture medium of no 50ng/ml PMA, and collecting cell and culture medium respectively, the antibody that utilizes MIRF is by Westernbolts analyzing and testing MIRF albumen.

The specific embodiment

Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.

If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment.

The proteic clone of embodiment 1 MIRF, expression and purification

Make primer with 5 '-ggggacaagtttgtacaaaaaagcaggcttaATGACCAAGGCCCGGCTGTTCCGG and 5 '-ggggaccactttgtacaagaaagctgggtcTCAGTCTCGGAGGAGGTTTTCGGG, PCR from IMAGE library (The I.M.A.G.E.Consortium) (pre-degeneration: 94 ℃, 5 minutes; Degeneration: 94 ℃, 50 seconds, annealing: 50 ℃, 1 minute, extend: 72 ℃, 90 seconds; From degeneration after extend 30 circulations, 72 ℃, 10 minutes) obtain the proteic cDNA of coding MIRF, insert among the prokaryotic expression carrier pET21DEST (Invitrogen company) by the Gateway clone technology proteic cDNA of MIRF that will encode, this carrier is held at its N and is had a histone (His ₆) label.The proteic cDNA of the MIRF that perhaps will encode inserts among the prokaryotic expression carrier pGEX4T-1 (GE company), and this carrier has a glutamyl transpeptidase albumen (GST) label at its N end.

Expression vector is transformed in e. coli bl21 (DE3) strain.Bacterium liquid after transforming is coated on the plating medium (1% sodium chloride, 1% peptone, 0.5% yeast extract, 1.5% agar powder) that contains ampicillin, cultivated 12 hours for 37 ℃.

Choose the monoclonal bacterium colony and contain in 20ml in the LB culture medium (1% sodium chloride, 1% peptone, 0.5% yeast extract) of ampicillin, cultivate after 12 hours for 37 ℃, amplification culture continues to cultivate 4 hours at 37 ℃ to the 1L culture medium, reaches OD to bacterium liquid density _0.6-0.8The time, the IPTG (isopropyl-b-d-thiogalactoside) of adding 1mM, induced protein is expressed under 18 ℃ of conditions, the centrifugal receipts of 5000g antibacterial after about 16 hours.

Antibacterial usefulness BufferA after centrifugal (20mM Tris, 500mM NaCl, pH7.5) resuspended.Ultrasonic degradation cell (the 200W 5s that works, 10s works 30 times at interval), after the carrying out ultrasonic bacteria breaking, collect and filter supernatant, supernatant is injected the nickel post, with Buffer B (20mM Tris, 500mM NaCl, 100mM imidazole, pH7.5) the weak bonded albumen of flush away at first, use Buffer C (20mMTris then, 500mMNaCl, 500mM imidazole pH7.5) washes destination protein.After concentrating, be further purified with GE Superdex75 molecular sieve, by desalting column albumen replaced in the phosphate buffer solution then, be concentrated into 10mg/ml, packing is stored in-80 ℃ of refrigerators.The MIRF albumen that is used to handle the purification that cell uses at first with clean polymyxin B resin albumen incubation to remove endotoxin (Nadesalingam, Doddset al.2005).

The proteic purification of MIRF also can be by the method purification of GST affinity chromatograph.Specifically: choose the monoclonal bacterium colony and contain in the LB culture medium (1% sodium chloride, 1% peptone, 0.5% yeast extract) of ampicillin in 20ml, cultivate after 12 hours for 37 ℃, amplification culture continues to cultivate 4 hours at 37 ℃ to the 1L culture medium, reaches OD to bacterium liquid density _0.6-0.8The time, the IPTG of adding 1mM, induced protein is expressed under 18 ℃ of conditions, the centrifugal receipts of 5000g antibacterial after about 16 hours.Antibacterial after centrifugal is used PBS Buffer (10mM Na ₂HPO ₄, 1.8mMKH ₂PO ₄, 2.7mM KCl, 140mM NaCl, pH7.3) resuspended.Ultrasonic degradation cell (the 200W 5s that works, interval 10s, work 30 times), after the carrying out ultrasonic bacteria breaking, collect and filter supernatant, supernatant is injected GST Sepharose 4B post (Amersham Pharmacia Biotech), with PBS Buffer flush away foreign protein at first, use GST Elution Buffer (50mMTris, 500mMNaCl then, 20mM reduced glutathione, pH8.0) eluting destination protein.Follow-up purification step please refer to above.

The polyclonal antibody of MIRF obtains by immune rabbit, and utilizes NBr-activatedSepharose 4B (Amersham Pharmacia Biotech) to carry out further purification by the antigen affinity chromatograph.

