CN105085656B - The application of P19/Ebi3 compounds and its polyclonal antibody in systemic loupus erythematosus diagnosis and treatment - Google Patents
The application of P19/Ebi3 compounds and its polyclonal antibody in systemic loupus erythematosus diagnosis and treatment Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39516—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum from serum, plasma
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
Abstract
The invention discloses the purposes of P19/Ebi3 compounds and its polyclonal antibody in systemic lupus erythematosus diagnosis, treatment.Experiment proves, compared with normal mouse, P19/Ebi3 compound high level expressions in systemic loupus erythematosus mouse, and the expression of mouse internal antibody and the number of immunocyte can be reduced using the polyclonal antibody of anti-P19/Ebi3 albumen, showing can be by preparing the antibody of anti-P19/Ebi3 come systemic lupus erythematosus, therefore P19/Ebi3 compounds and its inhibitor are used to prepare diagnosis and treatment system lupus erythematosus medicine, have good development prospect.
Description
Technical field
The present invention relates to biological technical field, more particularly it relates to P19/Ebi3 compounds and its Anti-TNF-α
Purposes of the body in systemic lupus erythematosus diagnosis, treatment.
Background technology
Systemic loupus erythematosus (Systemic Lupus Erythematosus, SLE) is that a kind of serious threat mankind are good for
The autoimmune disease of health and life, there is higher lethality and disability rate.Its cause and onset of disease mechanism remains unknown, generally
It is considered that environmental factor acts on body (including histocompatbility, cell factor, cytokine receptor etc. of certain genetic background
The different types of expression) and the result of formation.Its immunopathogenesis is characterized in producing a variety of heights in vivo, pathogenic itself is anti-
Body, while Lymphocyte Apoptosis.Discharge a large amount of nucleus contents and ribose corpusculum and become autoantigen；Antigen-antibody knot
Conjunction forms immune complex, and the deposition of immune complex can cause target cell damage and inflammation, cause each tissue and organ hair
Sick change.In the pathologic process, B cell is in advanced activation state, and the process is closely related with T cell, have T cell according to
Lai Xing.
Complicated cytokine network regulatory T-cell and growth, differentiation and the function of B cell, these cell factors can be with
It is divided into two major classes：Th1 types (main inducing cellular immune) and Th2 types (mainly promoting antibody generation).Interleukin 12 (IL-
12) play an important role in cytokine network adjusting, it is mainly secreted by mononuclear macrophage produces.IL-12 families
By a α-chain (P19, P28 or P35) and beta chain (P40 or Ebi3) heterodimer subunit composition (Fig. 1), itself and some from
The morbidity of the cell-mediated autoimmunity disease of body immunological diseases, especially Th1 is related.
The clinical diagnosis for systemic loupus erythematosus relies primarily on clinical manifestation, laboratory examination, histopathology at present
And imageological examination.Since SLE clinical manifestation illnesss vary with each individual, it sometimes appear that mistaken diagnosis.And treatment system erythema wolf
The conventional method of sore is to use Anti-Malarial, anti-inflammatory agent and immunosuppressive drug, when symptom is difficult to control, to non-steroidal anti-inflammatory agent
Supplement is with cortin；Although in symptom management, preventing have remarkable effect in disease development, there is obvious adverse reaction more, and
To some patientss unsatisfactory curative effect.B cell and T cell play an important role in the pathogenesis of SLE, for immunocyte
Biological therapy is by as the focus for the treatment of SLE.
The content of the invention
In order to make up for the deficiencies of the prior art, examined it is an object of the invention to provide one kind available for systemic loupus erythematosus
The compound P19/Ebi3 and its polyclonal antibody controlled.Compared to the diagnostic and therapeutic method of traditional systemic loupus erythematosus, make
The specificity for being interacted or being corrected it with polyclonal antibody and the pathologic immune response for causing auto-antibody excessively to produce
Treatment advantageously will such as have higher specificity and accuracy, relatively low side effect, so as to fulfill systemic erythema is cured
Lupus and/or in long-term basis improve patient quality of the life causal treatment.
To achieve these goals, the present invention adopts the following technical scheme that：
The present invention provides a kind of separated P19/Ebi3 compounds.
Further, compound mentioned above is made of two subunits of P19 and Ebi3.
Further, P19 subunits mentioned above include such as SEQ ID NO:The albumen of amino acid sequence shown in 1, or
Its conservative variation's albumen.
Further, Ebi3 subunits mentioned above include such as SEQ ID NO:The albumen of amino acid sequence shown in 2, or
Its conservative variation's albumen.
Further, subunit recited above is by polynucleotide encoding, and the nucleotide sequence of the P19 subunits is such as
SEQ ID NO:Shown in 3；The nucleotide sequence of the Ebi3 subunits such as SEQ ID NO:Shown in 4.
The present invention provides a kind of recombinant vector, it contains polynucleotide sequence recited above.
Further, also containing the expression regulation being operatively connected with the polynucleotide sequence in the recombinant vector
Sequence.
The carrier of the present invention for carrying gene is various carriers known in the art, such as commercially available carrier including plasmid,
Clay, bacteriophage, virus etc..
Viral vector can be any appropriate carrier, include but not limited to retroviral vector, adenovirus vector, gland
Viral related viral vectors, herpesviral (such as herpes simplex virus, vaccinia virus and Epstein-Barr virus) carrier, alphavirus vectors.
Carrier for expression of eukaryon can be any appropriate expression vector, include but not limited to pCMV-Myc expression vectors,
PcDNA3.0 expression vectors, pcDNA3.1 expression vectors, pEGFP expression vectors, pEFBos expression vectors, pTet expression vectors,
PTRE expression vectors, pReceiver-Lv18 expression vectors, pZE-LV122X expression vectors or the base in known expression vector
Engineered carrier on plinth, such as pBin438, pCAMBIA1301 etc..
Preferably, the expression vector is carrier for expression of eukaryon.Preferably, the carrier for expression of eukaryon is pReceiver-
Lv18 and pEZ-LV122X expression vectors.
The present invention provides a kind of host cell, it contains the recombinant vector, or is integrated with its genome described
Polynucleotides.
Preferably, the host cell is Chinese hamster ovary celI.
The present invention provides a kind of preparation method of P19/Ebi3 compounds, it comprises the following steps:
(a) the culture host cell；
(b) compound is isolated from culture.
The present invention provides a kind of antibody, the antibody specificity identifies and/or with reference to the compound.
Preferably, the antibody is polyclonal antibody.
