CN105085656A - P19/Ebi3 compound and application of polyclonal antibody of P19/Ebi3 compound in SLE (systemic lupus erythematosus) diagnosis and treatment - Google Patents

Abstract

The invention discloses a P19/Ebi3 compound and an application of a polyclonal antibody of the P19/Ebi3 compound in SLE (systemic lupus erythematosus) diagnosis and treatment. Experiments prove that compared with normal mice, mice with SLE have high-level expression of the P19/Ebi3 compound, with the application of the polyclonal antibody resistant to P19/Ebi3 protein, the expression level of antibodies and the number of immune cells in the mice can be reduced, and SLE can be treated through preparation of the antibodies resistant to P19/Ebi3, so that the P19/Ebi3 compound and an inhibitor thereof are used for preparing drugs for diagnosing and treating SLE and have excellent development prospects.

Description

P19/Ebi3 mixture and the application of polyclonal antibody in systemic lupus erythematous diagnosis and treatment thereof

Technical field

The present invention relates to biological technical field, more specifically, the present invention relates to P19/Ebi3 mixture and the purposes of polyclonal antibody in systemic lupus erythematosus diagnosis, treatment thereof.

Background technology

Systemic lupus erythematous (SystemicLupusErythematosus, SLE) is the autoimmune disorder of a kind of serious threat human health and life, has higher lethality rate and disability rate.Its cause and onset of disease mechanism does not still understand, generally believes it is that environmental factors acts on the body (comprising the different types that histocompatibility, cytokine, cytokine receptor etc. are expressed) of certain genetic background and the result that formed.Its immunopathogenesis feature produces multiple height, pathogenic autoantibodies in body, Lymphocyte Apoptosis simultaneously.Discharge a large amount of nucleus content and ribose corpusculum and become autoantigen; Antigen-antibody combines and forms immunocomplex, and the deposition of immunocomplex can cause target cell to damage and inflammation, causes each tissue and organ generation pathology.In this pathologic process, B cell is in advanced activation state, and this process and T cell closely related, have T cell dependency.

Complicated cytokine network regulatory T-cell and the growth of B cell, differentiation and function, these cytokines can be divided into two large classes: Th1 type (main inducing cellular immune) and Th2 type (main enhancing antibody generation).Interleukin 12 (IL-12) plays an important role in cytokine network regulates, and it produces primarily of mononuclear macrophage secretion.IL-12 family is by a α-chain (P19, P28 or P35) and beta chain (P40 or Ebi3) heterodimer subunit composition (Fig. 1), itself and some autoimmune diseases, especially the morbidity of the autoimmune disease that Th1 is cell-mediated is relevant.

Clinical diagnosis at present for systemic lupus erythematous mainly relies on clinical manifestation, laboratory examination, histopathology and imaging examination.Because SLE clinical manifestation illness varies with each individual, there is mistaken diagnosis sometimes.And the ordinary method of systemic lupus erythematosus uses antimalarial drug, antiphlogiston and immunosuppressive drug, when symptom is difficult to control, non-steroidal anti-inflammatory agent is supplemented with cortin; Although have remarkable effect in symptom management, prevention disease progression, there is obvious adverse reaction more, and to some patients unsatisfactory curative effect.B cell and T cell play an important role in the pathogenesis of SLE, and the biological therapy for immunocyte will become the focus for the treatment of SLE.

Summary of the invention

In order to make up the deficiencies in the prior art, the object of the present invention is to provide a kind of the mixture P19/Ebi3 and the polyclonal antibody thereof that can be used for systemic lupus erythematous diagnosis and treatment.Compare the Diagnosis and Treat method of traditional systemic lupus erythematous, using polyclonal antibody to respond with the pathologic immune causing autoantibody excessively to produce the specific treatment interacting or corrected will advantageously, as having higher specificity and accuracy, lower side effect, thus the causal treatment realizing the quality of the life of curing systemic lupus erythematous and/or improving patient in long-term basis.

To achieve these goals, the present invention adopts following technical scheme:

The invention provides a kind of P19/Ebi3 mixture of separation.

Further, the mixture mentioned above is made up of P19 and Ebi3 two subunits.

Further, above, the P19 subunit mentioned comprises the albumen of the aminoacid sequence as shown in SEQIDNO:1 or its conservative variation's albumen.

Further, above, the Ebi3 subunit mentioned comprises the albumen of the aminoacid sequence as shown in SEQIDNO:2 or its conservative variation's albumen.

Further, subunit recited above is by polynucleotide encoding, and the nucleotide sequence of described P19 subunit is as shown in SEQIDNO:3; The nucleotide sequence of described Ebi3 subunit is as shown in SEQIDNO:4.

The invention provides a kind of recombinant vectors, it contains polynucleotide sequence recited above.

Further, also containing the expression regulation sequence be connected with described polynucleotide sequence operability in described recombinant vectors.

The carrier carrying gene of the present invention is various carrier known in the art, as commercially available carrier, comprises plasmid, clay, phage, virus etc.

Virus vector can be any suitable carrier, includes but not limited to retroviral vector, adenovirus carrier, adeno-associated virus (AAV) carrier, simplexvirus (such as hsv, vaccinia virus and Epstein-Barr virus) carrier, alphavirus vectors.

Carrier for expression of eukaryon can be any suitable expression vector, include but not limited to pCMV-Myc expression vector, pcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP expression vector, pEFBos expression vector, pTet expression vector, pTRE expression vector, pReceiver-Lv18 expression vector, pZE-LV122X expression vector or the carrier through transforming on the basis of known expression vector, such as pBin438, pCAMBIA1301 etc.

Preferably, described expression vector is carrier for expression of eukaryon.Preferably, described carrier for expression of eukaryon is pReceiver-Lv18 and pEZ-LV122X expression vector.

The invention provides a kind of host cell, it contains described recombinant vectors, or is integrated with described polynucleotide in its genome.

Preferably, described host cell is Chinese hamster ovary celI.

The invention provides a kind of preparation method of P19/Ebi3 mixture, it comprises the following steps:

Host cell described in (a) cultivation;

B () isolates described mixture from culture.

