CN108948178A - A kind of people IL-37 albumen of improvement and its application - Google Patents
A kind of people IL-37 albumen of improvement and its application Download PDFInfo
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Abstract
The present invention relates to biomedicine fields, and in particular, to a kind of people IL-37 albumen of improvement and its application.People's IL-37 albumen of improvement of the invention is successively to connect people IL-37 albumen with cell-penetrating peptides and the Fc of IgG antibody section, forms IL-37-CPP-IgG Fc albumen.People's IL-37 albumen of improvement of the present invention has the amino acid sequence as shown in SEQ ID NO.1, or replacing, missing or adding by one or several amino acid residues, or with different IgG hypotypes, and there is the active protein as derived from SEQ ID NO.1 same or similar with SEQ ID NO.1.The people IL-37 albumen of improvement of the invention can polarization with from inducing macrophage to M2, while the activation of AMPK in macrophage can be obviously promoted.The people IL-37 albumen of the improvement of the application has the advantages that dosage is small, Half-life in vivo is long, Small side effects, curative effect is better compared with commercially available rIL-37 albumen.
Description
Technical field
The present invention relates to biomedicine fields, and in particular, to a kind of people IL-37 albumen of improvement and its application.
Background technique
Interleukin 37 (Interleukin-37, IL-37) is the newcomer of IL-1 family, and be otherwise known as IL-IF7.
IL-37 is that one kind of discovered in recent years can adjust the cell factor of inherent immunity and Acquired immune response, in various inflammatories
Have in disease and immunity disease and inhibits inflammatory effect.Compared to the IL-1 family member of other proinflammatory diseases, IL-37 is current
Uniquely has the cell factor of immunosuppressive activity in the family.IL-37 can be expressed in various kinds of cell, mainly be expressed in macrophage
Cell, lymphocyte, monocyte, tissue epithelial cell, synovial cell, interstitial cell, gland cell, keratinocyte and tree
Prominent shape cell etc..Mature IL-37 can be secreted into extracellularly with two receptors of mouse macrophage surface SIGIRR and IL-18R α
Industry base junction, which is closed, carries out immune negative regulation by receptor pathway, and wherein SIGIRR also plays immune negative regulator in various inflammation diseases and makees
With；In addition mature IL-37 may further enter into the nucleus of A549 cell and THP-1 cell, with Smad3 protein-interacting, lead to
It crosses nuclear factor approach and inhibits immune response.In the past research shows that IL-37 has immunoregulation effect to macrophage, but
Whether IL-37 influences the polarization of macrophage and mechanism of action has not been reported at present.Further, since commercially available IL-37 cell
The design of the factor does not embody the effect of its transcription factor, and the druggability of the cell factor may be underestimated, and cannot from turn
Record is because of subangle research IL-37.
Summary of the invention
In view of the deficienciess of the prior art, it is an object of the present invention to provide a kind of people's IL-37 albumen of improvement.
It is a further object of the present invention to provide the encoding genes of people's IL-37 albumen of above-mentioned improvement.
To achieve the above object, the specific technical solution of the present invention is as follows:
The people IL-37 albumen (human interleukin -37, hIL-37) of improvement of the invention, be by people IL-37 albumen successively
The Fc section of cell-penetrating peptides (cell-penetrating peptide, CPP) and IgG antibody are connected, IL-37-CPP-IgG is formed
Fc albumen.
People's IL-37 albumen of the improvement according to the present invention, if it is the intracorporal research of mouse for convenience, so that external source
The albumen of injection is not removed rapidly by host, and the best way is using source of mouse analog.However, IL-37 molecule is in mouse
There is no corresponding homologue.The present invention still uses people's IL-37 albumen, and the Fc section of IgG antibody is then using the IgG of mouse.
And when the application is used for human body, all sequence of mouse source, which must all change, makees corresponding source of people sequence, i.e., replaces with the IgG of mouse
The IgG of source of people.People's IL-37 albumen of the improvement according to the present invention, wherein the cell-penetrating peptides CPP can be ability
Domain is well known, and there is guidance foreign protein to pass through any one small peptide that cell membrane enters endochylema.
People's IL-37 albumen of the improvement according to the present invention, wherein preferably, people's IL-37 albumen of the improvement is in CPP
The WEHD amino acid sequence that Caspase restriction enzyme site is identified is added between IgG Fc.People's IL-37 albumen of the improvement
Caspase restriction enzyme site can be the recognition site of any one enzyme in Caspase family series, such as Caspase-1 digestion
Site.
People's IL-37 albumen of the improvement according to the present invention, wherein the IgG of people's IL-37 albumen of the improvement, it can be with
It is IgG1, IgG2a, IgG2b, IgG2c or IgG3 of mouse, is also possible to IgG1, IgG2, IgG3 or IgG4 of people；Wherein, excellent
It is selected as the IgG of people.
People's IL-37 albumen of the improvement according to the present invention, wherein the IgG of people's IL-37 albumen of the improvement, it can be with
It is wild type, more preferably after amino acid mutation, has further changed ADCC cell killing vigor and CDC complement swashs
While still alive the mutant of power.