The present invention provides a kind of method for preparing antibody, the described method includes：Animal is immunized with the compound, from
The specific antibody of the compound is isolated in animal body after immune.
Preferably, the antibody is polyclonal antibody, is prepared with following the method：
Mixed with the compound with complete Freund's adjuvant (according to volume ratio 1:2~2:1 mixing, preferably according to body
Product ratio 1:1 mixing) mouse is immunized afterwards, mixed after 2 weeks with the compound with incomplete Freund's adjuvant (according to volume ratio 1:2
~2:1 mixing, preferably according to volume ratio 1:1 mixing) reinforced immunological mouse afterwards, reinforced immunological 2- again in the same fashion
3 times, polyclonal antibody is isolated after 1-2 weeks from serum.
The present invention provides P19/Ebi3 compounds to prepare application of the suppression B cell into plasma cell differentiation medicine.
Further, the thick liquid cell is the (GL7 of B cell differentiation+) thick liquid cell.
Further, the specific antibody of P19/Ebi3 compounds, the tumor vaccine based on P19/Ebi3 compounds, for pressing down
The protein of P19/Ebi3 complex activities processed, suppresses the compound of P19/Ebi3 complex activities.
The present invention provides a kind of application of P19/Ebi3 compounds in diagnostic system lupus erythematosus product is prepared.
Further, diagnostic products mentioned above include：By immune detection, co-immunoprecipitation, ELISA or chip come
Detect the horizontal product with diagnostic system lupus erythematosus of P19/Ebi3 compounds.
Further, it is described to be included with immune detection come the product of diagnostic system lupus erythematosus：With P19/Ebi3 compounds
The antibody of specific binding；It is described to be included with co-immunoprecipitation come the product of diagnostic system lupus erythematosus：Answered with P19/Ebi3
The antibody that two subunits are specifically bound at the same time in compound；It is described to be included with ELISA come the product of diagnostic system lupus erythematosus：
ELISA kit；ELISA kit includes the antibody with the specific binding of P19/Ebi3 compounds；It is described to be diagnosed with chip
The product of systemic loupus erythematosus includes：Protein chip；Wherein protein chip includes specifically binding with P19/Ebi3 compounds
Antibody.
Preferably, the product includes kit and chip.
The present invention provides application of the P19/Ebi3 complex inhibitors in medicament for treating systemic lupus erythematosus is prepared.
Further, inhibitor of the present invention includes：The specific antibody of P19/Ebi3 compounds, answered based on P19/Ebi3
The tumor vaccine of compound, the protein for suppressing P19/Ebi3 complex activities, suppress the change of P19/Ebi3 complex activities
Compound.
Further, the specific antibody of the P19/Ebi3 compounds includes monoclonal antibody, polyclonal antibody.It is described
The specific antibody of P19/Ebi3 compounds includes complete antibody molecule, any fragment of antibody or modification (for example, inosculating antibody
Body, scFv, Fab, F (ab ') 2, Fv etc..As long as the fragment can retain the binding ability with P19/Ebi3 compounds.
Preferably, the specific antibody of the P19/Ebi3 compounds is polyclonal antibody.
Further, the medicine includes the antibody or its active fragment.
Further, medicine of the invention further includes pharmaceutically acceptable carrier, and this kind of carrier includes (but being not limited to)：
Diluent, excipient such as water etc., filler such as starch, sucrose etc.；Adhesive such as cellulose derivative, alginates, gelatin and poly-
Vinylpyrrolidone；Wetting agent such as glycerine；Disintegrant such as agar, calcium carbonate and sodium acid carbonate；Sorbefacient quaternary ammonium compound；
Surfactant such as hexadecanol；Absorption carrier such as kaolin and soap clay；Lubricant such as talcum powder, calcium stearate and magnesium, gather
Ethylene glycol etc..
The medicine of the present invention can also be with the drug combination of other treatment systemic loupus erythematosus, and multi-medicament is used in combination can
To greatly improve the success rate for the treatment of.
" P19/Ebi3 compounds " includes any functional equivalent of P19/Ebi3 compounds and P19/Ebi3 compounds.
The functional equivalent includes P19/Ebi3 compound conservative variation protein or its active fragment, or its reactive derivative,
Allelic variant, natural mutation, induced mutants, can be with the DNA hybridization of P19/Ebi3 under high or low high stringency conditions
The encoded protein of DNA.
Preferably, P19/Ebi3 compounds are the protein for having following amino acid sequences：
(1) by SEQ ID NO in sequence table:1 and SEQ ID NO:The protein of amino acid sequence composition shown in 2；
(2) by SEQ ID NO:1 and/or SEQ ID NO:Amino acid sequence shown in 2 passes through one or several amino acid
The substitution of residue and/or missing and/or addition and with SEQ ID NO:1 and/or SEQ ID NO:Amino acid sequence tool shown in 2
Have identical function by SEQ ID NO:1 and/or SEQ ID NO:Protein derived from amino acid sequence shown in 2.Substitution, lack
Lose or the number of the amino acid of addition is usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10.
(3) with SEQ ID NO:1 and/or SEQ ID NO:Amino acid sequence shown in 2 has at least 80% homology (again
Referred to as sequence identity), it is highly preferred that with SEQ ID NO:1 and/or SEQ ID NO:Amino acid sequence shown in 2 is at least about
The polypeptide that 90% to 95% homology is often 96%, the amino acid sequence of 97%, 98%, 99% homology is formed.
In specific embodiments of the present invention, the P19/Ebi3 compounds are with SEQ ID NO:1 and/or SEQ
ID NO:The protein of amino acid sequence shown in 2.
It is known that, conventionally, the modification of one or more amino acid does not interfere with the function of protein in a protein.
Those skilled in the art can approve the amino acid that changes single amino acids or small percentage or indivedual additions to amino acid sequence,
Missing, insertion, replacement are conservative modifications, and the change of wherein protein produces the protein with identity function.Function phase is provided
As the Conservative substitution tables of amino acid be well known in the art.
By adding the protein of an amino acid or more amino acid modification, as long as the albumen of gained retains P19/
The biological activity of Ebi3 compounds.
The P19/Ebi3 compounds of the present invention also include to SEQ ID NO:1 and/or SEQ ID NO:Amino acid shown in 2
The non-conservative modification of sequence, as long as the protein by modification remains able to the biological activity of reservation P19/Ebi3 compounds i.e.