The invention provides a kind of antibody, described antibodies specific identification and/or the mixture described in combination.

Preferably, described antibody is polyclonal antibody.

The invention provides a kind of method of Dispersal risk, described method comprises: with described mixture immune animal, isolates the specific antibody of described mixture in the animal body after immunity.

Preferably, described antibody is polyclonal antibody, prepares by the following stated method:

Mix (according to volume ratio 1:2 ~ 2:1 mixing with complete Freund's adjuvant with described mixture, preferably according to volume ratio 1:1 mixing) immune mouse afterwards, mix (according to volume ratio 1:2 ~ 2:1 mixing with incomplete Freund's adjuvant with described mixture after 2 weeks, preferably according to volume ratio 1:1 mixing) reinforced immunological mouse afterwards, according to identical mode reinforced immunological 2-3 time again, 1-2 isolates polyclonal antibody after week from serum.

The invention provides P19/Ebi3 mixture suppresses B cell to the application in plasma cell differentiation medicine in preparation.

Further, described plasmocyte is the (GL7 of B cell differentiation ⁺) plasmocyte.

Further, the specific antibody of P19/Ebi3 mixture, based on the tumor vaccine of P19/Ebi3 mixture, for suppressing the protein of P19/Ebi3 complex activity, suppress the compound of P19/Ebi3 complex activity.

The invention provides a kind of P19/Ebi3 mixture and prepare the application in diagnositc system lupus erythematosus product.

Further, the diagnostic products mentioned above comprises: detected the level of P19/Ebi3 mixture by immunodetection, co-immunoprecipitation, ELISA or chip with the product of diagnositc system lupus erythematosus.

Further, the product that described immunodetection carrys out diagnositc system lupus erythematosus comprises: with the antibody of P19/Ebi3 mixture specific binding; The product that described co-immunoprecipitation carrys out diagnositc system lupus erythematosus comprises: with the antibody of the specific binding simultaneously of two subunits in P19/Ebi3 mixture; The product that described ELISA carrys out diagnositc system lupus erythematosus comprises: ELISA kit; ELISA kit comprises the antibody with P19/Ebi3 mixture specific binding; The product that described chip carrys out diagnositc system lupus erythematosus comprises: protein chip; Wherein protein chip comprises the antibody with P19/Ebi3 mixture specific binding.

Preferably, described product comprises test kit and chip.

The invention provides P19/Ebi3 complex inhibitor and prepare the application in medicament for treating systemic lupus erythematosus.

Further, inhibitor of the present invention comprises: the specific antibody of P19/Ebi3 mixture, based on the tumor vaccine of P19/Ebi3 mixture, for suppressing the protein of P19/Ebi3 complex activity, suppress the compound of P19/Ebi3 complex activity.

Further, the specific antibody of described P19/Ebi3 mixture comprises monoclonal antibody, polyclonal antibody.The specific antibody of described P19/Ebi3 mixture comprises complete antibody molecule, any fragment of antibody or modification (such as, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc.As long as described fragment can retain the binding ability with P19/Ebi3 mixture.

Preferably, the specific antibody of described P19/Ebi3 mixture is polyclonal antibody.

Further, described medicine comprises described antibody or its active fragments.

Further, medicine of the present invention also comprises pharmaceutically acceptable carrier, and this kind of carrier comprises (but being not limited to): thinner, vehicle are if water etc., weighting agent are as starch, sucrose etc.; Tackiness agent is as derivatived cellulose, alginate, gelatin and polyvinylpyrrolidone; Wetting agent is as glycerine; Disintegrating agent is as agar, calcium carbonate and sodium bicarbonate; Absorption enhancer quaternary ammonium compound; Tensio-active agent is as cetyl alcohol; Absorption carrier is as kaolin and soap clay; Lubricant is as talcum powder, calcium stearate and magnesium, polyoxyethylene glycol etc.

Medicine of the present invention also can with the drug combination of other treatment systemic lupus erythematous, multi-medicament conbined usage can improve the success ratio for the treatment of greatly.

" P19/Ebi3 mixture " comprises any function equivalent of P19/Ebi3 mixture and P19/Ebi3 mixture.Described function equivalent comprises P19/Ebi3 mixture conservative variation's protein or its active fragments, or its reactive derivative, allelic variant, natural mutation, induced mutants, can with the protein coded by the DNA of the DNA hybridization of P19/Ebi3 under high or low high stringency conditions.

Preferably, P19/Ebi3 mixture is the protein with following amino acid sequences:

(1) protein be made up of the aminoacid sequence shown in SEQ ID NO:1 and SEQIDNO:2;

(2) aminoacid sequence shown in SEQIDNO:1 and/or SEQIDNO:2 had the protein derivative by the aminoacid sequence shown in SEQIDNO:1 and/or SEQIDNO:2 of identical function with the aminoacid sequence shown in SEQIDNO:1 and/or SEQIDNO:2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.The amino acid whose number replacing, lack or add is generally 1-50, preferably 1-30, and more preferably 1-20,1-10 is individual best.

(3) with the aminoacid sequence shown in SEQIDNO:1 and/or SEQIDNO:2, there is at least 80% homology (being also called sequence iden), more preferably, with the aminoacid sequence shown in SEQIDNO:1 and/or SEQIDNO:2 at least about 90% to 95% homology, be often 96%, 97%, 98%, 99% homology aminoacid sequence form polypeptide.

In specific embodiment of the invention scheme, described P19/Ebi3 mixture is the protein with the aminoacid sequence shown in SEQIDNO:1 and/or SEQIDNO:2.

Usually, it is known that in a protein one or more amino acid whose modification can not affect the function of protein.Those skilled in the art can approve the amino acid that changes single amino acids or little per-cent or be conservative modifications to indivedual interpolations of aminoacid sequence, disappearance, insertion, replacement, and wherein the change of protein produces the protein with identity function.Intimate amino acid whose Conservative substitution tables is provided to be well known in the art.

By adding the protein of an amino acid or multiple Modification of amino acid residues, as long as the albumen of gained retains the biologic activity of P19/Ebi3 mixture.