Embodiment 2 albumen crystallization and structure determinations

The MIRF albumen of purification is concentrated to 10mg/ml, uses meteorological diffusion sessile drop method (Protein Crystallography.T.L Blundell) to carry out crystalline screening then, the result is at 100mM Bis-Tris pH 6.5,2M NH ₄SO ₄, 10mM MPD, 100mM MgCl ₂Obtain crystal under the condition.The crystal diffraction data use MAR 325 ccd detectors to collect at Stanford Univ USA's synchrotron radiation laboratory.Glycerol with 20% is anti-icing fluid, and crystal corner with 1 ° under the 100K temperature carries out diffraction.Data after the collection are handled by Mosflm and CCP4 program.Crystallographic parameter and data collection statistics provide in table 1.

The collection and the statistics of table 1.MIRF crystallographic parameter

^*Numerical value in the bracket is the value under the high-resolution

Adopt normal equation to define different R-values, no longer in note, define here.

By selenium-single wavelength anomalous scattering method (Se-SAD method, Brodersen, de La Fortelle etal.2000)) structure of MIRF has been carried out resolving (PDB code: 3BXW).Selenic phase place is to utilize 30

To 3

Data by Phenix program (Adams, Grosse-Kunstleve et al.2002) definite.The preliminary correction of structure is by monitoring R _FreeThe factor and electron density map utilize the CNS program to finish.Have 50% aminoacid to be built out automatically approximately, remaining structure division is to utilize Coot (Emsley and Cowtan 2004) to build and structure refinement by manual.Final structure is by PROCHECK, and PYMOL and COOT program are carried out structural analysis.The Ramachandran figure of structure shows that 85.8% and 14.2% residue respectively in the core and the permission zone of MIRF structure, does not have residue in non-permission zone.

The detection of embodiment 3MIRF and saccharide and combined with lipopolysaccharide

Utilize BIAcore 3000 (Amersham Phramacia Biotech) to detect MIRF and comprise glucamine, galactosamine, acetylglucosamine, acetylgalactosamine, mannose, glucose and ribose etc. are at the binding specificity of interior different monosaccharide.The MIRF (200 μ g/ml) of the purification in 10mM sodium acetate (pH 5.0) solution and contrast are fixed on respectively by the activatory sensor chip of former amine (sensor chip CM5, Amersham PhramaciaBiotech)) surface, excessive N-Hydroxysuccinimide base ester group is blockaded by the ethylaminoethanol hydrochloride of 1M.The different monosaccharide of HBS buffer (10mM HEPES with150mM NaCl, 3mM EDTA, and 0.05%Tween-20) and LPS be will be dissolved in and the surface of chip and real-time monitored binding curve will be expelled to respectively with the speed of 30 μ l/min.The HBS-ET buffer is used to cessation reaction.Utilize BIA evaluation software 4.1 computational dynamics data (Pharmacia Biosensor AB).

MIRF and insoluble sugar are analyzed by the pulldown experiment as Peptidoglycan (PGN) and chitinous combination.Insoluble PGN and chitin etc. are resuspended in respectively among the PBS, add MIRF with it 4 ℃ in conjunction with 3h, after PBS washed 3 times, precipitation and supernatant were taken a sample respectively and are carried out SDS-PAGE electrophoresis and Western Blot analysis.

Its result as shown in Figure 2, (a) comparison chart of MIRF and HCgp39-N-acetyl glucosamine amine compound (1LG1) structure, shown sugar in conjunction with the crack.N-acetylglucosamine (NAG ₆) with bar-shaped expression.MIRF trunk (backbone) is represented with Dark grey.The HCgp39 complex is represented with light gray.On the fissured surface of MIRF, there are several aromatic amino acids (Tyr62, Trp66, Tyr239, Tyr241, Tyr280 and Trp358) in HCgp39 albumen, also to guard, these residues may participate in MIRF and sugared combining.(b) MIRF albumen and NAG ₆The molecular surface sketch map of butt joint.Combining between surface resonance energy transfer analysis checking different sugar (c) and lipopolysaccharide LPS (d) and the MIRF.Insoluble PGN and Chitin are verified (e) with combining by pull down analysis of MIRF.

Embodiment 4MIRF is in the THP-1 cell of PMA-differentiation and the detection of undifferentiated THP-1 cell surface expression

People's mononuclear cell THP-1 in containing the RPMI1640 culture medium of 10% hyclone, at 37 ℃, 5%CO ₂Cultivate under the condition.Processing was divided into macrophage after 48 hours to mononuclear cell THP-1 through 50ng/ml PMA.