People's IL-37 albumen of the improvement according to the present invention, wherein people's IL-37 albumen of the improvement can also be sugar
The albumen of base, the glycosylation albumen can be wild type, be also possible to the engineering cell strain being transformed by glycosyl production
Glycosyl Optimization-type.
People's IL-37 albumen of the improvement according to the present invention, wherein preferably, people's IL-37 albumen of the improvement has
The amino acid sequence as shown in SEQ ID NO.1；Or between IL-37, CPP, IgG structural domain, have not more than three it is non-dredge
The amino acid residue of water non-band charge replaces, misses or adds, or with different IgG hypotypes, and has and SEQ ID
The same or similar active protein as derived from SEQ ID NO.1 of NO.1.
The encoding gene of people's IL-37 albumen of improvement provided by the invention, with one of following nucleotide sequences:
1) there is the nucleotide sequence for encoding any of the above-described albumen；
2) encoding gene has the nucleotide sequence as described in SEQ ID NO.2, or has and SEQ ID NO.2
The nucleotide sequence of at least 60% homology.
The present invention also provides the expression vectors for containing above-mentioned encoding gene.Preferably, the expression vector is
pDirect4.0。
The present invention also provides the expression cell system of people's IL-37 albumen of improvement, above-mentioned volume is contained in the expression cell system
Code gene.Preferably, the expression cell system is CHO cell line.
Further, present invention also provides the people IL-37 albumen of above-mentioned improvement and encoding gene exempts from preparation treatment
Epidemic disease (the especially autoimmune diseases such as rheumatoid arthritis, rigid spine), virus disease (such as virus hepatitis etc.),
Application in parasitic infection (hepatic disease caused by infection by Schistosoma etc.) or the drug of tumour.
The present invention also provides above-mentioned expression cells to tie up to preparation treatment immunological diseases, virus disease, parasitic infection
Or the application in the drug of tumour.
In people's IL-37 albumen of improvement of the invention containing cell-penetrating peptides (cell-penetrating peptide,
CPP), facilitate albumen entrance to play a role into the cell；It is to extend the Half-life in vivo of IL-37 albumen simultaneously with IgG Fc
Be conducive to protein purification；The Caspase with WEHD amino acid sequence is devised also between IL-37-CPP and IgGFc simultaneously
Restriction enzyme site.IL-37-CPP-IgG Fc albumen can enter intracellular from the extracellular effect transduction by CPP in this way.Into thin
After born of the same parents, so that IL-37 is detached from IgG under Caspase effect and IL-37 can be delivered in nucleus, play functional transcription factor.
That is IL-37-CPP-IgG Fc albumen of the invention not only transmissibility cell-surface signal but also transmissibility be intracellular and core because
Subsignal.
The people IL-37 albumen of improvement of the invention can polarization with from inducing macrophage to M2, while can be obviously promoted
The activation of AMPK in macrophage.The people IL-37 albumen of the improvement of the application has medication compared with commercially available rIL-37 albumen
Dosage is small, Half-life in vivo is long, Small side effects, the advantages of curative effect is better.IL-37-CPP-IgG Fc albumen of the invention is not only
Hepatic fibrosis of schistosomiasis can be effectively prevented, can also be Future Development IL-37-CPP- by the comparison of different dosage forms
Various diseases caused by IgG fusion protein is activated as original treatment because of excessive immune, lay the basis of mechanism study.
Currently, various diseases caused by treating because of excessive immune activation, generally by the various inflammatory cytokines of injection
Neutralizing antibody (such as Xiu Meile antibody of anti-TNF alpha) or cytokine receptor antagonist (such as TNFR-Fc fusion protein
Benefit match is general).But the internal dosage of the antibody of blocking property or receptor antagonist fusion protein generally require it is very big, to patient and
Medical system poses a big pressure.Compared to the IL-1 family member of other proinflammatory diseases, IL-37 is unique in the current family
Has the cell factor of immunosuppressive activity.Theoretically, the cell factor of a small amount of inhibition need to only be provided, so that it may reverse inflammation
Reaction, not only more physiological regulating control, and also the production cost of medical protein and toxic side effect can all greatly reduce.It can be pre-
Meter can be worked in outside by IL-37 receptor simultaneously, and be can enter and worked after nucleus by regulatory transcription
IL-37-CPP-IgG fusion protein, in preparation treatment immunological diseases, (especially rheumatoid arthritis, rigid spine etc. are self
Immunological disease), virus disease (such as virus hepatitis etc.) or parasitic infection (hepatic disease caused by infection by Schistosoma etc.)
Drug in, application prospect will be had more than conventional IL-37-Ig fusion protein (i.e. IL-37-no CPP-IgG).
Detailed description of the invention
Fig. 1 is the improvement people's IL-37 egg identified in expressing cho cell supernatant with Western Blot protein blot method
It is white.A, B, E are IL-37-no CPP-mIgG2b；C, D, F IL-37-CPP-mIgG2b.In all subsequent embodiments, all marks
That no CPP-IL-37 is IL-37-no CPP-mIgG2b, and that indicate CPP-IL-37 is IL-37-CPP-mIgG2b.
Fig. 2 is that IL-37 content in Acute Schistosomiasis patients serum is significantly raised in the embodiment of the present invention；Wherein,
AA: blood fluke acute infection patients serum；CN: non-Endemic Area Healthy Human Serum；*, p < 0.05.
