CN103275222B - Monoclonal antibody for blocking 12p40 function of interleukin as well as coding gene and application thereof - Google Patents
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Abstract
The invention discloses a monoclonal antibody for blocking 12p40 function of interleukin as well as a coding gene and an application thereof. The monoclonal antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as the bit 20 to bit 468 of SEQ ID NO.3, and the amino acid sequence of the light chain is shown as the bit 20 to bit 233 of SEQ ID NO.4. The invention discloses a monoclonal antibody Ab123FR1 for blocking 12p40 function and a coding gene thereof. The monoclonal antibody Ab123FR1 can be combined with p40 antigen specificity, and the half effective concentration EC50 is 0.03992nM. Therefore, the monoclonal antibody Ab123FR1 can be used as a blocker of an IL-12 passage, and thus becomes a new drug for treating antoimmune diseases (such as plaque psoriasis).
Description
Technical field:
The invention belongs to Biochemistry and Molecular Biology field, be specifically related to a kind of monoclonal antibody and encoding gene and application of blocking interleukin 12 p40 function.
Background technology:
Interleukin 12 (Interleukin12, IL-12), have another name called cytotoxic lymphocyte maturation factor(CLMF) (cytotox-ic lymphocyte maturation factor, CLMF), or NKSF (natural killer cell stimulation factor, NK SF), be a member in interleukin family.It first the Wistar of the Shi You U.S. from Epstein-Barr virus(EB virus) find the nutrient solution of transfected with human B lymph matricyte system RPMI-8866, by its called after NKSF, the B clone NC37 nutrient solution that Shi You Hoffman research group transforms from Epstein-Barr virus subsequently, identify, and name CLMF, until Economical Purification after both and clone gene, find that NKSF and CLMF are same cytokines, and name as IL-12.
Interleukin 12 is mainly by dendritic cell, scavenger cell, bone-marrow-derived lymphocyte and some other antigen presenting cell (APC) produce, and can strengthen the cytotoxicity of NK cell (natural killer cell) and Tc cell (cytotoxic T cell); Stimulate T cell and NK Cells Interferon Production γ (IFN-γ) static or activation; Promote Th0 to the differentiation of Th1.Promote Th1 emiocytosis IFN-γ and IL-2, mediated cell immunne response; Inducing T cell and NK cell produce IFN-γ; Increase extramedullary hemopoiesis function; IL-12 has anti-angiogenic formation effect in vivo, and it does not directly act on vascular endothelial cell, by the generation of induction IFN-γ, thereby suppresses vasculogenesis, and the effect of this kind of angiogenesis inhibitor contributes to suppress growth and the transfer of tumour.IL-12 has all played the part of epochmaking role in the adaptive immunity process of the early stage non-specific immunity of body and antigen-specific subsequently, is a multi-functional immune-regulating factor.
On the one hand, IL-12 participates in antitumor, the antianaphylaxis of body, anti-infective process, and on the other hand, its excessive generation causes again Organism immunoregulation unbalance and cause autoimmune disease.Think that in the past autoimmunization is to be mediated by antibody and immunocomplex, and think now particularly organizing specific systemic autoimmune of autoimmunization, be cell-mediated by T or be T cell dependency.Research shows, the immunopathogenesis of many autoimmune diseases is all relevant with the generation of antigen specific T h1 cell, and IL-12 impels Th0 to the key cytokines of Th1 differentiation, therefore that IL-12 plays a part in the developing of this type of disease is very important.
IL-12 induces and promotes cell-mediated autoimmunization, and this fact is confirmed widely in Nonobese diabetes (NOD) mouse model, experimental colitis, collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveoretinitis (EAU) animal model.The pathological model of above disease equal reproducible not in the mouse of IL-12 deficient mice or IL-12 antibody neutralization.And there is EAE in the mouse that IL-12 neutralizing antibody can prevent inheritance to accept myelin basic protein primed lymphocyte.