P19/Ebi3 mixture of the present invention also comprises the non-conservative modification to the aminoacid sequence shown in SEQIDNO:1 and/or SEQIDNO:2, as long as the protein through modifying still can retain the biologic activity of P19/Ebi3 mixture.The amino acid number suddenlyd change in this type of modifying protein normally 10 or less, such as 6 or less, such as 3 or less.

Advantage of the present invention and beneficial effect:

Late Cambrian of the present invention P19/Ebi3 mixture and the relation with systemic lupus erythematous thereof, by detecting the level of P19/Ebi3 mixture in experimenter, can judge whether it suffers from systemic lupus erythematous, thus instruct thus instruct clinicist to provide prevention scheme or treatment plan to experimenter.

Present invention finds P19/Ebi3 mixture specific antibody can systemic lupus erythematosus, compare existing treatment means, the method accuracy is high, side effect is low, is conducive to the causal treatment realizing the quality of the life of curing systemic lupus erythematous and/or improving patient in long-term basis.

Accompanying drawing explanation

Fig. 1 shows IL-12 family molecule subunit pairing situation;

Fig. 2 shows people IL-12 family cytokine subunit P19 and Ebi3 can form mixture;

Fig. 3 shows mouse IL-12 family cytokine subunit P19 and Ebi3 can form P19/Ebi3 mixture; Fig. 4 shows purifying and the Mass Spectrometric Identification of mouse P19/Ebi3 mixture; Left figure shows the coomassie brilliant blue staining of purifying protein; Right figure shows the mass spectroscopy of purifying protein;

Fig. 5 shows mouse P19/Ebi3 complex biological activity research; The Ebi3-Flag/P19-His of left figure immunoblotting assay purifying can stimulate the phosphorylation of STAT1 in B cell; The Ebi3-Flag/P19-His of right figure immunoblotting assay purifying can stimulate the phosphorylation of STAT3 in B cell;

Fig. 6 shows the B cell high expression level P19/Ebi3 of LPS activation;

Fig. 7 shows the P19/Ebi3 of the B cell secreting high levels of LPS activation; Left figure shows the situation using ELISA method to detect the B cell secretory antibody that LPS stimulates; Right figure shows the situation using double crush syndrome method to detect the B cell secretory antibody that LPS stimulates;

Fig. 8 shows to plasma cell differentiation (GL7 ⁺) B cell high expression level P19/Ebi3;

Fig. 9 shows in SLE mouse model to plasma cell differentiation (GL7 ⁺) B cell and (CD138 ⁺) cell high expression level P19/Ebi3;

Figure 10 shows the qualification of P19/Ebi3 polyclonal antibody;

Figure 11 shows anti-P19/Ebi3 polyclonal antibody and reduces systemic lupus erythematous mouse retention albumen;

Figure 12 shows anti-P19/Ebi3 polyclonal antibody and reduces the enlargement of systemic lupus erythematous mice spleen;

Figure 13 shows anti-P19/Ebi3 polyclonal antibody and reduces systemic lupus erythematous mouse antibodies level;

Figure 14 shows anti-P19/Ebi3 polyclonal antibody and reduces systemic lupus erythematous mouse lymphocyte number.

Concrete embodiment

Below in conjunction with drawings and Examples, the present invention is further detailed explanation.Embodiments of the invention are only not used in for explaining the present invention and limit the scope of the invention.

The experimental technique of unreceipted actual conditions in following embodiment, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.

The material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Embodiment 1 people IL-12 family cytokine subunit P19 and Ebi3 can form P19/Ebi3 mixture

One, material

People Ebi3 albumen purchased from Origene company (article No. TP723769), people P19 albumen purchased from Origene company (article No. TP309680), anti-Ebi3 antibody purchased from SantaCruzBiotech company (article No. sc-32869), anti-P40 antibody purchased from SantaCruzBiotech company (article No. sc-7926), anti-P19 antibody purchased from SantaCruzBiotech company (article No. sc-50303), anti-c-Jun antibody purchased from SantaCruzBiotech company (article No. sc-44)

Two, method

1, immunoprecipitation

1) Agrose-proteinA/G cleaning: get in 60 μ lAgrose-proteinA/G to EP pipes (cutting rifle head), add the PBS of 500 μ l precoolings, 4 DEG C, the centrifugal 2min of 5000rpm (washing twice).

2) antibody and Agrose-proteinA/G preincubate: 1 μ g anti-Ebi3 antibody, anti-p40 antibody, anti-P19 antibody, anti-c-Jun antibody are joined in 15 washed μ lAgrose-proteinA/G respectively, 4 DEG C of upset 1h.

3) 5ng/ml people Ebi3 albumen and 5ng/ml people P19 albumen are mixed in PBS.

4) 1 μ g anti-Ebi3 antibody, anti-P40 antibody, anti-P19 antibody, anti-c-Jun antibody are joined step 3 with 30 μ lproteinA/G-beads respectively) described in mixed solution in, 4 DEG C are slowly rocked overnight incubation.

5) antigen-antibody is hatched: step 4) after carry out centrifugal (5000rpm, 2min), Ebi3 antibody, P40 antibody, P19 antibody, c-Jun antibody are joined respectively in the EP pipe containing the good corresponding antibodies of preincubate and Agrose-proteinA/G, 4 DEG C of overnight incubation.

6) wash IP mixture: 5000rpm, centrifugal 2min, sops up supernatant, adds IP lysate (generally without inhibiting) 500 μ l, plays even rear 4 DEG C of rocking-turn 5min, repeats 6 times.

7) add SDS60 μ l to detect.

2, immunoblotting assay

1) glue is joined

Table 110% separation gel and 4% concentrated glue

	10% separation gel	4% concentrated glue
			Ultrapure 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 persulphate)	50μl	25μl
			TEMED	5μl	5μl

After adding TEMED, mixing immediately gets final product encapsulating.

First carefully spread 10% separation gel with 1ml rifle head, above add a little Virahol, after glue to be separated is done and coagulated, steal Virahol, wash unnecessary unpolymerized glue with water, filter paper blots.Repave 4% concentrated glue, note not producing bubble, comb on oblique cutting, for subsequent use after glue is dry, take out comb.