Can.The amino acid number being mutated in such modifying protein is typically 10 either less such as 6 or less, such as 3
It is a or less.
The advantages of the present invention：
Present invention firstly discovers that P19/Ebi3 compounds and its relation with systemic loupus erythematosus, tested by detecting
The level of P19/Ebi3 compounds in person, it can be determined that whether it suffers from systemic loupus erythematosus, so as to instruct so as to instruct to face
Bed doctor provides prevention scheme or therapeutic scheme to subject.
Can be with systemic lupus erythematosus, compared to existing present invention finds P19/Ebi3 compounds specific antibody
Treatment means, this method accuracy is high, side effect is low, is advantageously implemented and cures systemic loupus erythematosus and/or in long-term basis
The causal treatment of the upper quality of the life for improving patient.
Brief description of the drawings
Fig. 1 shows IL-12 family molecules subunit pairing situation；
Fig. 2 shows that people IL-12 families cell factor subunit P19 and Ebi3 can form compound；
Fig. 3 shows that mouse IL-12 families cell factor subunit P19 and Ebi3 can form P19/Ebi3 compounds；Fig. 4 is shown
Purifying and the Mass Spectrometric Identification of mouse P19/Ebi3 compounds are shown；Left figure shows the coomassie brilliant blue staining of purifying protein；It is right
Figure shows the mass spectral analysis of purifying protein；
Fig. 5 shows mouse P19/Ebi3 complex biological activity research；The Ebi3- of left figure immunoblotting assay purifying
Flag/P19-His can stimulate the phosphorylation of STAT1 in B cell；The Ebi3-Flag/P19- of right figure immunoblotting assay purifying
His can stimulate the phosphorylation of STAT3 in B cell；
Fig. 6 shows the high expression P19/Ebi3 of the B cell of LPS activation；
Fig. 7 shows the P19/Ebi3 of the B cell secreting high levels of LPS activation；Left figure is shown to be examined using ELISA method
Survey the situation for the B cell secretory antibody that LPS is stimulated；Right figure shows the B stimulated using double crush syndrome method detection LPS
The situation of cell secretory antibody；
Fig. 8 is shown to plasma cell differentiation (GL7+) the high expression P19/Ebi3 of B cell；
Fig. 9 is shown in SLE mouse models to plasma cell differentiation (GL7+) B cell and (CD138+) the high expression of cell
P19/Ebi3；
Figure 10 shows the identification of P19/Ebi3 polyclonal antibodies；
Figure 11 shows that anti-P19/Ebi3 polyclonal antibodies reduce systemic loupus erythematosus mouse Urine proteins；
Figure 12 shows that anti-P19/Ebi3 polyclonal antibodies reduce systemic loupus erythematosus mouse splenomegaly；
It is horizontal that Figure 13 shows that anti-P19/Ebi3 polyclonal antibodies reduce systemic loupus erythematosus mouse antibodies；
Figure 14 shows that anti-P19/Ebi3 polyclonal antibodies reduce systemic loupus erythematosus mouse lymphocyte number.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.The embodiment of the present invention is only used for solving
Release the present invention rather than limit the scope of the invention.
The experimental method of actual conditions is not specified in following embodiments, usually according to normal condition, such as Sambrook etc.
People, molecular cloning：Laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) described in
Condition, or according to the condition proposed by manufacturer.
Material, reagent used in following embodiments etc., are commercially available unless otherwise specified.
Embodiment 1 people IL-12 families cell factor subunit P19 and Ebi3 can form P19/Ebi3 compounds
First, material
People Ebi3 albumen is purchased from Origene companies (article No. purchased from Origene companies (article No. TP723769), people P19 albumen
TP309680), anti-Ebi3 antibody is purchased from purchased from SantaCruzBiotech companies (article No. sc-32869), anti-P40 antibody
SantaCruzBiotech companies (article No. sc-7926), anti-P19 antibody are purchased from SantaCruzBiotech companies (article No. sc-
50303), anti-c-Jun antibody is purchased from SantaCruzBiotech companies (article No. sc-44)
2nd, method
1st, precipitation is immunized
1) Agrose-proteinA/G is cleaned：Take in 60 μ l Agrose-proteinA/G to EP pipes and (cut pipette tips), add
The PBS of 500 μ l precoolings, 4 DEG C, 5000rpm centrifugation 2min (washing twice).
2) antibody and Agrose-proteinA/G preincubates：By the anti-Ebi3 antibody of 1 μ g, anti-p40 antibody, anti-P19 antibody,
Anti- c-Jun antibody is added separately in 15 washed μ l Agrose-proteinA/G, 4 DEG C of upset 1h.
3) 5ng/ml people Ebi3 albumen and 5ng/ml people's P19 albumen are mixed in PBS.
4) by the anti-Ebi3 antibody of 1 μ g, anti-P40 antibody, anti-P19 antibody, anti-c-Jun antibody respectively with 30 μ l proteinA/
G-beads is added in the mixed liquor described in step 3), and 4 DEG C are slowly rocked overnight incubation.
5) antigen-antibody is incubated：(5000rpm, 2min) is centrifuged after step 4), by Ebi3 antibody, P40 antibody, P19
Antibody, c-Jun antibody are added separately in the EP pipes containing the good corresponding antibodies of preincubate and Agrose-proteinA/G, and 4 DEG C incubate
Educate overnight.
6) IP compounds are washed：5000rpm, centrifuges 2min, sops up supernatant, adds IP lysates (generally without inhibiting)
500 μ l, play even rear 4 DEG C of rocking-turn 5min, are repeated 6 times.
7) 60 μ l of SDS detections are added.
2nd, immunoblotting assay
1) glue is matched somebody with somebody
1 10% separation gel of table and 4% concentration glue
| 10% separation gel | 4% concentration glue | |
| Ultra-pure water | 4.85ml | 3.16ml |
| 40%Acr/Bic (37.5:1) | 2.5ml | 0.5ml |
| 1.5mol/LTris·HCl(PH8.8) | 2.5ml | --- |
| 0.5mol/LTris·HCl(PH6.8) | --- | 1.26ml |
| 10%SDS | 100μl | 50μl |
| 10%AP (ammonium persulfate) | 50μl | 25μl |
| TEMED | 5μl | 5μl |
After adding TEMED, mixing immediately can encapsulating.
First with careful 10% separation gel of paving of 1ml pipette tips, above plus a little isopropanol, glue to be separated do it is solidifying after, steal isopropanol,
Unnecessary unpolymerized glue is washed with water, filter paper blots.4% concentration glue is repaved, is careful not to produce bubble, comb on oblique cutting, is treated
Glue is done rear spare, takes out comb.