The effect of IL-12 in autoimmunization Induction Process shows as: (1) promotes antigen specific T h1 cytodifferentiation to breed and produces cytokine profiles, strengthen the immune response of Th1 type.Known Th1 plays a crucial role in the process of bringing out the various autoimmune diseases such as EAE, EAU, CIA, and IL-12 monoclonal antibody or blocker all can weaken its Th1 type reaction, improve the state of an illness.(2) stimulate mononuclear macrophage to produce various active medium, strengthen the cytotoxicity of immunologically competent cell, cause autologous tissue's damage.(3) IL-12 also participates in antibody-mediated autoimmunization.Many research report IL-12 participate in the pathogenesis of systemic lupus erythematosus, represent that animal model is MRL
lpr/Lprwith NZB mouse model.In lupus nephritis, the expression level of IL-12 increases, and the IL-12 increasing stimulates NK cell, T cell to produce IFN-γ, activates nitricoxide synthase (NOS) and produces NO, and result causes the pathological lesion of kidney.
Therefore IL-12 is playing a part very importantly aspect autoimmune induction and immunoreactive maintaining, block generation or the signal conduction of IL-12, and generation and the development of autoimmune disease can be prevented or stop to any one link all.
IL-12 has unique heterodimer structure, is by the covalently bound glycosylation peptide chain forming of disulfide linkage (relative molecular mass is 75000 dalton) by p40 and two subunits of p35.Its heavy chain (p40) is comprised of 306 amino acid, comprises 10 cysteine residues and 4 potential glycosylation sites, and light chain (p35) is comprised of 197 amino acid, comprises 7 cysteine residues and 3 potential glycosylation sites.The monoclonal antibody of specific binding p40 subunit can be used as the blocker of IL-12 path, thereby becomes a kind of novel drugs of autoimmune disease (for example patchiness psoriatic) treatment.
Summary of the invention:
The object of this invention is to provide a kind of monoclonal antibody Ab123FR1 and encoding gene and application of blocking interleukin 12 p40 function.
The monoclonal antibody Ab123FR1 of blocking-up interleukin 12 p40 function of the present invention, it is characterized in that, comprise heavy chain and light chain, the aminoacid sequence of described heavy chain is as the 20th of SEQ ID NO.3 the to as shown in the of 468, and the aminoacid sequence of described light chain is as the 20th of SEQ ID NO.4 the to as shown in the of 233.
SEQ ID NO.3 and SEQ ID NO.4 the 1st to 19 amino acids sequences are signal peptide sequences.
The gene of a kind of monoclonal antibody Ab123FR1 of the blocking-up interleukin 12 p40 function of encoding, it is characterized in that, comprise that coding has as the nucleotide sequence of the 20th of SEQ ID NO.3 the heavy chain to aminoacid sequence as shown in the of 468, and coding has as the nucleotide sequence of the 20th of SEQ ID NO.4 the light chain to aminoacid sequence as shown in the of 233.
The nucleotide sequence of described encoding heavy chain is preferably as the 58th of SEQ ID NO.1 the to the nucleotide sequence as shown in the of 1407.
The nucleotide sequence of described coding light chain is preferably as the 58th of SEQ ID NO.2 the to the nucleotide sequence as shown in the of 702.
SEQ ID NO.1 and SEQ ID NO.2 the 1st to 57 bit base sequences are nucleotide sequences of coded signal peptide sequence.
The present invention also provides the application of monoclonal antibody Ab123FR1 in the blocker of preparation IL-12 path.
New discovery of the present invention a kind of monoclonal antibody Ab123FR1 and the encoding gene thereof that can block interleukin 12 p40 function, this monoclonal antibody Ab123FR1 can be combined with p40 antigen-specific, medium effective concentration EC50 is 0.03992nM, therefore blocker that can be using monoclonal antibody Ab123FR1 of the present invention as IL-12 path, thus a kind of novel drugs of autoimmune disease (for example patchiness psoriatic) treatment become.