Fig. 3 is that schistosoma japonicum infection mouse liver general appearance is obviously improved after IL-37 processing in the embodiment of the present invention；
Wherein, A, Normal group；B infects control group；C, rIL-37 processing group；D, no CPP-IL-37 processing group；E, CPP-IL-
37 processing groups.
Fig. 4 is that schistosoma japonicum infection mouse liver egg granulomas synergentic area is bright after IL-37 processing in the embodiment of the present invention
It is aobvious to reduce；Wherein, A, Normal group；B infects control group；C, rIL-37 processing group；D, no CPP-IL-37 processing group；E,
CPP-IL-37 processing group；Mouse liver HE coloring pathological section, n=5.F, each group mouse liver egg granulomas synergentic area/liver
Gross area statistical chart.
Fig. 5 is that the serum aminotransferase levels at commencement of schistosoma japonicum infection mouse after IL-37 processing in the embodiment of the present invention is obvious
It reduces；Wherein, * p < 0.05, each group is compared with infected group；* p < 0.05, no CPP-IL-37 group and CPP-IL-37 group with
RIL-37 group is compared；N=5.
Fig. 6 be the embodiment of the present invention in IL-37 processing after to schistosome-infected mice hepatic tissue macrophage cytokines mRNA
Expression influence；Wherein, the gene expression dose of the group of cells factor is compared with infected group, * p < 0.01, n=5.
Fig. 7 is the ratio of flow cytometer showed each group mouse liver M1 and M2 type macrophage in the embodiment of the present invention；Wherein, A,
Flow cytometer detection mouse liver Granuloma cells iNOS and CD206 result represents figure；B, flow cytometer showed M2 ration statistics figure.*, with NC
Than p < 0.01；*, with IC ratio, p < 0.01；* *, with rIL-37 ratio, p < 0.01.
Fig. 8 is flow cytometer showed each group mouse peritoneal M1 and M2 type macrophage ratio in the embodiment of the present invention；Wherein, A, stream
Formula detects Turnover of Mouse Peritoneal Macrophages iNOS and CD206 result and represents figure；B, flow cytometer showed M2 ration statistics figure.*, with NC ratio, p
< 0.01；*, with IC ratio, p < 0.01；* *, with rIL-37 ratio, p < 0.01.
Fig. 9 is to detect IL-37 albumen experiment in vitro inducing mouse peritoneal macrophage using FACS in the embodiment of the present invention
To the polarized ratio of M2；Wherein, cell used be Turnover of Mouse Peritoneal Macrophages, cell use respectively rIL-37, no CPP-IL-37,
CPP-IL-37+SIS3 or CPP-IL-37 albumen co-cultures 12h, and cell is marked with F4/80 and CD206.Different batches experiment weight
Again three times.*, with PBS ratio, p < 0.01；*, with other processing group ratios, p < 0.01.
Figure 10 is the activation that IL-37 promotes AMPK in macrophage in the embodiment of the present invention；Wherein, cell used is mouse
Peritoneal macrophage is co-cultured with rIL-37, no CPP-IL-37, CPP-IL-37+SIS3 or CPP-IL-37 albumen respectively
12h.Cell is collected, its total protein is extracted, carries out Western blot.Anti-pAMPK antibody and anti-AMPK concentration are 1
: 1000, anti-β-actin concentration is that the goat anti-rabbit igg concentration of 1: 200, HRP- label is 1: 20,000.Different batches experiment weight
Again three times.*, with PBS ratio, p < 0.01；*, with other processing group ratios, p < 0.05.
Specific embodiment
Technical solution of the present invention is described in detail below with reference to embodiment.The reagent and biomaterial used below
It if not otherwise specified, is commercially produced product.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer suggests
Condition carry out.Do not make the experimental methods of molecular biology illustrated in embodiment, referring to " Molecular Cloning:A Laboratory guide "
Listed specific method carries out in one book of (third edition) J. Pehanorm Brooker, or carries out according to kit and product description.
Expression of the 1 IL-37-CPP-mIgG2b and IL-37-no CPP-mIgG2b albumen of embodiment in Chinese hamster ovary celI and
Purifying
We have been synthesized by Chinese hamster ovary celI codon optimization comprising people IL-37's according to the sequence NM_014439 of GenBank
The DNA fragmentation of coding CPP-mIgG2b Fc or coding mIgG2b Fc under code area (CDS) and same reading frame.The former is
IL-37-CPP-mIgG2b, amino acid sequence is as shown in SEQ ID NO.1, wherein signal peptide sequence is
MWWRLWWLLLLLLLLWPMVWAS, DNA sequence dna is as shown in SEQ ID NO.2.The latter, that is, IL-37-no CPP-mIgG2b,
Amino acid sequence is as shown in SEQ ID NO.3, wherein signal peptide sequence MWWRLWWLLLLLLLLWPMVWAS, DNA sequence dna is such as
Shown in SEQ ID NO.4.