2) loading
Loading sample is taken out into 0.5ml centrifuge tubes.5 × SDS sample-loading buffers are added to final concentration of 1 × SDS.(on
Sample cumulative volume is usually no more than 15 μ l, and well is 20 μ l samples to greatest extent).Sample is boiled in boiling water before loading
5min makes albuminous degeneration.
Fill up enough electrophoresis liquids and begin preparing for loading.With the adherent pipette samples of micro sample adding appliance, it is careful not to produce bubble.
Sample injector syringe needle is inserted to well and is slowly added to sample.When adding next sample, injector need to be in water jacket electrophoretic buffer
It is middle to wash 3 times, in order to avoid cross contamination.
3) electrophoresis
100V, after bromjophenol blue goes to separation gel bottom, about 1.5h rear electrophoresis terminate.
4) transferring film
Gel carefully is removed, filter paper and nitrocellulose filter equilibrated in transferring film buffer solution in advance are cut into and gel
It is onesize, it is stacked together in order, in 4 DEG C, 100V voltage transferring film 1h, nitrocellulose filter is taken out, with 1 × the beginning of spring red dye
Liquid dye 5min (in being shaken on decolorization swinging table).Then rinsed to film and turned white with TBST.
5) close
Skimmed milk power 1g is weighed, 20ml is dissolved into 1 × TBS, it is 5% to make skimmed milk power concentration.Nitrocellulose membrane is soaked
In 20ml confining liquids, room temperature shakes 2h.
6) primary antibody hybridization incubation
1 × TBS solution containing 5% skimmed milk power is used to dilute primary antibody, it is 1 to make antibody concentration:1000；The film that will be closed
It is placed in primary antibody dilution, 4 DEG C overnight；Then inhale and abandon primary antibody dilution, washed 3 times with 1 × TBST, each 5min.
7) secondary antibody hybridization incubation
1 × TBS solution containing 5% skimmed milk power is used to dilute the secondary antibody of horseradish peroxidase connection, it is 1 to make concentration:
2000, and add the anti-biotin antibodies (concentration 1 of horseradish peroxidase connection:1000) film, is placed in secondary antibody dilution
In, shaken at room temperature is incubated 1h；Then inhale and abandon secondary antibody dilution, washed 3 times with 1 × TBST, each 5min.
8) develop
Film is inserted into 10ml LumiGLO solution (formula：0.5ml 20×LumiGLO、0.5ml 20×Peroxide、
9.0ml Milli-Qwater), room temperature jog 1min, notices that lucifuge operates；Cut in darkroom with an equal amount of film of film,
It is pressed in magazine, about 1min；Film is placed on developer solution lunch and washes 1-5min；Water rinses；2-5min is washed in fixing solution；Water rushes
Wash, purpose band can be seen in relevant position.
9) gel image analysis
Film is scanned or taken pictures, molecular weight and net optical density with gel images processing system analysis object tape
Value.
3rd, result
Experimental result is as shown in Fig. 2, immunoblotting assay finds that people IL-12 families cell factor subunit P19 and Ebi3 can
Form P19/Ebi3 compounds.
Embodiment 2 mouse IL-12 families cell factor subunit P19 and Ebi3 can form P19/Ebi3 compounds
First, material
Plasmid and bacterial strain：
P19/pReceiver-Lv18, Ebi3/pEZ-LV122X expression plasmid, JM109 bacterial strains
2nd, method
1st, design of primers
Expand P19ORF primers in P19/Ebi3 compounds
Positive-sense strand：5’-ATGCTGGATTGCAGAGCAGT-3’(SEQ ID NO:5)
Antisense strand：5’-GTAAGCTGTTGGCACTAAGG-3’(SEQ ID NO:6)
Plus the positive-sense strand after EcoRI sites and protection base：
5’-CCGGAATTCATGCTGGATTGCAGAGCAGT-3’(SEQ ID NO:7)
Plus the antisense strand after XhoI sites and protection base：
5’-CCGCTCGAGGTAAGCTGTTGGCACTAAGG-3’(SEQ ID NO:8)
Expand Ebi3ORF primers in P19/Ebi3 compounds
Positive-sense strand：5’-ATGTCCAAGCTGCTCTTCCT-3’(SEQ ID NO:9)
Antisense strand：5’-TCAGGGCTTATGGGGTGCAC-3’(SEQ ID NO:10)
Plus the positive-sense strand after EcoRI sites and protection base：
5’-CCGGAATTCATGTCCAAGCTGCTCTTCCT-3’(SEQ ID NO:11)
Plus the antisense strand after XhoI sites and protection base：
5’-CCGCTCGAGTCAGGGCTTATGGGGTGCAC-3’(SEQ ID NO:12)
2nd, the structure of P19, Ebi3, P19/Ebi3 gene cloning and recombinant plasmid
1) extraction of total mRNA
DC cells are sub-elected out of 9 week old C57BL/6 Mice Bodies by crossing CD11c immunomagnetic beadses (Mei Tian Ni companies), every 1
×107The TRIZOL of 1ml is added in cell, sample is transferred in the EP pipes of 1.5ml, 5min is placed at 15-30 DEG C.
The chloroform of 0.2ml is added, acutely rocks 15s, 2-3min is placed at 15-30 DEG C, 2-8 DEG C, 12,000g centrifuge
15min。
Upper strata aqueous phase is transferred in another clean EP pipes, 0.5ml isopropanols is added, is stored at room temperature 10min, 2-8 DEG C,
12,000g centrifuges 10min.
Supernatant is removed, adds 75% ethanol of 1ml washing RNA precipitate, oscillator mixes, 2-8 DEG C, 12,000g centrifugation 5min.
RNA precipitate is resuspended with no RNase water or 0.5%SDS solution, is blown and beaten repeatedly with pipette tips several times, it is quiet at 55-60 DEG C
Put 10min.- 20 DEG C of preservations after survey concentration.
Denaturing formaldehyde agarose electrophoresis, determines extracting RNA integrality and DNA pollution situation.