Accompanying drawing explanation:
Fig. 1 is by the bacterium colony PCR checking electrophorogram transforming after cDNA insertion vector pcDNA3.1/myc-his (-) A of p40 after escherichia coli DH5a;
Fig. 2 is the aminoacid sequence after C end is translated with the reading frame of the p40 gene of his6 label, the wherein abbreviation of alphabetical represented amino acid;
Fig. 3 is the SDS-PAGE figure of the antigen p40 albumen with his after purifying;
Fig. 4 is that the bacterium colony PCR transforming after escherichia coli DH5a after cDNA insertion vector pcDNA3.1/myc-his (-) A of heavy chain verifies that the bacterium colony PCR transforming after escherichia coli DH5a after cDNA insertion vector pcDNA3.1/myc-his (-) A of electrophorogram and light chain verifies electrophorogram;
Fig. 5 is the aminoacid sequence after the reading frame of the cDNA of heavy chain is translated, the wherein abbreviation of alphabetical represented amino acid, and concrete sequence is as shown in SEQ ID NO.3;
Fig. 6 is the aminoacid sequence after the reading frame of the cDNA of light chain is translated, the wherein abbreviation of alphabetical represented amino acid, and concrete sequence is as shown in SEQ ID NO.4;
Fig. 7 is the SDS-PAGE figure of the antibody A b123FR1 after purifying;
Fig. 8 is the medium effective concentration EC50 test pattern of P40 antibody A B123FR1.
Embodiment:
Following examples are to further illustrate of the present invention, rather than limitation of the present invention.
Embodiment 1:
One, the structure of p40 screening antigen presentation carrier
1. from Aureal Dongyuan County, Beijing company, buy the cDNA(SC124116 of p40, IL12B (NM_002187) human cD NA clone) as the gene source of antigen p40.
2. take SC124116 as masterplate, with F1(5 '-TCTAGAGCCGCCACCATGTGTCACCAGCAGTTG-3 '), R1 (5 '-GTGGTGGTGGTGATGACTGCAGGGCACAGATGC-3 ') be primer, carry out first round pcr amplification, using the product of first round pcr amplification as template, with F2 (5 '-CCCAAGCGAGCTCTAGAGCCGC CACCATGT-3 '), R2 (5 '-CGCGGATCCTCAGTGGTGGTGGTGGTGATGACTG-3 ') as amplimer, carry out second and take turns pcr amplification, after p40 gene, add his6 purification tag, and at two ends introducing Xba I, two restriction enzyme sites of Bam H I.With sepharose, reclaim the amplified production that pcr amplification is taken turns in test kit (TIANGEN DP209-03) recovery second.
3. the pcr amplification product reclaiming and carrier pcDNA3.1/myc-his (-) A(are purchased from Invitrogen) respectively through restriction enzyme Xba I, BamH I (purchased from NEB company) double digestion, with sepharose, reclaim test kit (TIANGEN DP209-03) recovery enzyme and cut product, utilize DNA Ligation Kit Ver.2.0 (TAKARA D6022S) to carry out ligation, by the Xba I of pcr amplification product insertion vector pcDNA3.1/myc-his (-) A, between BamH I restriction enzyme site.Then will connect product and transform sky, competent escherichia coli cell DH5a(Beijing root biochemistry, CB101), coat on LB culture medium flat plate.
4. the mono-clonal on picking LB culture medium flat plate carries out a small amount of cultivation with LB liquid nutrient medium, with primers F the 3(5 '-CCAAAAACGAGGAGGATT-3 ' on carrier), R3(5 '-CATATGCCTTCCGAGTGAGAG-3 ') carry out bacterium colony PCR checking (Fig. 1), the clone who contains Insert Fragment (clone 4, clone 5 and clone 8) positive clone, the plasmid DNA of extracting positive colony, and send invitrogen to carry out sequence verification.
5. sequencing result shows, C end has inserted carrier pcDNA3.1/myc-his (-) A(purchased from Invitrogen with the p40 gene of his6 label) in, aminoacid sequence after reading frame translation is completely shown in Fig. 2, obtain thus his fusion expression vector, it is that the pcr amplification product of step 2 has been inserted in carrier pcDNA3.1/myc-his (-) A.
Two, the Expression and purification of p40 screening antigen (p40-his6)
1. order-checking is identified to correct his fusion expression vector 50ug transfection 5 * 10
7individual 293f cell (purchased from invitrogen), 37 degree, 8%CO2,130rpm/min cultivated after 7 days, centrifugal collection supernatant.