The THP-1 cell was cultivated 48 hours in the culture medium that contains or do not contain 50ng/ml PMA, then respectively by immunofluorescence analysis and flow cytometry (fluorescence-activated cell sorting FACS) detects, and is specific as follows:

For immunofluorescence analysis, cell is at first washed 3 times with ice-cold PBS buffer, under 25 ℃, fix 20 minutes with 3.7% paraformaldehyde afterwards, after blockading with 3% BSA, cell is the 4 ℃ times polyclonal antibody overnight incubation with MIRF, afterwards with the anti-rabbit igg antibody of FITC labelled goat 37 ℃ of incubations 30 minutes, cell is redyed with DAPI, observes down at fluorescence microscope (Olympus IX51-A11PH) then and takes a picture.

In order to carry out facs analysis, PMA induces the macrophage of differentiation at first to hatch in the PBS that contains 50mM EDTA so that cell separates from culture dish, wash 3 times with ice-cold PBS buffer, afterwards with the MIRF polyclonal antibody of 10 μ g/ml or rabbit igg antibody 4 ℃ of incubations 1 hour, then, analyze with FACScan flow cytometer at once with the link coupled goat anti-rabbit igg of FITC incubation 30 minutes under same temperature.

Real-time quantitative RT-PCR is analyzed

Cell is not having LPS to handle, perhaps 1 μ g/ml LPS handles, perhaps 125 μ g/ml PGN, or 1 μ g/ml LPS (perhaps 125 μ g/ml PGN) and 10ng-10 μ g/ml MIRF albumen are common handles after 24 hours, utilize TRIZOL reagent (Invitrogen company) to extract the full RNA of cell.It is cDNA that the full RNA of 1 μ g is used to reverse transcription, and reverse transcription reaction utilizes the Reverse Transcription System test kit of Promega company to carry out, and concrete operations are carried out according to the description of test kit.

Obtain cDNA with reverse transcription reaction and make template, according to MIRF, IL-1 β, IL-6, IL-8, TNF α gene order design primer carries out quantitative real time pcr analysis.The primer sequence is as follows:

MIRF?Forward?primer?5’-GGGGCTCAACTTCTATGGTATGGA

Reverse?primer?5’-TCCCACTGCGGCTCTTCTTGT

TNFαForward?primer?5′-ATCACCTTTTTGGACCGCC

Reverse?primer?5′-GCTTGTAGGTGCCCAGGAGA

IL-6?Forward?primer?5’-AACCTGAACCTTCCAAAGATGG

Reverse?primer?5’-TCTGGCTTGTTCCTCACTACT

IL-1β?Forward?primer?5′-CGATCACTGAACTGCACGCTC

Reverse?primer?5′-CAACACGCAGGACAGGTACAGA

IL-8Forward?primer?5′-ATACTCCAAACCTTTCCACCC

Reverse?primer?5′-AAAACTTCTCCACAACCCTCTG

Actin?Forward?primer?5’-AAGTGTGACGTGGACATCCGC

Reverse?primer?5’-CCGGACTCGTCATACTCCTGCT

Utilize SYBR Green qPCR test kit (Finnzymes),, on Bio-Rad MJ quantitative real time PCR Instrument, carry out Real Time PCR by real-time quantitative RT-PCR, detect mRNA expression and the preceding inflammatory factor TNF α of mirf, IL-1 β, IL-6, the expression of and IL-8.β-actin is as last sample quantitative criterion albumen.200nM forward primer, 200nMreverse primer, 10 μ l, 2 * SYBR Green PCR Mix, 0.5-2 μ l cDNA adds water to 20 μ l.

The PCR condition: 95 ℃ of 10min → 95 ℃ 15sec → 64 ℃ of second step of 20sec → 72 ℃ 20sec to the 4th steps are carried out 44 circulations.

According to following method obtaining data are analyzed: in the PCR reaction, thereby the product of each amplification cycles makes product be Exponential growth as next circulation substrate.But along with the increase of period, since the increase of template number, the following degradation reason of the reduction of primer amount and Taq enzymatic activity, and amplification efficiency descends and platform effect occurs, and the interior period ability actual response template amount of the range of linearity what therefore have only.Pcr amplification curve according to fluorescence signal reflects defines a thresholding (threshold) in linear limit increase, the pairing period of the intersection point of this thresholding and amplification curve is called the Ct value.We adopt 2-Δ Δ Ct method [Livak andSchmittgen, 2001] to come analytical data, the method is a kind of method of the relative expression quantity of analyzing gene easily.