2) RT-PCR expands P19 and Ebi3ORF sequences in P19/Ebi3 compounds
2 RT reaction systems (first step) of table：About 20min
| Volume | |
| RNA extracts | 5μl |
| Radome primers | 2μl |
| RNAsin | 0.5μl |
Ice bath is immediately placed in after 65 DEG C of 15min
3 RT reaction systems (second step) of table：About 2h
| Volume | |
| RNAsin | 0.5μl |
| 10mM dNTP | 1μl |
| 5 × RT buffer solutions | 4μl |
| 25mM MgCl2 | 4μl |
| AMV reverse transcriptases | 3μl |
37℃1.5h；94℃5-10min；Reactant is stored in -20 DEG C or carries out PCR
4 PCR reaction systems of table：About 4.5h
| Volume | |
| 25mM MgCl2 | 2μl |
| 10 × PCR buffer solutions | 5μl |
| 10mM dNTP | 1μl |
| Upstream and downstream primer 10pmol/ μ l | 2.5μl×2 |
| CDNA templates | 2.5μl |
| ddH2O | 34μl |
| Taq enzyme | 0.5μl |
| Light paraffinic oil | 50μl |
94 DEG C of 2min, 55 DEG C of 1min, 72 DEG C of 2min
94 DEG C of 45s, 55 DEG C of 40s, 72 DEG C of 1min, 30 circulations
72 DEG C of 10min, 4 DEG C of 5min.
- 20 DEG C of preservations or electrophoresis
Electrophoresis detection P19 and Ebi3 genetic fragment
3) structure of recombinant expression carrier
P19 and Ebi3ORF is inserted respectively into by EcoRI and XhoI restriction enzyme sites in P19/Ebi3 compounds
On pReceiver-Lv18 and pEZ-LV122X carriers
5 EcoRI and XhoI double digestion times of table and its system：
| Volume | |
| EcoRI | 1μl |
| XhoI | 1μl |
| 10×Buffer | 2μl |
| Plasmid or PCR product | 5μl |
| ddH2O | 11μl |
37 DEG C of reaction 20h, with the purification column kits double digestion product of TAKARA.
The connection of PCR product and carrier after digestion, recycling
Conversion：
A, full dose (10 μ l) is added into 100 μ l JM109 competent cells, and 30min is placed in ice.
B, after 42 DEG C of heating 45s, then 1min is placed in ice.
C, 890 μ l AMP Negative medias, 37 DEG C of shaken cultivation 60min are added.
D, 100 μ l bed boards are taken.
E, it is used to be sequenced through PCR and digestion identification positive colony.
3rd, the induced expression of mouse restructuring P19, Ebi3, P19/Ebi3 compound
P19/pReceiver-Lv18 and Ebi3/pEZ-LV122X expression vector corotation transfected cho cells
1) by 0.5-2 × 105 cell inoculation into 24 well culture plates, and 500 μ l antibiotic-free culture mediums are added, culture
24h。
2) P19/pReceiver-Lv18 and Ebi3/pEZ-LV122X are added to the low blood of 50 μ l serum-frees Opti-MEMI
(or other serum free mediums) is diluted in clear culture medium, and is gently mixed.
3) take corresponding amount to be added in Opti-MEM culture mediums the LipofectamineTM2000 gently mixed to carry out
Dilution, stands 5min at room temperature.
4) dilution of LipofectamineTM2000 and DNA is mixed.Gently mix and stand 20min at room temperature.
5) culture medium in culture plate is sucked, and is washed twice with serum free medium.
6) 100 μ l of mixture are added into culture hole, and the culture hole that rocks back and forth makes to be evenly distributed.
7) transfect 24h after by cell with 1:10 (or dilution factors of higher) are transferred in fresh growth medium.
8) in Selective agar medium Puromycin (P19/pReceiver-Lv18 carriers carry) and Neomycin (Ebi3/
PEZ-LV122X carriers carry) in coexpression P19/Ebi3 Chinese hamster ovary celI.
9) Chinese hamster ovary celI of P19/Ebi3 is co-expressed in 37 DEG C, CO2Cultivate 48-72h in incubator, collect supernatant with carrying out
Protein purification.
4th, precipitation and immunoblotting assay is immunized
Experimentation is as described in Example 1
3rd, result
Results of immunoblot analysis is as shown in figure 3, mouse IL-12 families cell factor subunit P19 and Ebi3 can be formed
P19/Ebi3 compounds.
3 mouse P19/Ebi3 complex purifications of embodiment and Mass Spectrometric Identification
First, method
1st, the coexpression of mouse Ebi3-Flag/P19-His
Experimentation is with reference to embodiment 2.
2nd, the purifying of Ebi3-Flag/P19-His
1) nickel column is handled
By in 1ml nickel column suction dead level analysis pipe, level pad 3ml is added after liquid flow therein is complete.
2) 10ml supernatants are added on equilibrated nickel column, and the sample for crossing column is repeated into loading 3 times, take 20 μ l to cross column
Sample afterwards, in case running glue.
3rd, coomassie brilliant blue staining
1) albumen after purification is subjected to SDS-PAGE, experimentation is as described in Example 1.Soak in fixer
In at least 30min.
2) by the gel after fixation, in the dyeing liquor of heat, (0.29g Coomassie brilliant blues are dissolved in 250ml destainers, are used
Front stirring change be heated to 60 DEG C) in immersion 10min, then with distilled water by gel elute once.
3) destainer (250ml ethanol, 80ml glacial acetic acid, adds distilled water to 1L) is repeatedly converted, until gel background purifies,
, can temperature slightly to accelerate to decolourize；Each step, which is placed on shaking table, above operates.
4) it is cracked in order to prevent after gel drying, the gel for sloughing background colour soaks 30min in liquid is preserved.Then will be solidifying
Glue is put on a glass, then with preservation liquid is soaked glassine paper and encased gel and dry at room temperature.
4th, Mass Spectrometric Identification
2nd, result
Experimental result as shown in figure 4, (left figure) in spite of foreign protein band, P19/Ebi3 purity is more than 70%；(right figure)
Mass Spectrometric Identification shows that P19-His molecular weight is about 18KD, and Ebi3-Flag molecular weight is about 36KD, and Ebi3-Flag/P19-His divides
Son amount is about 53.5KD.