By supernatant in the centrifugal 20min of 6000rpm, then use 0.2um membrane filtration;
3. filtrate is changed liquid to Buffer A(50mM Tris, 300mM NaCl, 10mM Imidazole, pH8.0 through Millipore labscale ultrafiltration and concentration) in, concentrated rear sample obtained;
4. concentrated rear sample loading after 0.2um filters has extremely been used the good 1ml HisTrap HP post of PBS (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4,2mM KH2PO4, pH7.4) balance;
5. after complete on sample, with Buffer A, rinse, flow velocity 1ml/min, ultraviolet monitoring is level.
6. with 0-60%Buffer B(50mM Tris, 300mM NaCl, 500mM Imidazole, pH8.0), 15 column volume linear elutions, flow velocity 1ml/min, collects eluting peak, and carries out SDS-PAGE detection (Fig. 3)
7. merge corresponding elution peak, and change liquid to PBS with ultrafiltration and concentration pipe is concentrated, obtain thus antigen p40-his.
Three, the structure of p40 antibody A b123FR1 expression vector
1. its sequence of the cDNA(of the light chain of synthetic antibody Ab123FR1 is as shown in SEQ ID NO.2, wherein 1-57 bit base is the nucleotide sequence of coded signal peptide sequence, 58-699 is the base sequence of coding light chain, 700-702 is terminator), and add EcoR I restriction enzyme site and Kozak sequence at the cDNA of light chain 5 ' end, at 3 ' end, add Hind III restriction enzyme site, specifically at 5 ' end, connect GAATTCgccgccacc, at 3 ' end, connect AAGCTT, the whole EcoR I restriction enzyme site that comprises, Kozak sequence, the sequence of the cDNA of light chain and Hind III restriction enzyme site can direct labor be synthesized.And then its sequence of the cDNA(of synthetic heavy chain is as shown in SEQ ID NO.1, wherein 1-57 bit base is the nucleotide sequence of coded signal peptide sequence, 58-1404 is the base sequence of encoding heavy chain, 1405-1407 is terminator), and add EcoR I restriction enzyme site and Kozak sequence at the cDNA5 ' of heavy chain end, at 3 ' end, add Hind III restriction enzyme site, specifically at 5 ' end, connect GAATTCgccgccacc, at 3 ' end, connect AAGCTT, the whole EcoR I restriction enzyme site that comprises, Kozak sequence, the sequence of the cDNA of heavy chain and Hind III restriction enzyme site can direct labor be synthesized.
2. by the cDNA of the heavy chain with restriction enzyme site and carrier pcDNA3.1/myc-his (-) A(Invitrogen) respectively through restriction enzyme EcoR I, Hind III (purchased from NEB company) double digestion, with sepharose, reclaiming test kit (TIANGE N DP209-03) reclaims respectively enzyme and cuts product, utilize DNA Ligation Kit Ver.2.0 (TAKARA D6022S) to carry out ligation, by cDNA insertion vector pcDNA3.1/myc-his (-) A(Invitrogen of heavy chain) EcoR I, between Hind I II restriction enzyme site.Then will connect product and transform sky, competent escherichia coli cell DH5a(Beijing root biochemistry, CB101), coat on LB flat board.The mono-clonal of transformed competence colibacillus cell DH5a on picking LB flat board carries out a small amount of with LB substratum and cultivates, with primers F 4(5 '-TAATACGACTCACTATAGGG-3 '), R4(5 '-TAGAAGGCACAGTC GAGG-3 ') carry out bacterium colony PCR checking (Fig. 4), using the clone who contains Insert Fragment (clone 1 and clone 2) as positive colony, the plasmid DNA of extracting positive colony, and send invitrogen to carry out sequence verification.Sequencing result demonstration, the cDNA of the heavy chain of Ab123FR1 has inserted in carrier pcDNA3.1/myc-his (-) A, inserts correctly, and the aminoacid sequence after reading frame translation is completely shown in Fig. 5, obtains thus Ab123FR1 heavy chain expression carrier.