2) loading

Take out loading sample in 0.5ml centrifuge tube.Adding 5 × SDS sample-loading buffer to final concentration is 1 × SDS.(loading cumulative volume is generally no more than 15 μ l, well be 20 μ l samples to greatest extent).Sample to be boiled in boiling water 5min before loading and make protein denaturation.

Add enough electrophoresis liquid to start to prepare loading.With the adherent pipette samples of micro sample adding appliance, note not producing bubble.Sample injector syringe needle is inserted to well and slowly adds sample.When adding next sample, sampler need wash 3 times, in order to avoid crossed contamination in water jacket electrophoretic buffer.

3) electrophoresis

100V, bromjophenol blue is gone to after bottom separation gel, and about 1.5h rear electrophoresis terminates.

4) transferring film

Carefully take off gel, filter paper equilibrated in transferring film damping fluid in advance and nitrocellulose filter are cut into gel onesize, be stacked together in order, at 4 DEG C, 100V voltage transferring film 1h, take out nitrocellulose filter, with red dye liquor dye 5min (shaking on decolorization swinging table) in 1 × the beginning of spring.Then turn white to film with TBST rinsing.

5) close

Take skim-milk 1g, be dissolved into 20ml with 1 × TBS, make skim-milk concentration be 5%.Be immersed in by nitrocellulose membrane in 20ml confining liquid, room temperature shakes 2h.

6) primary antibodie hybridization incubation

By the 1 × TBS solution dilution of primary antibodie containing 5% skim-milk, antibody concentration is made to be 1:1000; Be placed on by the film closed in primary antibodie diluent, 4 DEG C are spent the night; Then inhale and abandon primary antibodie diluent, wash 3 times with 1 × TBST, each 5min.

7) two anti-hybridization incubation

The two anti-use connected by horseradish peroxidase are containing 1 × TBS solution dilution of 5% skim-milk, concentration is made to be 1:2000, and adding the anti-biotin antibodies (concentration is 1:1000) of horseradish peroxidase connection, film is placed in two anti-diluents, shaken at room temperature hatches 1h; Then inhale and abandon two anti-diluents, wash 3 times with 1 × TBST, each 5min.

8) develop

Film is inserted (formula: 0.5ml20 × LumiGLO, 0.5ml20 × Peroxide, 9.0mlMilli-Qwater) in 10mlLumiGLO solution, room temperature jog 1min, note lucifuge operation; In darkroom, cut the film onesize with film, be pressed in magazine, about 1min; Film is placed on developing solution lunch and washes 1-5min; Water rinses; 2-5min is washed in stop bath; Water rinses, and can see object band in corresponding position.

9) gel image analysis

Film is carried out scanning or taking pictures, by molecular weight and the clean optical density value of gel images treatment system evaluating objects band.

Three, result

As shown in Figure 2, immunoblotting assay finds experimental result, and people IL-12 family cytokine subunit P19 and Ebi3 can form P19/Ebi3 mixture.

Embodiment 2 mouse IL-12 family cytokine subunit P19 and Ebi3 can form P19/Ebi3 mixture

One, material

Plasmid and bacterial strain:

P19/pReceiver-Lv18, Ebi3/pEZ-LV122X expression plasmid, JM109 bacterial strain

Two, method

1, design of primers

P19ORF primer in amplification P19/Ebi3 mixture

Positive-sense strand: 5 '-ATGCTGGATTGCAGAGCAGT-3 ' (SEQIDNO:5)

Antisense strand: 5 '-GTAAGCTGTTGGCACTAAGG-3 ' (SEQIDNO:6)

Add the positive-sense strand after EcoRI site and protection base:

5’-CCGGAATTCATGCTGGATTGCAGAGCAGT-3’(SEQIDNO:7)

Add the antisense strand after XhoI site and protection base:

5’-CCGCTCGAGGTAAGCTGTTGGCACTAAGG-3’(SEQIDNO:8)

Ebi3ORF primer in amplification P19/Ebi3 mixture

Positive-sense strand: 5 '-ATGTCCAAGCTGCTCTTCCT-3 ' (SEQIDNO:9)

Antisense strand: 5 '-TCAGGGCTTATGGGGTGCAC-3 ' (SEQIDNO:10)

Add the positive-sense strand after EcoRI site and protection base:

5’-CCGGAATTCATGTCCAAGCTGCTCTTCCT-3’(SEQIDNO:11)

Add the antisense strand after XhoI site and protection base:

5’-CCGCTCGAGTCAGGGCTTATGGGGTGCAC-3’(SEQIDNO:12)

2, the structure of P19, Ebi3, P19/Ebi3 gene clone and recombinant plasmid

1) extraction of total mRNA

DC cell was sub-elected in C57BL/6 Mice Body, every 1 × 10 from 9 week age by crossing CD11c immunomagnetic beads (Mei Tian Ni company) ⁷add the TRIZOL of 1ml in cell, sample is transferred in the EP pipe of 1.5ml, place 5min at 15-30 DEG C.

Add the chloroform of 0.2ml, acutely rock 15s, place 2-3min, 2-8 DEG C at 15-30 DEG C, the centrifugal 15min of 12,000g.

By upper water phase transition in another clean EP pipe, add 0.5ml Virahol, room temperature leaves standstill 10min, 2-8 DEG C, the centrifugal 10min of 12,000g.

Remove supernatant, add 1ml75% washing with alcohol RNA and precipitate, vibrator mixes, the centrifugal 5min of 2-8 DEG C, 12,000g.

With without RNase water or the resuspended RNA precipitation of 0.5%SDS solution, repeatedly blow and beat several times with rifle head, at 55-60 DEG C, leave standstill 10min.-20 DEG C of preservations after survey concentration.

Denaturing formaldehyde agarose electrophoresis, determines extracting RNA integrity and DNA pollution situation.