Δ Ct=Ct _{Genes of interest}-Ct _{Confidential reference items}

Δ Δ Ct=Δ Ct _Sample-Δ Ct _Contrast

The result as shown in Figure 4.

Western?Blot

Utilize MIRF antibody (embodiment 1 prepares), by proteic expression of Western Blot analyzing and testing MIRF and secretion situation.

Proteinic Western Blot analyzes specific as follows:

Protein is transferred to the nitre fibrous membrane from the SDS-PAGE gel, conventional configuration transfering buffering liquid (5.8g Tris, 2.9g Gly, 0.375g SDS, 200ml methanol), 100mA transferase 12 h under the ice bath (glue towards negative electrode, the film chongyang utmost point); Place TBS T buffer (1.21g Tris with shifting the fine film of successful nitre, 4g NaCl, the polysorbas20 of 1/1000 volume) the middle flushing 2-3 time, change 10ml sealing buffer (0.5g defatted milk powder over to, 1ml 10 * TBS, 10 μ l polysorbas20s) room temperature sealing 1h or 4 ℃ spend the night; Adding antibody diluent (0.5g BSA, 1ml 10 * TBS, 10 μ l Tween-20) is diluted to an anti-solution of working concentration, and incubated at room 2h, TBST give a baby a bath on the third day after its birth inferior, 10min/ time; Then add antibody diluent and be diluted to two anti-solution of working concentration, incubated at room 2h, TBST give a baby a bath on the third day after its birth time, 10min/ time; After adding chemical luminescence for liquid 3min at last, tabletting in the darkroom is developed a film, observation signal.

The result is shown in Fig. 3 and 4.Fig. 3 shows that MIRF induces the THP-1 cell surface of differentiation to combine with PMA-.(a) immunofluorescence detects the combining of THP-1 cell that MIRF and monokaryon THP-1 cell and PMA-induce differentiation.(b) facs analysis detects the combining of THP-1 cell that endogenous and reorganization MIRF albumen and monokaryon THP-1 cell and PMA-induce differentiation.The numeral that the upper right corner shows is the number in conjunction with cell.(c) MIRF induces the THP-1 cell of differentiation to combine with the time-dependent form with PMA-.PMA induces collecting cell after the different time, hatches with 80 μ g/ml MIRF albumen, and flow cytometry MIRF is proteic in conjunction with strong and weak behind the two anti-labellings of anti-MIRF and FITC labelling.Every pictures upper right corner shown respectively PMA induction time (on) and surface combination the proteic cell number percentage ratio of MIRF (descending) is arranged.

Fig. 4 shows that MIRF suppresses the expression of inflammatory factor in the cell.(a) RT-PCR analyzes the influence (left side) of MIRF pair cell factor TNF α and IL-6 expression.That show among the figure is 1.5% agarose gel electrophoresis figure.This tests independent triplicate.Real time RT-PCR analyzes the influence (right side) of variable concentrations MIRF pair cell factor TNF alpha expression.Experimental result is the meansigma methods of 4 independent experiments, and standard deviation as shown in the figure.(b) MIRF suppresses the expression of inductive TNF α of LPS and IL-6 significantly in PMA-induces the THP-1 cell of differentiation.What the result showed is the meansigma methods of 4 independent experiments, and standard deviation is P＜0.01, ^*Expression is handled with independent LPS and is compared, and TNF α that MIRF+LPS produces and IL-6 express the difference in change heteropole it is remarkable.(c) PGN or PGN+MIRF handle the THP-1 cell of PMA differentiation, and ctrl. is as untreated contrast.Extract total RNA and carry out reverse transcription reaction, the expression of real time pcr analysis inflammation relevant cell factor.Block diagram has shown the meansigma methods of 4 independent experiments, and standard deviation as shown in the figure. ^*Expression is compared with independent PGN processing, and the TNF alpha expression that MIRF+PGN produces changes significant difference (P＜0.05); ^*Expression is compared with independent PGN processing, and the expression difference in change heteropole of the IL-6 that MIRF+PGN produces is (P＜0.01) significantly.