4 mouse P19/Ebi3 compounds of embodiment can induce B cell STAT3 and STAT1 phosphorylation
First, material
P-STAT1, p-STAT3, p-STAT4, p-STAT5, p- are bought from CellSignalTechnology companies
STAT6, STAT1, STAT3, STAT4, STAT5, STAT6, β-actin (article No. is respectively 9167,9145,4134,9314,
9364、9172、4904、2653、9363、9362、4967)
2nd, method
1st, the expression and purifying of Ebi3-Flag, P19-His, Ebi3-Flag/P19-His albumen
2nd, B220 immunological magnetic bead sortings B cell
1) immunomagnetic beads is cleaned
Immunomagnetic beads is resuspended in bottle (vortex is no less than 30s)；
The immunomagnetic beads of appropriate volume is shifted in pipe；
Isometric dissociating buffer or at least 1ml are added, is resuspended；
Pipe is placed in 1min in magnet, discards supernatant；
Pipe is removed from magnet, washed magnetic bead is resuspended with the dissociating buffer with same volume in above-mentioned steps.
2) sample is prepared
Prepare individual cells suspension (lymphoid organ), use dissociating buffer so that cell final concentration of 1 × 107
A/ml.
3) sorting of B cell
The cell of 1ml is added into pipe, then adds the immunomagnetic beads in 25 μ l steps 1)；
20min is incubated at 4 DEG C, is placed on shaking table and jiggles；
Pipe is placed in magnet 2min；
Carefully discard supernatant；
Pipe is taken out from magnet, adds 1ml dissociating buffers thereto, is blown and beaten 2-3 times (or vortex 2-3s), then
The pipe is placed in 2min in magnetic field, carefully discards supernatant；
Repeat previous step at least once, clean the B cell connected with magnetic bead.
3rd, the priming reaction of B cell
LPS stimulates B cell
Prepare the suspension of single B cell, be 1 × 10 with RPMI1640 complete mediums culture adjustment B cell density6
A/ml, is added in 96 orifice plates, adds 10ug/ml LPS, 37 DEG C of culture 72-96h.
4th, immunoblotting assay
3rd, result
For experimental result as shown in figure 5, the Ebi3-Flag/P19-His albumen of purifying has biological activity, it can be effective
Ground stimulates the phosphorylation of STAT1, STAT3 in B cell.
The high expression P19/Ebi3 of B cell of embodiment 5LPS activation
First, material
Anti- P19 antibody is purchased from eBioscience purchased from eBioscience companies (article No. 50-7023-82), anti-P28 antibody
Company (article No. 12-7285-80), anti-P35 antibody are purchased purchased from eBioscience companies (article No. 50-7352-80), anti-P40 antibody
R＆D companies (article No. IC18341C) are purchased from from eBioscience companies (article No. 12-7123-81), anti-Ebi3 antibody
2nd, method
1st, LPS stimulates B cell
2nd, Fc acceptors are blocked：(being used to eliminate nonspecific combination dyeing)
Using the antibody (CD16/32) of the FcII/III acceptors of purifying, according to 1 μ g/106The dosage of cell, it is slow in dyeing
4 DEG C of incubation 15min in fliud flushing, after PBS cleaning, directly progress next step dyeing.
3rd, intracellular elements dyeing and detection
1) it is fixed：ICFix/Perm Buffer100 μ l are added, are mixed, are incubated at room temperature 20min.
2) 1 time (2000rpm, 10min) is washed with 1 × PermWashBuffer 1ml, topples over rear filter paper and blot mouth of pipe liquid
Body.
3) rupture of membranes：1 × PermWashBuffer 1ml are added, are mixed, 4 DEG C of incubation 45min.
4) (2000rpm, 10min) is centrifuged, topples over rear filter paper and blot mouth of pipe liquid.
5) cytokine antibodies, 4 DEG C of incubation 45min are added.
6) washed 1 time, after toppling over 1 × PermWashBuffer 1ml, add 300 μ l PBS and cell is resuspended.
7) machine testing on.
3rd, result
Experimental result is as shown in fig. 6, the B cell of LPS activation expresses a small amount of P28, P35, P40, and height expresses P19/Ebi3.
The P19/Ebi3 of the B cell energy secreting high levels of embodiment 6LPS activation
First, material
Purchased from the anti-Ebi3 antibody (article No. sc-32869) of SantaCruzBiotech companies, anti-P40 antibody (article No. sc-
7926), anti-P19 antibody (article No. sc-50303), anti-P28 antibody (article No. sc-27490), anti-P35 antibody (article No. sc-9350)
2nd, method
1st, the activation of B cell
2nd, ELISA is tested
1) it is coated with process (paying attention to setting blank control, negative control)
Antigen used coating diluted is arrived into suitable 10 μ g/ml, often hole antigen adds 100 μ l, is placed in 37 DEG C, 4h or
4 DEG C overnight；BSA solution can be coated with or only add coating buffer as blank control.
2) sealase mark reacting hole
Discard liquid in hole, 200 μ l confining liquids added per hole, 37 DEG C of wet box close 6h or 4 DEG C overnight, after with washing
Liquid washs 3-5 times, and in gently being patted dry on blotting paper.
3) detected sample is added
Certain diluted 100 μ l of measuring samples are added to add PBST as blank control in above-mentioned coated reacting hole.Put
37 DEG C, it is incubated 45min.It is washed out 3-5 times, and in gently being patted dry on blotting paper.
4) enzyme labelled antibody is added
In each reacting hole, the enzyme labelled antibody (dilution factor after titration of diluted fresh is added：1:More than 1000 dilute)
100μl.37 DEG C of incubation 45min, are washed 3 times, and in gently being patted dry on blotting paper.
5) substrate solution (matching while using) is added
Add the tmb substrate solution 100 μ l of Extemporaneous in each reacting hole, 37 DEG C, 10~30min.
6) reaction is terminated
50 μ l of 2M sulfuric acid are added in each reacting hole, in determination experiment result in 20min.
7) result judges
Each hole OD values are surveyed after 450nm is returned to zero with blank control wells with Bio-Rad microplate reader, it is right if more than defined feminine gender
It is positive (size of numerical value is depending on specific testing requirements) according to 2.1 times of OD values.
3rd, double crush syndrome is tested
With reference to above-mentioned experimental procedure.
3rd, result
Experimental result is as shown in fig. 7, the B cell of (left figure) LPS activation secretes a small amount of P28, P35, P40, and secretes Gao Shui
Flat P19, Ebi3；B cell secretion a small amount of IL-23, the IL-27 of (right figure) LPS activation, and secreting high levels P19/Ebi3.
Embodiment 7 is to plasma cell differentiation (GL7+) the high expression P19/Ebi3 of B cell
First, method
Method of the experimental implementation with reference to embodiment 5.