2) P19 and Ebi3ORF sequence in RT-PCR amplification P19/Ebi3 mixture

Table 2RT reaction system (the first step): about 20min

	Volume
		RNA extract	5μl
Radome primer	2μl
		RNAsin	0.5μl

Ice bath is put into immediately after 65 DEG C of 15min

Table 3RT reaction system (second step): about 2h

	Volume
		RNAsin	0.5μl
10mM dNTP	1μl
		5 × RT damping fluid	4μl
25mM MgCl 2	4μl
		AMV reversed transcriptive enzyme	3μl

37 DEG C of 1.5h; 94 DEG C of 5-10min; Reactant is stored in-20 DEG C or carry out PCR

Table 4PCR reaction system: about 4.5h

	Volume
		25mM MgCl 2	2μl
10 × PCR damping fluid	5μl
		10mM dNTP	1μl
Upstream and downstream primer 10pmol/ μ l	2.5μl×2
		CDNA template	2.5μl
ddH 2O	34μl
		Taq enzyme	0.5μl
Light paraffinic oil	50μl

94℃2min，55℃1min，72℃2min

94 DEG C of 45s, 55 DEG C of 40s, 72 DEG C of 1min, 30 circulations

72℃10min，4℃5min。

Preserve or electrophoresis for-20 DEG C

Electrophoresis detection P19 and Ebi3 gene fragment

3) structure of recombinant expression vector

In P19/Ebi3 mixture, P19 and Ebi3ORF is inserted on pReceiver-Lv18 and pEZ-LV122X carrier respectively by EcoRI and XhoI restriction enzyme site

Table 5EcoRI and XhoI double digestion time and system thereof:

	Volume
		EcoRI	1μl
XhoI	1μl
		10×Buffer	2μl
Plasmid or PCR primer	5μl
		ddH 2O	11μl

37 DEG C of reaction 20h, with the purification column kits double digestion product of TAKARA.

Enzyme is cut, reclaim after PCR primer and the connection of carrier

Transform:

A, full dose (10 μ l) are added in 100 μ lJM109 competent cells, place 30min in ice.

After b, 42 DEG C of heating 45s, then place 1min in ice.

C, add 890 μ lAMP Negative media, 37 DEG C of shaking culture 60min.

D, get 100 μ l bed boards.

E, through PCR and enzyme cut qualification positive colony for order-checking.

3, the abduction delivering of mouse restructuring P19, Ebi3, P19/Ebi3 mixture

P19/pReceiver-Lv18 and Ebi3/pEZ-LV122X expression vector cotransfection Chinese hamster ovary celI

1) 0.5-2 × 105 cell is inoculated in 24 well culture plates, and adds 500 μ l antibiotic-free substratum, cultivate 24h.

2) P19/pReceiver-Lv18 and Ebi3/pEZ-LV122X is joined (or other serum free medium) in the low blood serum medium of 50 μ l serum-free Opti-MEMI to dilute, and mix gently.

3) LipofectamineTM2000 mixed gently is got corresponding amount to join in Opti-MEM substratum and dilute, left at room temperature 5min.

4) diluent of LipofectamineTM2000 and DNA is mixed.Mixing also at room temperature leaves standstill 20min gently.

5) suck the substratum in culture plate, and wash twice with serum free medium.

6) mixture 100 μ l is added culture hole, and the culture hole that rocks back and forth makes to be evenly distributed.

7) after transfection 24h, cell is transferred in fresh growth medium with 1:10 (or higher extent of dilution).

8) Chinese hamster ovary celI of coexpression P19/Ebi3 in Selective agar medium Puromycin (P19/pReceiver-Lv18 carrier with) and Neomycin (Ebi3/pEZ-LV122X carrier with).

9) Chinese hamster ovary celI of coexpression P19/Ebi3 is in 37 DEG C, CO ₂cultivating 48-72h in incubator, collecting supernatant with carrying out protein purification.

4, immunoprecipitation and immunoblotting assay

Experimentation as described in Example 1

Three, result

As shown in Figure 3, mouse IL-12 family cytokine subunit P19 and Ebi3 can form P19/Ebi3 mixture to results of immunoblot analysis.

Embodiment 3 mouse P19/Ebi3 complex purification and Mass Spectrometric Identification

One, method

1, the coexpression of mouse Ebi3-Flag/P19-His

Experimentation is with reference to embodiment 2.

2, the purifying of Ebi3-Flag/P19-His

1) nickel post process

1ml nickel post is sucked dead level analyses in pipe, adds level pad 3ml after liquid stream is wherein complete.

2) 10ml supernatant is added on equilibrated nickel post, and the sample crossing post is repeated loading 3 times, get 20 μ l cross post after sample, in order to running glue.

3, coomassie brilliant blue staining

1) albumen after purifying is carried out SDS-PAGE, experimentation as described in Example 1.Soak at least 30min in stationary liquid.

2) gel after fixing is soaked 10min in the staining fluid (0.29g Xylene Brilliant Cyanine G is dissolved in 250ml destainer, uses front to stir change and is heated to 60 DEG C) of heat, then with distilled water by gel drip washing once.

3) repeatedly converting destainer (adding distil water is to 1L for 250ml ethanol, 80ml Glacial acetic acid), until gel background purifies, is accelerate decolouring, can temperature slightly; Each step is all placed on shaking table and operates above.

4) chap after preventing gel drying, the gel sloughing background colour soaks 30min in conserving liquid.Then gel is put on a glass, then soak glassine paper with conserving liquid and encase gel and at room temperature dry.

4, Mass Spectrometric Identification

Two, result

As shown in Figure 4, (left figure), although there is foreign protein band, P19/Ebi3 purity is more than 70% for experimental result; (right figure) Mass Spectrometric Identification display P19-His molecular weight is about 18KD, and Ebi3-Flag molecular weight is about 36KD, and Ebi3-Flag/P19-His molecular weight is about 53.5KD.

Embodiment 4 mouse P19/Ebi3 mixture can elicit B cell STAT3 and STAT1 phosphorylation

One, material

P-STAT1, p-STAT3, p-STAT4, p-STAT5, p-STAT6, STAT1, STAT3, STAT4, STAT5, STAT6, β-actin (article No. is respectively 9167,9145,4134,9314,9364,9172,4904,2653,9363,9362,4967) is bought from CellSignalTechnology company

Two, method

1, the expression and purification of Ebi3-Flag, P19-His, Ebi3-Flag/P19-His albumen

2, B220 immunological magnetic bead sorting B cell

1) immunomagnetic beads is cleaned

Resuspended immunomagnetic beads (vortex is no less than 30s) in bottle;

The immunomagnetic beads of transfer appropriate volume is in pipe;

Add isopyknic dissociating buffer or at least 1ml, resuspended;

Pipe is placed in magnet 1min, discards supernatant;

Pipe is removed from magnet, with and above-mentioned steps in the resuspended washed magnetic bead of dissociating buffer of same volume.