Above-mentioned cell-penetrating peptides (cell penetrating peptide, CPP) RQIKIWFQNRRMKWK is derived from black abdomen fruit
Antennapedia homeodomain albumen (Turner, J.et al.Nucleic the Acids Res 33,27 of fly
(2005).).Its left and right sides is connected with GG with GGS respectively, to reduce steric hindrance.WEHD is that Caspase-1 digestion identifies position
Point.DEVD is Caspase-3 and Caspase-7 digestion recognition site.RGP is proteolytic cleavage recognition site in cytoplasm.IL-
After IL-37 membrane receptor and the excitation signal conduction on 37-CPP-mIgG2b combination cell surface, acid is entered by receptoe mediated endocytosis
Property lysosome (endosome).Under a variety of Caspase albumen enzyme effects in lysosome, the Fc of IL-37-CPP and mIgG2b
It is partially separated.For IL-37 under the action of CPP cell-penetrating peptides, cross-film enters cytoplasm, then again in CPP cell-penetrating peptides
Under the action of, enter nucleus across nuclear membrane, plays the role of adjusting transcription.It a small amount of is not cut by Caspase digestion even if having
But still enter the IL-37-CPP-mIgG2b of cytoplasm by CPP cross-film, it can also be known by proteolytic cleavage in RGP cytoplasm
It is not dissociated with the part mIgG2b Fc.Multiple design may insure that IL-37-CPP-mIgG2b can exist as cell factor in this way
It is extracellular to work, it can also work in core as transcription factor.IL-37-no CPP-mIgG2b then because can not cross-film, only
It can work as cell factor extracellular.The design of this novel improvement IL-37 has no document report.
The method that we then use PCR, IL-37-CPP-mIgG2b the and IL-37-no CPP-mIgG2b gene of synthesis
Both ends all add NheI and XhoI restriction enzyme site respectively, are connected into pDirect4.0 expression vector (Antagen company).Use Neon
Electroporation electroporation (LifeTech company) converts CHO-K1 cell strain (CCL-62, ATCC).Electricity turns condition
1620v, 10ms, 3 subpulse.DNA dosage is 8 μ g, and Chinese hamster ovary celI dosage is 2,000,000.After electricity turns, Chinese hamster ovary celI is containing 5%FBS
DMEM culture medium in cultivate, and add 300 μ g/mL Zeocin (InVivogen company) screening.After two weeks, drug resistance gram
Grand formation, cell use pancreatin to digest after as mixing library, 250mL shaking flask is transferred to, with HyCell serum free medium culture 3 weeks.
After harvesting supernatant, the filter of 1.1 μm of filtration is to remove cell fragment.Supernatant is added to Protein A affinity column
In (GenScript company).In conjunction with fusion protein washed with the PBS of 10 times of column volumes after, with the 0.1M's of pH2.5
Glycine-HCl elution, and neutralized with the Tris-HCl buffer of the 1M of pH8.0 rapidly.0.22 μm of filter sterility of albumen
After filtering, with OD280 spectrophotometric determination concentration, for testing in next step.
As the identification of albumen, we are thin the CHO of expression IL-37-CPP-mIgG2b and IL-37-no CPP-mIgG2b
Born of the same parents' supernatant walks SDS-PAGE electrophoresis, and Western Blot western blot is examined with the sheep anti-mouse antibody of 1: 4000 diluted HRP label
It surveys.Fig. 1 shows that the molecular weight of the albumen with CPP shows that albumen constructs than without the high of CPP, molecular weight all meets expection
With express successfully.
Further, when the people IL-37 of the improvement of the present embodiment is used for human body, the mIgG2b of all source of mouse is changed to source of people
hIgG2。
We have been synthesized by Chinese hamster ovary celI codon optimization comprising people IL-37's according to the sequence NM_014439 of GenBank
The DNA fragmentation of coding CPP-hIgG2 Fc or coding hIgG2 Fc under code area (CDS) and same reading frame.The former is
IL-37-CPP-hIgG2, amino acid sequence is as shown in SEQ ID NO.5, wherein signal peptide sequence is
MWWRLWWLLLLLLLLWPMVWAS, DNA sequence dna is as shown in SEQ ID NO.6.The latter, that is, IL-37-no CPP-hIgG2, ammonia
Base acid sequence is as shown in SEQ ID NO.7, wherein signal peptide sequence MWWRLWWLLLLLLLLWPMVWAS, DNA sequence dna is such as
Shown in SEQ ID NO.8.
2 IL-37-CPP-mIgG2b albumen of embodiment adjusts the test that macrophage inhibits inflammatory reaction
1, materials and methods
The preparation of 1.1 schistosoma japonicum infection animal models and IL-37 albumen are given
C57BL/6 mouse is randomly divided into five groups, every group of 5 mouse.Wherein four groups of mouse routinely preparation Schistosoma japonicum sense
Model is contaminated, and is randomly divided into infection control group, CPP-IL-37 (the people IL-37 of improvement of the invention) administration group, no respectively
CPP-IL-37 (the people IL-37 relative to improvement of the invention is free of CPP) administration group and rIL-37 (commercially available) administration group.According to
Grouping starts the CPP-IL-37 albumen (5ng/kg/ times) that tail vein injection is sterile respectively, no for situation the 24th day after infection
CPP-IL-37 albumen (10ng/kg/ times) or rIL-37 albumen (20ng/kg/ times, R&D company), it is primary every injection in 3 days, directly
The 6th week after to infection；Control group mice the 24th day sterile PBS of beginning tail vein injection (100 μ l) after infection is infected, every 3d
Injection is primary, until the 6th week after infection.It cuts open and kills mouse, leave and take serum and liver, collect adult and count, it is spare.The experiment weight
It is multiple primary.