In like manner, by the cDNA of the light chain with restriction enzyme site and pcDNA3.1/myc-his (-) A(Invitrogen) respectively through restriction enzyme EcoR I, Hind III (purchased from NEB company) double digestion, with sepharose, reclaiming test kit (TIANGE N DP209-03) reclaims respectively enzyme and cuts product, utilize DNA Ligation Kit Ver.2.0 (TAKARA D6022S) to carry out ligation, by cDNA insertion vector pcDNA3.1/myc-his (-) A(Invitrogen of light chain) EcoR I, between Hind I II restriction enzyme site.Then will connect sky, product transformed competence colibacillus cell DH5a(Beijing root biochemical, CB101), coat on L B flat board.The mono-clonal of transformed competence colibacillus cell DH5a on picking LB flat board carries out a small amount of and cultivates, with primers F 4(5 '-TAATACGACTCACTATAGGG-3 '), R4(5 '-TAGAAGGCACAGTCGAGG-3 ') carry out bacterium colony P CR checking (Fig. 4), using the clone who contains Insert Fragment (clone 6 and clone 7) as positive colony, the plasmid DNA of extracting positive colony, and send invitrogen to carry out sequence verification.Sequencing result demonstration, the cDNA of the light chain of Ab123FR1 has inserted in carrier pcDNA3.1/myc-his (-) A, and gene inserts correct, and the aminoacid sequence after reading frame translation is completely shown in Fig. 6, obtains thus Ab123FR1 light chain expression vector.
Four, the Expression and purification of p40 antibody A b123FR1
1. order-checking is identified to correct Ab123FR1 heavy chain expression carrier and light chain expression vector (1:1) 50ug cotransfection to 5 * 10
7in individual 293f cell (purchased from invitrogen), 37 degree, 8%CO2,130rpm/min cultivated after 7 days, centrifugal collection supernatant.
2. by the centrifugal 20min of supernatant 6000rpm, and use 0.2um membrane filtration, collect filtrate;
3. filtrate loading is to using the 1ml HiTrap protein A HP post that PBS (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4,2mM KH2PO4, pH7.4) balance is good;
4. after complete on sample, with PBS, rinse, flow velocity 1ml/min, ultraviolet monitoring is baseline;
5. use 100%0.1M Citric (pH3.5) wash-out instead, flow velocity 1ml/min, collects eluting peak, and carries out SDS-PAG E detection (Fig. 7)
6. merge corresponding elution peak, and change liquid to PBS with ultrafiltration and concentration pipe is concentrated, obtain thus antibody A b123FR1.
Five, with indirect ELISA (enzyme-linked immunosorbent assay), measure the medium effective concentration EC50 of P40 antibody A B123FR1
1. enzyme plate is coated with antigen p40-his, hatches 2h for 37 ℃.Add 4 ℃ of overnight incubation of confining liquid.
2. the antibody A b123FR1 that adds gradient concentration is primary antibodie, 37 ℃ of incubation 30min.
3. use Goat Anti Human IgG, HRP(Jackson, catalog number (Cat.No.): 109-035-088) do two and resist, hatch 30min for 37 ℃.
4.TMB developer, surveys OD value (in Table 1) at 450nm place by microplate reader.
Data transcription is carried out to data analysis to software GraphPad Prism5, and calculating medium effective concentration EC50 is that 0.03992nM(is shown in Fig. 8).
Table 1:
Claims (5)
1. the recombinant antibodies Ab123FR1 in conjunction with the p40 subunit of interleukin 12, it is characterized in that, by heavy chain and light chain, formed, the aminoacid sequence of described heavy chain is as the 20th of SEQ ID NO.3 the to as shown in the of 468, and the aminoacid sequence of described light chain is as the 20th of SEQ ID NO.4 the to as shown in the of 233.
One kind coding recombinant antibodies Ab123FR1 claimed in claim 1 gene.
3. gene according to claim 2, is characterized in that, the nucleotide sequence of encoding heavy chain is as the 58th of SEQ ID NO.1 the to as shown in the of 1407.
4. gene according to claim 2, is characterized in that, the nucleotide sequence of coding light chain is as the 58th of SEQ ID NO.2 the to as shown in the of 702.
Recombinant antibodies Ab123FR1 claimed in claim 1 in preparation in conjunction with the application in the preparation of the p40 subunit of interleukin 12.
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