2) sample is prepared

Prepare individual cells suspension (lymphoid organ), use dissociating buffer to make the final concentration of cell be 1 × 10 ⁷individual/ml.

3) sorting of B cell

In pipe, add the cell of 1ml, then add 25 μ l steps 1) in immunomagnetic beads;

Hatch 20min at 4 DEG C, be placed on shaking table and jiggle;

Pipe is placed in magnet 2min;

Discard supernatant carefully;

Pipe is taken out from magnet, adds 1ml dissociating buffer wherein, blow and beat 2-3 time (or vortex 2-3s), then this pipe is placed in magnetic field 2min, discard supernatant carefully;

Repeat previous step at least one times, clean the B cell be connected with magnetic bead.

3, the priming reaction of B cell

LPS stimulates B cell

Prepare the suspension of single B cell, cultivating adjustment B cell density with RPMI1640 perfect medium is 1 × 10 ⁶individual/ml, joins in 96 orifice plates, adds 10ug/mlLPS, cultivates 72-96h for 37 DEG C.

4, immunoblotting assay

Three, result

As shown in Figure 5, the Ebi3-Flag/P19-His albumen of purifying has biologic activity to experimental result, and it can stimulate the phosphorylation of STAT1, STAT3 in B cell effectively.

The B cell high expression level P19/Ebi3 of embodiment 5LPS activation

One, material

Anti-P19 antibody purchased from eBioscience company (article No. 50-7023-82), anti-P28 antibody purchased from eBioscience company (article No. 12-7285-80), anti-P35 antibody purchased from eBioscience company (article No. 50-7352-80), anti-P40 antibody purchased from eBioscience company (article No. 12-7123-81), anti-Ebi3 antibody purchased from R & D company (article No. IC18341C)

Two, method

1, LPS stimulates B cell

2, Fc acceptor is blocked: (for eliminating nonspecific combination dyeing)

Use the antibody (CD16/32) of the FcII/III acceptor of purifying, according to 1 μ g/10 ⁶the consumption of cell, hatches 15min for 4 DEG C in dye solution, after PBS cleaning, directly carries out next step dyeing.

3, intracellular elements dyeing and detection

1) fixing: to add ICFix/PermBuffer100 μ l, mixing, incubated at room 20min.

2) wash 1 time (2000rpm, 10min) with 1 × PermWashBuffer1ml, topple over rear filter paper and blot mouth of pipe liquid.

3) rupture of membranes: add 1 × PermWashBuffer1ml, mixing, hatches 45min for 4 DEG C.

4) centrifugal (2000rpm, 10min), topples over rear filter paper and blots mouth of pipe liquid.

5) add cytokine antibodies, hatch 45min for 4 DEG C.

6) wash 1 time, after toppling over 1 × PermWashBuffer1ml, add 300 μ lPBS re-suspended cells.

7) upper machine testing.

Three, result

As shown in Figure 6, the B cell of LPS activation expresses a small amount of P28, P35, P40 to experimental result, and high expression level P19/Ebi3.

The P19/Ebi3 of the B cell energy secreting high levels of embodiment 6LPS activation

One, material

Purchased from the anti-Ebi3 antibody (article No. sc-32869) of SantaCruzBiotech company, 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)

Two, method

1, the activation of B cell

2, ELISA experiment

1) bag is by process (attention arranges blank, negative control)

Wrap antigen used by diluted to suitable 10 μ g/ml, every hole antigen adds 100 μ l, is placed in 37 DEG C, and 4h or 4 DEG C spends the night; Can wrap by BSA solution or only add coating buffer as blank.

2) sealase mark reacting hole

Discard liquid in hole, every hole adds 200 μ l confining liquids, and 37 DEG C of wet boxes are closed 6h or 4 DEG C and spent the night, and terminates rear washings washing 3-5 time, and pats dry gently on thieving paper.

3) detected sample is added

The measuring samples 100 μ l adding certain dilution has wrapped in the reacting hole of quilt in above-mentioned, adds PBST as blank.Put 37 DEG C, hatch 45min.Then wash 3-5 time, and pat dry gently on thieving paper.

4) enzyme labelled antibody is added

In each reacting hole, add enzyme labelled antibody (extent of dilution after titration: more than 1:1000 dilutes) the 100 μ l of diluted fresh.Hatch 45min for 37 DEG C, wash 3 times, and pat dry gently on thieving paper.

5) substrate solution (matching while using) is added

The tmb substrate solution 100 μ l of Extemporaneous is added, 37 DEG C, 10 ~ 30min in each reacting hole.

6) termination reaction

2M sulfuric acid 50 μ l is added, determination experiment result in 20min in each reacting hole.

7) result judges

After 450nm is with blank control wells zeroing, survey each hole OD value by Bio-Rad microplate reader, if be greater than 2.1 times of the negative control OD value of regulation, be the positive (size of numerical value is determined according to concrete testing requirement).

3, double crush syndrome experiment

With reference to above-mentioned experimental procedure.

Three, result

As shown in Figure 7, the B cell that (left figure) LPS activates secretes a small amount of P28, P35, P40 to experimental result, and P19, Ebi3 of secreting high levels; The B cell that (right figure) LPS activates secretes a small amount of IL-23, IL-27, and secreting high levels P19/Ebi3.

Embodiment 7 is to plasma cell differentiation (GL7 ⁺) B cell high expression level P19/Ebi3

One, method

Experimental implementation is with reference to the method for embodiment 5.

Two, result

As shown in Figure 8, from figure, we can find out to plasma cell differentiation (GL7 experimental result ⁺) B cell high expression level P19/Ebi3.