1.2 mice serum Liver function grades
Each group mice serum a part is for detecting each group mouse liver injury index: glutamic-pyruvic transaminase (ALT) and millet straw turn
Adnosine deaminase (AST), detection are carried out according to the concrete operation step of kit operational manual.
1.3 egg count
Each group mouse liver lobus sinister hepatic tissue (weight is in 0.1g or so) is left and taken, is disappeared in 37 DEG C of incubators with 5% KOH
After changing 3h, each sample takes 0.1ml × 5 time smear on glass slide, then counts schistosoma japonice ovum under the microscope, counts
Analyze every gram of hepatic tissue worm's ovum number of every group of every mouse.
1.4 HE dyeing
Take each group mouse part hepatic tissue respectively, materials whens is taken as far as possible with a piece of lobuli hepatis.It is put into 10% formal
It is fixed in woods more than for 24 hours.Conventional dehydration, transparent, saturating wax, embedding, slice and HE dyeing.Each group model liver damage of microscopically observation
Evil situation, the number and type of granuloma area, inflammatory cell infiltration.
Separation, stimulation and the culture of 1.5 liver egg granulomas synergentic cells
The isolated Granuloma cells of conventional method, with 1 × 106/ ml is inoculated in 24 orifice plates, and the SEA that 1ml is added (is used
RPMI-1640 culture medium dilutes SEA to 50 μ g/ml), is put into 37 DEG C of 5%CO2It cultivates in incubator, adds when culture is to 4h
Enter the BFA of the PMA of 25ng/ml, the ionomycin of 1 μ g/ml, 10ug/ml.Group of cells is collected after 48h after stimulation culture, it is standby
With.
The separation and culture of 1.6 Turnover of Mouse Peritoneal Macrophages
It, will be isolated small in order to further look at IL-37 to the polarized influence of Turnover of Mouse Peritoneal Macrophages and mechanism
After mouse peritoneal macrophage (M0) is seeded to 24 orifice plates, grouping is once tested.1)M0+PBS；2)M0+rIL-37；3)M0+
CPP-IL-37；4)M0+CPP-IL-37+SIS3；5)M0+no CPP-IL-37.Wherein rIL-37 concentration is 100ng/ml, CPP-
IL-37 concentration is 10ng/ml, and SIS3 concentration is 2 μM, and no CPP-IL-37 concentration is 20ng/ml, and action time is 12h.It receives
Cell after collection Fiber differentiation, a part are used for Flow cytometry M1 type and M2 type number of macrophages, and a part is for WB inspection
Survey the expression of p-MAPK and AMPK.
The gene expression dose of 1.7 real-time fluorescence quantitative PCRs detection correlation factor
The liver or cell left and taken in above-mentioned experiment, conventional to carry out RNA extraction, reverse transcription is at cDNA.It is interior with β-actin
Ginseng utilizes the gene expression dose of real-time fluorescence quantitative PCR detection iNOS, Fizzl, IL-17 and TGF-β.
1.8 Western blot detect the variation of p-AMPK in macrophage
Group of cells is collected, with suitable RIPA buffer lytic cell to discharge albumen, conventional line SDS-PAGE and egg
White transfer.NC film after transfer closes 3h with 5% skimmed milk power 100ml at room temperature；It is separately added into the diluted β-of confining liquid
Actin (1: 200), anti-pAMPK (1: 1000) or anti-AMPK (1: 1000), 4 DEG C of incubation 16h；PBST washing, 15min/
It is secondary × 4 times；The goat anti-rabbit igg (1: 20,000) of HRP- label, 37 DEG C of incubation 1h；PBST is washed again, and 15min/ times × 4
It is secondary；Uniform fold is changed on chemiluminescence imaging instrument on NC film after being mixed in equal volume with A liquid in ECL luminescent solution and B liquid
The colour developing that shines is learned, develop the color result row gray scale scanning.
1.9 Flow cytometries and analysis
1.9.1 the surface antigen dyeing of cell
Collect group of cells, 1 × PBS washing, adjustment cell density to every pipe 1 × 106A cell；Routine rat serum envelope
It closes；FITC-CD4, APC-CD25, FITC-CD11b, PE-iNOS2, PerCP/Cy5.5-F4/ are separately added into according to experimental group
80, APC-CD206, it is protected from light, 4 DEG C, 30min；Washing: be added 1 × PBS 1mL, 4 DEG C, 3500rpm, be centrifuged 5min, × 2 times.
1.9.2 factor dyeing intracellular
Fixed, permeabilized cells: 200 μ L Fix/Perm buffer of addition, resuspension cell precipitation, 4 DEG C, 60min；It is penetrating to wash
Wash: be added 1ml Perm/Wash buffer, 7500rpm, 4 DEG C, be centrifuged 2min, × 2 times；Intracellular antibody label: titration is added
The antibody of good concentration, FITC-IFN- γ, APC-IL-4, PE-IL-17, PE-Foxp3 are protected from light, and 4 DEG C, 40min；Washing: it is added
1mL Perm/Wash buffer, 7500rpm, 4 DEG C, be centrifuged 2min, × 2 times；It is fixed: 3% paraformaldehyde fixer to be added, often
Pipe 200 μ L, it is to be checked.