2nd, result
Experimental result is as shown in figure 8, we can see that to plasma cell differentiation (GL7 from figure+) the high expression of B cell
P19/Ebi3。
To plasma cell differentiation (GL7 in 8 systemic loupus erythematosus of embodiment (SLE) mouse model+) B cell and thick liquid cell
(CD138+) the high expression P19/Ebi3 of cell
First, material
The spontaneous sexual norm mouse of systemic loupus erythematosus：Female MRL/MpJ/lpr/lpr (MRL/lpr) mouse is purchased from Nanjing
Model organism research institute；MRL/MpJ/+ /+(MRL/++) mouse is purchased from Institute of Basic Medical Sciences of medical courses in general institute.
2nd, method
To plasma cell differentiation (GL7+) B cell, thick liquid cell (CD138+) acquisition and counting
1st, the acquisition of systemic loupus erythematosus Mouse spleen cells
1) mouse spleen is taken, is put into the culture dish for filling 5ml Hanks liquid.
2) pressure spleen (along same direction) is gently ground, splenocyte suspension is drawn with suction pipe, is put into test tube.
3) cell count under mirror：40 times of camera lenses, observe splenocyte.
2nd, the sorting of lymphocyte
1) splenocyte (splenocyte is diluted:Dilution=1:2).
2) Ficoll is added in centrifuge tube, diluted peripheral blood (Ficoll is slowly added into along inclined tube wall:Dilute spleen
Cell=1:2), 20 DEG C, 1500rpm, 30min is centrifuged.
3) along PBMC layers of another test tube of immigration of tube wall periphery gentle aspiration.
4) plus enough dilutions fully wash, and 1800rpm centrifugation 10min, abandon supernatant.
5) once, 1400rpm centrifugation 10min, abandon supernatant to repeated washing.
6) cell is resuspended in suitable culture medium, is mouse spleen lymphocyte suspension.
3rd, the counting of lymphocyte
Diluting cells, tally are put under microscope and are counted.
4th, cell streaming technical Analysis GL7+And CD138+Cell
1) the good primary antibody of suitable pre-dilution (fluorescent marker anti-B220, GL7, CD138 are added in each flow cytometer detection pipe
Antibody)；Added and the same amount of contrast agents of antibody in blank tube or Isotype control pipe.Then each pipe lucifuge ice bath or 4
30min is incubated in DEG C refrigerator.
2) at least 2ml CellStainingBuffer are added, then 350g centrifuges abandoning supernatant after 5min；Repetition is washed
Wash twice.
3) cell is resuspended with 0.5ml Cell Staining Buffer, is incubated 3-5min on ice.
5th, upper machine testing and analysis result.
2nd, result
Experimental result is as shown in figure 9, to plasma cell differentiation (GL7 in systemic loupus erythematosus (SLE) mouse model+) B
Cell and thick liquid cell (CD138+) cell and high expression P19/Ebi3 to plasma cell differentiation (GL7+) B cell and thick liquid cell
(CD138+) cell number significantly raises.
The anti-P19/Ebi3 polyclonal antibodies of embodiment 9 prepare and identification
First, method
1st, the preparation and purification of P19/Ebi3
Experimental implementation is with reference to embodiment 3.
2nd, the preparation of polyclonal antibody
1) selection of animal is immunized：The healthy male Balb/c mouse of 10 7-9 week old.
2) immunogene：Use the P19/Ebi3 compounds through ni-sepharose purification.Immune 100-200 μ g every time.
3) after mouse cultivates 3~4 days in new environment, 0.1ml tail veins are taken as negative control.
4) mouse is immune
Exempted from after being mixed in equal volume with complete Freund's adjuvant with the P19/Ebi3 compounds of purifying using back multi-point injection method
Epidemic disease mouse, 100 μ l of per injection, reinforced immunological after being mixed after 2 weeks with the compound with incomplete Freund's adjuvant equal proportion
Mouse (is injected) in above-mentioned position difference, reinforced immunological 2-3 times again in the same fashion, rear molding venous blood sampling inspection in 1 week
Survey antibody titer.
5) separation of serum
Centrifuge tube is positioned over 2h in 37 DEG C of baking ovens, 4 DEG C overnight, 10000RCF centrifugations 10min.NaN is added in serum3
To final concentration 0.02%, -20 DEG C of preservations after packing.
6) purifying (antigen affinity chromatography) of antibody
Crosslinking：Use Na2CO3-NaHCO3System, 10mg/ml P19/Ebi3 antigens are connected to beads, react at room temperature 4h.
The beads for being combined with antigen is filled into column.In order to ensure joint efficiency, sealed with the micromolecular compound urea containing amino
Close, after the completion of closing, remove confining liquid, column is washed three times with PBS.
The assessment of connection effect uses Coomassie Brilliant Blue (Bradford methods).
Cross column：The serum anticipated (10,000g centrifugation 5min, pass through 0.45 μm of membrane filtration) is diluted with PBS
One times, then in the good antigen pillar of the company of addition.
Elution：Eluted with the HCl (NaCl containing 150mM) of pH2.5 or so.One ultraviolet monitoring instrument detection outflow component with
Just immediately monitoring, elution process to the greatest extent in completed in low temperature, the component under eluting should place on ice chest, after eluting into
Part Na2HPO4Buffer solution.
Antibody preserves：Antibody after purification be placed in close in the buffer system of body fluid (PBS+0.02% Sodium azides+
5%BSA), -20 DEG C of preservations.
3rd, the identification of polyclonal antibody
Experimentation is with reference to embodiment 6
2nd, result
Experimental result is as shown in Figure 10, and the polyclonal antibody of mouse acquisition is immunized in P19/Ebi3 compounds, can be effective
Anti- P19 albumen, and antibody effects of the effect than positive control IL-23P19 are good.
The anti-P19/Ebi3 polyclonal antibodies of embodiment 10 reduce systemic loupus erythematosus mouse Urine proteins
First, method
1st, 16 test tubes are taken, 1 flag blank, 3 give over to unknown sample, remaining test tube is divided into two groups by order in table 6, divides
Not Jia Ru sample, water and reagent, i.e., be separately added into the standard protein solution of 1.0mg/ml to each test tube：0、0.01、0.02、
0.04th, 0.06,0.08,0.1ml, then adds to 0.1ml with deionized water.5.0ml, which is separately added into, in last each test tube examines horse
This bright orchid reagent of G -250, often adds a pipe, mixed immediately on vortex mixer (it is careful not to too acutely, in order to avoid produce big
Measure bubble and be difficult to eliminate).The 8th, 9,10 during the sample-adding amount of unknown sample see the table below manage.