To plasma cell differentiation (GL7 in embodiment 8 systemic lupus erythematous (SLE) mouse model ⁺) B cell and plasmocyte (CD138 ⁺) cell high expression level P19/Ebi3

One, material

The spontaneous pattern mouse of systemic lupus erythematous: female MRL/MpJ/lpr/lpr (MRL/lpr) mouse is purchased from Nanjing model animals institute; MRL/MpJ/+ /+(MRL/++) mouse is purchased from Institute of Basic Medical Sciences of medical courses in general institute.

Two, method

To plasma cell differentiation (GL7 ⁺) B cell, plasmocyte (CD138 ⁺) acquisition and counting

1, the acquisition of systemic lupus erythematous Mouse spleen cells

1) get mouse spleen, put into the culture dish filling 5mlHanks liquid.

2) grind pressure spleen (a good luck direction) gently, draw splenocyte suspension with suction pipe, put into test tube.

3) cell counting under mirror: 40 times of camera lenses, observes splenocyte.

2, lymphocytic sorting

1) splenocyte (splenocyte: diluent=1:2) is diluted.

2) in centrifuge tube, add Ficoll, slowly add the peripheral blood (Ficoll: dilution splenocyte=1:2) of dilution along the tube wall tilted, 20 DEG C, 1500rpm, centrifugal 30min.

3) move in another test tube along tube wall periphery gentle aspiration PBMC layer.

4) add enough diluents fully to wash, the centrifugal 10min of 1800rpm, abandons supernatant.

5) repeated washing once, and the centrifugal 10min of 1400rpm, abandons supernatant.

6) namely appropriate substratum re-suspended cell is mouse spleen lymphocyte suspension.

3, lymphocytic counting

Diluting cells, tally counts under being put into microscope.

4, cell streaming technical Analysis GL7 ⁺and CD138 ⁺cell

1) in each flow cytometer detection pipe, add the good primary antibodie of appropriate pre-dilution (anti-B220, GL7, CD138 antibody of fluorescent mark); The contrast agents of amount identical with antibody is added in blank tube or Isotype control pipe.Then each pipe lucifuge ice bath or hatch 30min in 4 DEG C of refrigerators.

2) at least 2mlCellStainingBuffer is added, then abandoning supernatant after the centrifugal 5min of 350g; Repeated washing twice.

3) use 0.5mlCellStainingBuffer re-suspended cell, hatch 3-5min on ice.

5, upper machine testing analytical results.

Two, result

Experimental result as shown in Figure 9, to plasma cell differentiation (GL7 in systemic lupus erythematous (SLE) mouse model ⁺) B cell and plasmocyte (CD138 ⁺) cell and high expression level P19/Ebi3 to plasma cell differentiation (GL7 ⁺) B cell and plasmocyte (CD138 ⁺) cell number significantly raises.

The anti-P19/Ebi3 polyclonal antibody preparation of embodiment 9 and qualification

One, method

1, the preparation and purification of P19/Ebi3

Experimental implementation is with reference to embodiment 3.

2, the preparation of polyclonal antibody

1) selection of immune animal: 10 male Balb/c mouse of 7-9 health in age in week.

2) immunogen: use the P19/Ebi3 mixture through ni-sepharose purification.Each immune 100-200 μ g.

3) after mouse cultivates 3 ~ 4 days in new environment, 0.1ml tail vein is got as negative control.

4) immunity of mouse

Back multi-point injection method immune mouse is adopted after mixing with complete Freund's adjuvant equal-volume with the P19/Ebi3 mixture of purifying, per injection 100 μ l, reinforced immunological mouse (difference is injected in above-mentioned position) after mixing with incomplete Freund's adjuvant equal proportion with described mixture after 2 weeks, according to identical mode reinforced immunological 2-3 time again, after 1 week, tail venous blood sampling detects antibody titer.

5) separation of serum

Centrifuge tube is positioned over 2h in 37 DEG C of baking ovens, 4 DEG C are spent the night, the centrifugal 10min of 10000RCF.NaN is added in serum ₃to final concentration 0.02% ,-20 DEG C of preservations after packing.

6) purifying (antigen affinity chromatography) of antibody

Crosslinked: to use Na ₂cO ₃-NaHCO ₃system, 10mg/mlP19/Ebi3 antigen is connected to beads, room temperature reaction 4h.The beads being combined with antigen is filled post.In order to ensure joint efficiency, close with containing amino micromolecular compound urea, after having closed, removing confining liquid, washes post three times with PBS.

The assessment connecting effect uses Coomassie Brilliant Blue (Bradford method).

Cross post: the serum of anticipating (the centrifugal 5min of 10,000g, by 0.45 μm of membrane filtration) is diluted one times with PBS, then add and connect in good antigen pillar.

Wash-out: with HCl (containing the 150mMNaCl) wash-out of about pH2.5.A ultraviolet monitoring instrument detects and flows out component so that immediately monitoring, and complete in low temperature elution process is most, the component under wash-out should be placed on ice chest, the composition Na after eluting ₂hPO ₄damping fluid.

Antibody is preserved: the antibody after purifying is placed in relatively close to the buffer system (PBS+0.02% sodium azide+5%BSA) of body fluid ,-20 DEG C of preservations.

3, the qualification of polyclonal antibody

Experimentation is with reference to embodiment 6

Two, result

As shown in Figure 10, the polyclonal antibody that P19/Ebi3 mixture immune mouse obtains, can effective anti-P19 albumen, and effect is better than the antibody effects of positive control IL-23P19 for experimental result.

The anti-P19/Ebi3 polyclonal antibody of embodiment 10 reduces systemic lupus erythematous mouse retention albumen

One, method

1,16 test tubes are got, 1 flag is blank, 3 give over to unknown sample, all the other test tubes are divided into two groups by order in table 6, add sample, water and reagent respectively, namely add to each test tube respectively with the standard protein solution of 1.0mg/ml: 0,0.01,0.02,0.04,0.06,0.08,0.1ml, then add to 0.1ml with deionized water.Add 5.0ml Coomassie brilliant blue G-250 reagent in last each test tube respectively, often add a pipe, mix on vortex mixer immediately (noting too inviolent, in order to avoid produce a large amount of bubble and be difficult to eliminate).The 8th, 9,10 pipes during the application of sample amount of unknown sample sees the following form.