1.9.3 flow cytometer detection and data analysis
FACSVerse instrument on sample to be tested is detected, analyzes the data obtained with 7.6 software of FlowJo.
1.10 ELISA detect IL-37 concentration in serum
According to Human IL-37/IL-IF7 ELISA Kit specification, Japanese blood is detected using double crush syndrome and is inhaled
The concentration of IL-37 in worm acute infection patient and the Endemic Area non-Healthy Human Serum.With anti-human IL-37 monoclonal antibody pre-coated elisa plate,
The diluted different human serums of appropriateness are added in every hole, and Interleukin-37 therein washes away free ingredient in conjunction with its monoclonal antibody；
Be added that biotinylated anti-human IL-37 is mostly anti-, anti-human IL-37 is mostly anti-in conjunction in conjunction with IL-37 onboard, wash away it is free at
Point；The avidin of HRP label is added, avidin and biotin are specifically bound, and wash away unbonded ingredient；Developing solution is added,
If someone IL-37 in reacting hole, horseradish peroxidase makes the aobvious blue of colourless color developing agent；Liquid in terminate liquid metapore is added to become
It is yellow.OD450 value is surveyed using full-automatic microplate reader, standard curve is drawn according to the OD450 value of standard items, calculates IL-37 in sample
Concentration.
1.11 statistical method
Data carry out statistical analysis using spss11.0, and measurement data is indicated with mean ± SD, between two groups of measurement data
Directly relatively, it when meeting normal distribution and homogeneity of variance, is examined using t.It is inspection level with α=0.05.
2, test result
2.1 IL-37 content in Acute Schistosomiasis patients serum is significantly raised
Collect 14 parts of Schistosoma japonicum acute infection patients serums and 14 parts of non-Endemic Area Healthy Human Serums, double antibodies sandwich
ELISA detects the expression of IL-37 in serum.As a result, it has been found that healthy control group low expression IL-37, and acute Schistosoma japonicum
The expression of IL-37 is significantly raised (p < 0.05, Fig. 2) in infected patient serum.
2.2 experiment in vivo prove that IL-37 is able to suppress the reaction of mouse liver japonice ovum granulomatous inflammation
2.2.1 IL-37 processing does not influence imago of blood fluke worm lotus, worm's ovum and liver weight
Because the difference of adult worm lotus and egg counts influences whether that the liver egg granulomas synergentic of schistosome-infected mice is scorching
The severity of disease reaction, in order to exclude influence of the infectiosity difference to experimental result, we to each group mouse at borer population and
Worm's ovum number is counted and does statistical analysis.As shown in table 1, to infecting mouse after the IL-37 albumen processing of different dosage forms
Worm lotus and ovum lotus statistically have not significant impact (p > 0.05)；Although display uses liver after the processing of IL-37 albumen in data
Weight is mitigated, but the liver weight of each group mouse does not have difference (p > 0.05).
1 each group mouse worm lotus of table, ovum lotus and liver weight (n=5)
2.2.2 the substantially reduced liver egg granulomas synergentic inflammation and hepatic injury of mouse after IL-37 processing
As shown in figure 3, liver gross specimen picture shows that IL-37 processing group mouse liver general appearance is obviously improved, liver
Dirty surface ova nodule is reduced；Especially most obvious with CPP-IL-37 albumen processing group liver appearance investigation, no CPP-IL-37 albumen
Mitigated with rIL-37 albumen processing group inflammatory reaction than model group, difference is unobvious between two groups.
The inflammatory infiltrated in the IL-37 albumen processing group mouse liver egg granulomas synergentic of HE coloration result display different dosage forms
Cell significantly reduces, and necrosis of liver cells is substantially reduced (Fig. 4).Compared with infecting control group, IL-37 processing group mouse liver is found
(p < 0.05) is obviously reduced in egg granulomas synergentic area；Especially reduced with CPP-IL-37 albumen processing group granuloma area the brightest
Aobvious, no CPP-IL-37 albumen and rIL-37 albumen processing group granuloma area ratio model group are also reduced, three groups of liver worms
Ovum granuloma area discrepancy does not have conspicuousness (p > 0.05) after statistical analysis.
Two kinds of enzyme values of AST and ALT in each group mice serum are detected, find the IL-37 albumen processing group mouse of different dosage forms
Transaminase AST and ALT level are significantly lower than infection control group (p < 0.05) in serum；Wherein CPP-IL-37 albumen and no
The mitigation of CPP-IL-37 albumen processing group hepatic disorder is the most obvious, and difference is not statistically significant (p > 0.05) between two groups；
RIL-37 albumen processing group hepatic disorder is also substantially reduced compared with infecting control group, but not as good as no CPP-IL-37 albumen and
Good (the p < 0.05, Fig. 5) of CPP-IL-37 albumen processing group liver function recovery.