Fig. 5 shows, compares with undifferentiated cell, induces the expression of MIRF in the THP-1 cell of differentiation obviously to improve at PMA-.(a) result of sxemiquantitative RT-PCR (left side) and real-time quantitative RT-PCR (right side).(b) Western blot result.Band 1, untreated monokaryon THP-1 cell; Band 2, PMA-are induced the THP-1 cell of differentiation.Actin is as the standard of last sample.(c) induce the proteic secretion of MIRF in the THP-1 cell of differentiation at monokaryon THP-1 cell and PMA-.The THP-1 cell having/cultivated 48 hours in the culture medium of no 50ng/ml PMA, and collecting cell and culture medium respectively, the antibody that utilizes MIRF is by Westernbolts analyzing and testing MIRF albumen.

List of references:

Adams，P.D.，R.W.Grosse-Kunstleve，et?al.(2002).″PHENIX：building?newsoftware?for?automated?crystallographic?structure?determination.″ Acta Crystallogr?D?Biol?Crystallogr?58(Pt?11)：1948-54.

Brodersen，D.E.，E.de?La?Fortelle，et?al.(2000).″Applications?of?single-wavelengthanomalous?dispersion?at?high?and?atomic?resolution.″ Acta?Crystallogr?D?Biol Crystallogr?56(Pt?4)：431-41.

Emsley，P.and K.Cowtan(2004).″Coot：model-building?tools?for?moleculargraphics.″ Acta?Crystallogr?D?Biol?Crystallogr?60(Pt?12Pt?1)：2126-32.

Nadesalingam，J.，A.W.Dodds，et?al.(2005).″Mannose-binding?lectin?recognizespeptidoglycan?via?the?N-acetyl?glucosamine?moiety，and?inhibitsligand-induced?proinflammatory?effect?and?promotes?chemokine?productionby?macrophages.″ J?Immunol?175(3)：1785-94.

Blundell，T.L?and?Johnson，L.N.(1976).Protein?Crystallography.Academic?Press.

The sequence table explanation:

SEQ ID NO.1 ﹠amp; The 2nd, the primer of amplification MIRF albumen cDNA from the IMAGE library, SEQ IDNO.3～14th, to MIRF, IL-1 β, IL-6, IL-8, TNF α carry out real-time quantitative PCR and analyze used primer.

Sequence table

＜110〉Peking University

＜120〉macrophage is regulated the application of albumen MIRF in the preparation anti-inflammatory drug

<130>

<160>14

<170>PatentIn?version?3.3

<210>1

<211>55

<212>DNA

＜213〉artificial sequence

<400>1

ggggacaagt?ttgtacaaaa?aagcaggctt?aatgaccaag?gcccggctgt?tccgg 55

<210>2

<211>54

<212>DNA

＜213〉artificial sequence

<400>2

ggggaccact?ttgtacaaga?aagctgggtc?tcagtctcgg?aggaggtttt?cggg 54

<210>3

<211>24

<212>DNA

＜213〉artificial sequence

<400>3

ggggctcaac?ttctatggta?tgga 24

<210>4

<211>21

<212>DNA

＜213〉artificial sequence

<400>4

tcccactgcg?gctcttcttg?t 21

<210>5

<211>19

<212>DNA

＜213〉artificial sequence

<400>5

atcacctttt?tggaccgcc 19

<210>6

<211>20

<212>DNA

＜213〉artificial sequence

<400>6

gcttgtaggt?gcccaggaga 20

<210>7

<211>22

<212>DNA

＜213〉artificial sequence

<400>7

aacctgaacc?ttccaaagat?gg 22

<210>8

<211>21

<212>DNA

＜213〉artificial sequence

<400>8

tctggcttgt?tcctcactac?t 21

<210>9

<211>21

<212>DNA

＜213〉artificial sequence

<400>9

cgatcactga?actgcacgct?c 21

<210>10

<211>22

<212>DNA

＜213〉artificial sequence

<400>10

caacacgcag?gacaggtaca?ga 22

<210>11

<211>21

<212>DNA

＜213〉artificial sequence

<400>11

atactccaaa?cctttccacc?c 21

<210>12

<211>22

<212>DNA

＜213〉artificial sequence

<400>12

aaaacttctc?cacaaccctc?tg 22

<210>13

<211>21

<212>DNA

＜213〉artificial sequence

<400>13

aagtgtgacg?tggacatccg?c 21

<210>14

<211>22

<212>DNA

＜213〉artificial sequence

<400>14

ccggactcgt?catactcctg?ct 22

Claims (3)

1, macrophage is regulated the application of albumen MIRF in the preparation anti-inflammatory drug.

2, application as claimed in claim 1 is characterized in that described anti-inflammatory drug is for suppressing the medicine of the inductive and inflammation that autoimmune disease causes of LPS, PGN.

3, a kind of anti-inflammatory drug, it contains the MIRF albumen of effective dose.

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