The application of sample amount of the different test tube of table 6

2, after adding reagent 2-5min, measure the absorbance value A595 of each sample at 595nm place with cuvette on spectrophotometer, blank is No. 1 test tube, i.e. 0.1mlH ₂o adds 5.0mlG-250 reagent.Attention: can not use quartz colorimetric utensil (because not easily washing away dyeing), available plastics or glass cuvette, the ethanol immediately with a small amount of 95% after using swings to be washed, to wash away dyeing.Plastics cuvette never can soak with ethanol or acetone for a long time.

Two, result

As shown in figure 11, the 7th day time, anti-P19/Ebi3 Anti-TNF-α physical efficiency reduces systemic lupus erythematous mouse retention protein content to experimental result significantly.

The anti-P19/Ebi3 polyclonal antibody of embodiment 11 reduces the enlargement of systemic lupus erythematous mice spleen

One, method

1, anti-P19/Ebi3 polyclonal antibody systemic lupus erythematosus mouse

1) selection of laboratory animal and grouping

The female systemic lupus erythematous MRL/lpr mouse 20 at 8 monthly ages of morbidity, PBS treatment group 10; Anti-P19/Ebi3 Antybody therapy group 10.

2) preparation of polyclonal antibody

Adopt immune mouse and obtain the anti-P19/Ebi3 polyclonal antibody of high-titer through affinity chromatography method, using PBS to be mixed with 4mg/ml.

3) polyclonal antibody treatment disease mice is used

Every injected in mice 0.1ml antibody or PBS, inject twice weekly, altogether injection 4 times.Latter 1 week for the treatment of, detects Antybody therapy effect.

2, obtain the spleen tissue of experiment mice, compare size

Two, result

Experimental result as shown in figure 12, adopts anti-P19/Ebi3 Antybody therapy systemic lupus erythematous mouse, has fundamentally recovered splenomegaly little, contributed to the recovery of splenic function like this.

The anti-P19/Ebi3 polyclonal antibody of embodiment 12 reduces systemic lupus erythematous mouse antibodies level

One, method

1, anti-P19/Ebi3 polyclonal antibody systemic lupus erythematosus mouse

2, the detection (double crush syndrome method) of antibody horizontal in Mice Body

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 to put in 4 DEG C of refrigerators 3 ~ 4h or spend the night.

3) after blood coagulation blood clot retraction, the centrifugal 10min of 4000rpm.

4) get supernatant in clean centrifuge tube, be stored in-20 DEG C or add sanitas and put in 4 DEG C of refrigerators and save backup.

5) IgM and IgGELISA detects:

Bag quilt: be buffered liquid with the bag of 0.05MPH9 and will catch IgM or IgG antibody to be diluted to protein content be 1 ~ 10 μ g/ml.In the reacting hole of each polystyrene board, add 100 μ l, 4 DEG C are spent the night.Discard solution in hole, wash 3 times with lavation buffer solution, each 3min.

Application of sample: the measuring samples 100 μ l adding certain dilution in above-mentioned wrapped by reacting hole in, put 37 DEG C and hatch 1h, then wash.(doing blank well, negative control hole and Positive control wells) simultaneously.

Add enzyme labelled antibody: in each reacting hole, the enzyme mark adding diluted fresh detects IgM or IgG antibody (extent of dilution after titration) 100 μ l.Hatch 0.5 ~ 1h for 37 DEG C, washing.

Add substrate solution colour developing: the tmb substrate solution 100 μ l adding Extemporaneous in each reacting hole, at 37 DEG C, react 10 ~ 30min.

Termination reaction: add 2M sulfuric acid 50 μ l in each reacting hole.

Survey OD value: on ELISA detector, in 450nm place, to survey each hole OD value after blank control wells zeroing, if be greater than 2.1 times of the negative control OD value of regulation, be the positive.

Two, result

Experimental result as shown in figure 13, adopts anti-P19/Ebi3 Antybody therapy systemic lupus erythematous mouse can reduce antibody horizontal in Mice Body significantly.

The anti-P19/Ebi3 polyclonal antibody of embodiment 13 reduces systemic lupus erythematous mouse lymphocyte number

One, method

1, anti-P19/Ebi3 polyclonal antibody systemic lupus erythematosus mouse

2, the detection of immunocyte in Mice Body

Experimental procedure is with reference to embodiment 8, and during cell streaming technical Analysis, the primary antibodie added in each flow cytometer detection pipe is fluorescent mark anti-CD11b, Gr-1, CD3, B220, GL7, IgG, IgM antibody.

Two, result

Experimental result as shown in figure 14, adopts anti-P19/Ebi3 Antybody therapy systemic lupus erythematous mouse effectively can reduce innate immune cells as neutrophil leucocyte (CD11b ⁺gr-1 ⁺), scavenger cell (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 just for understanding method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also will fall in the protection domain of the claims in the present invention.

Claims (10)

1. the P19/Ebi3 mixture be separated, is characterized in that, the heterodimer that described mixture is made up of P19 and Ebi3 albumen.

2. a preparation method for P19/Ebi3 mixture according to claim 1, comprises the following steps:

A () cultivates host cell;

B () isolates P19/Ebi3 mixture from culture.

3. an antibody, described antibodies specific identification and/or in conjunction with P19/Ebi3 mixture according to claim 1.

4. antibody according to claim 3, is characterized in that, described antibody is polyclonal antibody.

5. prepare a method for the antibody described in claim 3 or 4, it is characterized in that, described method comprises: with mixture immune animal according to claim 1, isolates the antibody that specificity resists mixture according to claim 1 in the animal body after immunity.

6.P19/Ebi3 mixture suppresses B cell to the application in plasma cell differentiation medicine in preparation.

7. application according to claim 6, is characterized in that, described plasmocyte is the GL7 broken up by B cell ⁺plasmocyte.

8.P19/Ebi3 mixture is preparing the application in diagnositc system lupus erythematosus product.

9.P19/Ebi3 complex inhibitor is preparing the application in medicament for treating systemic lupus erythematosus.

10. application according to claim 9, is characterized in that, described complex inhibitor is the antibody described in claim 3 or 4.