2.3 experiment in vivo prove that IL-37 can induce schistosome-infected mice liver and peritoneal macrophage to polarize to M2
After the IL-37 albumen processing group of different dosage forms compared with infecting control group, M1 type macrophage is thin in mouse liver tissue
The mRNA level in-site of born of the same parents' cell factor iNOS is obviously lowered, and the mRNA level in-site of M2 type Macrophage Cell factor Fizzl is higher than sense
It contaminates control group (p < 0.05, n=5, Fig. 6).
Facs analysis shows after the IL-37 albumen processing of different dosage forms the M2 type in Liver granuloma compared with infecting control group
Cell proportion is significantly more than infected group (p < 0.05)；Further analysis finds, CPP-IL-37 processing group M2 type number of macrophages
It increased significantly (p < 0.05, table 2).
Influence after 2 IL-37 of table processing to schistosoma japonicum infection mouse liver granuloma M2 type macrophage
Note: * p < 0.05, each group is compared with infected group；* p < 0.05, CPP-IL-37 processing group and other treatment group phase
Than；N=5.
Because M2 type number of macrophages increased significantly after IL-37 induction, therefore further pass through flow cytometer showed each group Mouse Liver
The ratio of M1 type and M2 type macrophage in dirty egg granulomas synergentic cell.It is found after being statistically analyzed, after infection by Schistosoma 6 weeks
The M2 type macrophage ratio that CD206 is expressed in mouse liver significantly improves compared with normally group mouse；When infected mice is through rIL-
M2 ratio further increases (p < 0.01) compared with infected group after the processing of 37 or no CPP-IL-37 albumen；And through CPP-IL-37
M2 ratio is raised at most after albumen processing, accounts for about 6.73% (p < 0.01) of macrophage sum.But infected mice is through difference
After the IL-37 albumen processing of dosage form, the ratio of M1 type macrophage is without significant change (p > 0.05, figure in Liver granuloma cell
7).As a result disclosing IL-37 can promote the macrophage in Liver granuloma cell to polarize to M2, but not influence M1 type macrophage
Ratio.
The peritoneal macrophage for further separating each group mouse, utilizes itself M1 and M2 type macrophage of flow cytometry
Ratio.It is found after being statistically analyzed, expresses the M2 type macrophage ratio of CD206 after infection by Schistosoma 6 weeks in mouse peritoneal
It is significantly improved compared with normally group mouse, up to 24.9%；When infected mice is after the processing of rIL-37 or no CPP-IL-37 albumen
M2 ratio is even more further to increase compared with infected group, and ratio is 34.6% and 36.9% (p < 0.01) respectively；Through CPP-
Mouse peritoneal M2 macrophage ratio has reached 56.6% after the processing of IL-37 albumen, it is raised at most (p < 0.01).But
Infected mice is after the processing of the IL-37 albumen of different dosage forms, and the ratio of M1 type macrophage is without significant change in peritoneal macrophage
(p > 0.05, Fig. 8).As a result it confirms that IL-37 can promote peritoneal macrophage to polarize to M2, but does not influence M1 type macrophage
Ratio.
2.4 experiment in vitro IL-37 are by promoting AMPK phosphorylation inducing macrophage to polarize to M2
After Turnover of Mouse Peritoneal Macrophages (M0) is seeded to 24 orifice plates, respectively with rIL-37,10ng/ml of 100ng/ml
The SIS3 and M0 of+2 μM of the CPP-IL-37 albumen of CPP-IL-37 albumen, the no CPP-IL-37 albumen of 20ng/ml or 10ng/ml
Co-culture 12h.Because CPP-IL-37 albumen has CPP, facilitates IL-37 albumen and enter in cell and nucleus to play a role, be
Effect of the observation CPP-IL-37 albumen as nuclear factor, in this experiment the inhibitor SIS3 of addition Smad3.
2.4.1 prove that IL-37 can induce peritoneal macrophage to polarize to M2 by flow cytometry
Cell after collection Fiber differentiation, Flow cytometry M2 type macrophage ratio.The results show that with rIL-37,
After no CPP-IL-37, CPP-IL-37+SIS3 or the protein induced macrophage 12h of CPP-IL-37, cell that M0 polarization is M2
Ratio increased significantly (p < 0.01) compared with the control group；Wherein rIL-37, no CPP-IL-37 and CPP-IL-37+SIS3 tri-
Group is compared, and the ratio for M2 is induced not have statistical difference (p > 0.05)；The induction of CPP-IL-37 albumen processing group is that M2 type is huge
The ratio of phagocyte is maximum, and up to 6.73%, difference all has conspicuousness (p < 0.01, Fig. 9) compared with other three groups.The reality
It tests result and illustrates that IL-37 can polarize by cell surface receptor signal path inducing macrophage to M2, while being also possible to lead to
Nuclear factor approach inducing macrophage is crossed to polarize to M2
2.4.2 Western blot has found IL-37 by promoting the induced expression M2 of macrophage pAMPK to polarize
Cell after collection Fiber differentiation, the expression of immune-blotting method group of cells pAMPK (1: 2000) and AMPK
(Figure 10).As the result is shown: after rIL-37, no CPP-IL-37 and the protein induced macrophage 12h of CPP-IL-37, pAMPK egg
White expression quantity significantly improves (p < 0.01) compared with PBS control group, the expression quantity and rIL-37 of pAMPK after SIS3 inhibits
It is substantially similar (p > 0.05) with no CPP-IL-37 albumen processing group, the expression of CPP-IL-37 albumen processing group pAMPK albumen
It measures higher compared with other processing groups (p < 0.05).Illustrate that IL-37 can promote the phosphorylation of AMPK in macrophage, then
Inducing macrophage polarizes to M2；Illustrate that CPP-IL-37 albumen can be by cell surface receptor signal path and consideration convey simultaneously
Record the activation of AMPK in Factor Pathway inducing macrophage.