The sample-adding amount of the different test tubes of table 6
2nd, after adding reagent 2-5min, light absorbs of each sample at 595nm are measured on spectrophotometer with cuvette
Value A595, blank control are No. 1 test tube, i.e. 0.1ml H2O adds the reagents of 5.0mlG -250.Pay attention to：Unusable quartz cuvette
Ware (because being not easy to wash away dyeing), can use plastics or glass cuvette, be swung and washed with a small amount of 95% ethanol immediately after use, to wash away
Dyeing.Plastic cuvette never can use ethanol or acetone to soak for a long time.
2nd, result
Experimental result is as shown in figure 11, and at the 7th day, anti-P19/Ebi3 Anti-TNF-αs physical efficiency significantly decreased systematicness
Lupus erythematosus mouse urine albumen amount.
The anti-P19/Ebi3 polyclonal antibodies of embodiment 11 reduce systemic loupus erythematosus mouse splenomegaly
First, method
1st, anti-P19/Ebi3 polyclonal antibodies systemic lupus erythematosus mouse
1) selection and packet of experimental animal
The female systemic loupus erythematosus MRL/lpr mouse 20 in the August age of morbidity, PBS treatment groups 10；Anti- P19/
Ebi3 Antybody therapies group 10.
2) preparation of polyclonal antibody
The anti-P19/Ebi3 polyclonal antibodies of high-titer are obtained using immune mouse and through affinity chromatography method, are matched somebody with somebody using PBS
4mg/ml is made.
3) using polyclonal antibody treatment disease mice
Every mouse injection 0.1ml antibody or PBS, are injected weekly twice, a co-injection 4 times.1 week after treatment, detection is anti-
Body therapeutic effect.
2nd, the spleen tissue of experiment mice is obtained, compares size
2nd, result
Experimental result is as shown in figure 12, using anti-P19/Ebi3 Antybody therapies systemic loupus erythematosus mouse, fundamentally
Recover that splenomegaly is small, so contributed to the recovery of splenic function.
It is horizontal that the anti-P19/Ebi3 polyclonal antibodies of embodiment 12 reduce systemic loupus erythematosus mouse antibodies
First, method
1st, anti-P19/Ebi3 polyclonal antibodies systemic lupus erythematosus mouse
2nd, the detection (double crush syndrome method) of mouse internal antibody level
1) the eyeball blood sampling of mouse is extractd.
2) blood in centrifuge tube is placed in 37 DEG C of incubators or water-bath 1h, also can room temperature 2h, then put in 4 DEG C of refrigerators 3~4h or
Overnight.
3) after blood clotting clot contraction, 4000rpm centrifugations 10min.
4) supernatant is taken in clean centrifuge tube, is stored in -20 DEG C or addition preservative is put in 4 DEG C of refrigerators and saved backup.
5) IgM and IgGELISA detections：
Coating：IgM will be captured with the coating buffer solution of 0.05M PH9 or IgG antibody is diluted to protein content as 1~10
μg/ml.Add 100 μ l in the reacting hole of each polystyrene board, 4 DEG C overnight.Solution in hole is discarded, 3 are washed with lavation buffer solution
It is secondary, each 3min.
Sample-adding：Add certain diluted 100 μ l of measuring samples in the above-mentioned reacting hole being coated with, put 37 DEG C of incubation 1h, so
After wash.(while doing blank well, negative control hole and Positive control wells).
Enzyme labeling antibody：In each reacting hole, the enzyme mark for adding diluted fresh detects IgM or IgG antibody (after titration
Dilution factor) 100 μ l.37 DEG C of 0.5~1h of incubation, washing.
Substrate solution is added to develop the color：The 100 μ l of tmb substrate solution of Extemporaneous are added in each reacting hole, are reacted at 37 DEG C
10~30min.
Terminate reaction：50 μ l of 2M sulfuric acid are added in each reacting hole.
Survey OD values：On ELISA detectors, at 450nm, each hole OD values are surveyed after returning to zero with blank control wells, if more than
2.1 times of defined negative control OD value, are the positive.
2nd, result
Experimental result is as shown in figure 13, can be significantly using anti-P19/Ebi3 Antybody therapies systemic loupus erythematosus mouse
Reduce the antibody level in Mice Body.
The anti-P19/Ebi3 polyclonal antibodies of embodiment 13 reduce systemic loupus erythematosus mouse lymphocyte number
First, method
1st, anti-P19/Ebi3 polyclonal antibodies systemic lupus erythematosus mouse
2nd, in Mice Body immunocyte detection
Experimental procedure is with reference to embodiment 8, and during cell streaming technical Analysis, the primary antibody added in each flow cytometer detection pipe is glimmering
Signal anti-CD11b, Gr-1, CD3, B220, GL7, IgG, IgM antibody.
2nd, result
Experimental result is as shown in figure 14, can effectively be dropped using anti-P19/Ebi3 Antybody therapies systemic loupus erythematosus mouse
Low innate immune cells such as neutrophil leucocyte (CD11b+Gr-1+), macrophage (CD11b+Gr-1-), T cell (CD3+), B cell
(B220+), activation B cell (GL7+B220+), antibody secreting cell (IgG+Or IgM+) level.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, some improvement can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
Claims (6)
1.P19/Ebi3 heterodimeric complex is preparing application of the suppression B cell into plasma cell differentiation medicine.
2. application according to claim 1, it is characterised in that the thick liquid cell is the GL7 broken up by B cell+Slurry is thin
Born of the same parents.
Application of the 3.P19/Ebi3 heterodimeric complex in diagnostic system lupus erythematosus product is prepared.
4.P19/Ebi3 application of the heterodimeric complex inhibitor in medicament for treating systemic lupus erythematosus is prepared.
5. application according to claim 4, it is characterised in that the P19/Ebi3 heterodimeric complex inhibitor is
Specific recognition and/or the antibody for combining P19/Ebi3 heterodimers.
6. application according to claim 5, it is characterised in that the antibody is polyclonal antibody.
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| IL-23生理病理学意义及信号通路;田红岭 等;《免疫学杂质》;20140601(第6期);全文 * |
| TLR3 drives IRF6-dependent IL-23p19 expression and p19/EBI3 heterodimer formation in keratinocytes;Divya Ramnath等;《Immunology and Cell Biology》;20150825(第93期);第771页摘要部分 * |
| 系统性红斑狼疮患者血清IL-23表达水平及临床相关性研究;李传静 等;《临床和实验医学杂质》;20150210(第3期);全文 * |
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