The invention shows the people IL-37 of the improvement with CPP is under kinds of experiments system, all than the people IL- without CPP
37 is more active.This illustrates that our design is successful.Such design can be used not only for described in embodiment to blood
The treatment of fluke disease, and the cell factor based on IL-37 as inhibitive ability of immunity unique in unique IL-1 superfamily,
Its indication applied (such as in treatment immunological diseases, especially rheumatoid arthritis, the autoimmune diseases such as rigid spine,
In the application of the virus diseases such as virus hepatitis or parasitic infection and tumour etc.), can through the invention in explain
The method stated, and CPP sequence, while the extracellular and intracellular two different mechanism of action of IL-37 are assigned, reach better treatment
Effect.
In addition it is emphasized that the present invention is because make model with mouse, to avoid repulsion of the mouse to non-self albumen, I
Used mouse mIgG2b sequence.This does not indicate that the present invention is only applicable to the mIgG2b sequence of mouse.The common skill of this field
Art personnel preferably use the sequence of complete people it should be understood that for the therapeutic antibodies for human body or the fusion protein with Fc segment
Column.Therefore the present invention is suitable for all improvement people IL-37 for using human IgG as fusion ingredient.
It should be noted last that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting.Although ginseng
It is described the invention in detail according to embodiment, it will be apparent to an ordinarily skilled person in the art that technical side of the invention
Case is modified or replaced equivalently, and without departure from the spirit and scope of technical solution of the present invention, should all be covered in the present invention
Scope of the claims in.
Claims (13)
1. a kind of people's IL-37 albumen of improvement, which is characterized in that people's IL-37 albumen of the improvement be by people IL-37 albumen according to
It is secondary to be connect with cell-penetrating peptides CPP and the Fc of IgG antibody section, form IL-37-CPP-IgG Fc albumen.
2. the people's IL-37 albumen improved according to claim 1, which is characterized in that people's IL-37 albumen of the improvement it is thin
Born of the same parents' penetrating peptide CPP is to pass through the small peptide that cell membrane enters endochylema with guidance foreign protein.
3. the people's IL-37 albumen improved according to claim 1, which is characterized in that people's IL-37 albumen of the improvement exists
The WEHD amino acid sequence that Caspase restriction enzyme site is identified is added between CPP and IgG Fc.
4. the people's IL-37 albumen improved according to claim 1, which is characterized in that people's IL-37 albumen of the improvement
IgG is IgG1, IgG2a, IgG2b, IgG2c or IgG3 of mouse, alternatively, being IgG1, IgG2, IgG3 or IgG4 of people.
5. the people's IL-37 albumen improved according to claim 1, which is characterized in that people's IL-37 albumen of the improvement
IgG is wild type, or after amino acid mutation, has further changed ADCC cell killing vigor and CDC complement swashs
While still alive the mutant of power.
6. the people's IL-37 albumen improved according to claim 1, which is characterized in that people's IL-37 albumen of the improvement is sugar
The albumen of base, the glycosylation albumen is wild type, or excellent by the glycosyl that the engineering cell strain that glycosyl was transformed produces
Change type.
7. people's IL-37 albumen of -6 any improvement according to claim 1, which is characterized in that people's IL-37 egg of the improvement
It is white that there is the amino acid sequence as shown in SEQ ID NO.1；Or there are not more than three between IL-37, CPP, IgG structural domain
Non-hydrophobic non-band charge amino acid residues replace, miss or add, or with different IgG hypotypes, and have and SEQ ID
The same or similar active protein as derived from SEQ ID NO.1 of NO.1.
8. a kind of encoding gene of people's IL-37 albumen of improvement, which is characterized in that the encoding gene has following nucleotides sequence
One of column:
1) there is the nucleotide sequence for encoding albumen as claimed in claim 1 to 7；
2) encoding gene has the nucleotide sequence as described in SEQ ID NO.2, or has with SEQ ID NO.2 at least
The nucleotide sequence of 60% homology.
9. the expression vector containing encoding gene described in claim 8.
10. expression vector according to claim 9, which is characterized in that the expression vector is pDirect4.0.
11. a kind of expression cell system of people's IL-37 albumen of improvement, which is characterized in that the expression cell system, which contains, to have the right to want
Seek 8 encoding genes.
12. expression cell system according to claim 11, which is characterized in that the expression cell system is CHO cell line.
13. encoding gene or claim 11 described in the people IL-37 albumen of any improvement of claim 1-7, claim 8
Or 12 the expression cell tie up to answering in the drug of preparation treatment immunological diseases, virus disease, parasitic infection or tumour
With.
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