CN103275222A - Monoclonal antibody for blocking 12p40 function of interleukin as well as coding gene and application thereof - Google Patents
Monoclonal antibody for blocking 12p40 function of interleukin as well as coding gene and application thereof Download PDFInfo
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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 the 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), perhaps NK cell stimulating factor (natural killer cell stimulation factor, NK SF), be a member in the interleukin family.It at first is that Wistar by the U.S. is from Epstein-Barr virus(EB virus) find the nutrient solution of transfection people B lymph matricyte system RPMI-8866, with its called after NKSF, be to be identified from the B clone NC37 nutrient solution that Epstein-Barr virus transforms by Hoffman research group subsequently, and name CLMF, find that NKSF and CLMF are with a kind of cytokine behind treated complete purifying both and the clone gene, and name and be IL-12.
Interleukin 12 is mainly by dendritic cell, scavenger cell, and 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 static or activated T cell and NK cell generation interferon-gamma (IFN-γ); Promote Th0 to the differentiation of Th1.Promote Th1 emiocytosis IFN-γ and IL-2, the mediated cell immunne response; Inducing T cell and NK cell produce IFN-γ; Increase the extramedullary hemopoiesis function; IL-12 has anti-angiogenic formation effect in vivo, and it does not directly act on vascular endothelial cell, by inducing the generation of IFN-γ, thereby suppresses vasculogenesis, and the effect of this kind angiogenesis inhibitor helps to suppress growth of tumor and transfer.IL-12 has all played the part of epochmaking role in the early stage non-specific immunity of body and the adaptive immunity process of 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 immunity of organism to regulate unbalance again and causes autoimmune disease.Thinking that in the past autoimmunization is to be mediated by antibody and immunocomplex, and think particularly organizing specific systemic autoimmune of autoimmunization now, is cell-mediated or be T cell dependency by T.Studies show that 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, so that IL-12 plays a part is very important in the developing of this type of disease.
IL-12 induces and promotes cell-mediated autoimmunization, and this fact is confirmed in non-non-insulin-dependent diabetes mellitus (NOD) mouse model, experimental colitis, collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis (EAU) animal model widely.The pathological model of above disease equal reproducible not in the mouse of IL-12 deficient mice or IL-12 antibody neutralization.And EAE takes place in the mouse that the IL-12 neutralizing antibody can prevent inheritance to accept the myelin basic protein primed lymphocyte.
IL-12 shows as in the autoimmunization effect in the process of inducing: (1) promotes antigen specific T h1 cytodifferentiation propagation and produces the various kinds of cell factor, strengthen the immune response of Th1 type.Known Th1 plays a crucial role in the process of bringing out 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 the various active medium, the cytotoxicity of enhancing immunity active cells causes 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, and representing animal model is MRL
Lpr/LprWith the NZB mouse model.The expression level of IL-12 increases in lupus nephritis, and the IL-12 that increases stimulates NK cell, T cell to produce IFN-γ, activates nitricoxide synthase (NOS) and produces NO, and the result causes the pathological lesion of kidney.
IL-12 autoimmune induce and immunoreactive keeping aspect play a part very importantly, therefore block generation or the signal conduction of IL-12, generation and the development of autoimmune disease all can be prevented or stop to any one link.
IL-12 has unique heterodimer structure, is by the covalently bound glycosylation peptide chain that forms of disulfide linkage (relative molecular mass is 75000 dalton) by p40 and two subunits of p35.Its heavy chain (p40) is made up of 306 amino acid, comprises 10 cysteine residues and 4 potential glycosylation sites, and light chain (p35) is made up of 197 amino acid, comprises 7 cysteine residues and 3 potential glycosylation sites.Specificity can be used as the blocker of IL-12 path in conjunction with the monoclonal antibody of p40 subunit, thereby becomes a kind of novel drugs of autoimmune disease (for example patchiness psoriatic) treatment.
Summary of the invention:
The purpose 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 as the 20th of SEQ ID NO.3 to shown in 468, the aminoacid sequence of described light chain as the 20th of SEQ ID NO.4 to shown in 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, comprising that coding has as the 20th of SEQ ID NO.3 and has the 20th nucleotide sequence to the light chain of aminoacid sequence shown in 233 as SEQ ID NO.4 to the nucleotide sequence of the heavy chain of aminoacid sequence shown in 468 and coding.
The nucleotide sequence of described encoding heavy chain preferably as the 58th of SEQ ID NO.1 to the nucleotide sequence shown in 1407.
The nucleotide sequence of described coding light chain preferably as the 58th of SEQ ID NO.2 to the nucleotide sequence shown in 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 the p40 antigen-specific, medium effective concentration EC50 is 0.03992nM, therefore can be with the blocker of monoclonal antibody Ab123FR1 of the present invention as the IL-12 path, thus a kind of novel drugs of autoimmune disease (for example patchiness psoriatic) treatment become.
Description of drawings:
Fig. 1 is with the bacterium colony PCR checking electrophorogram behind the transformed into escherichia coli DH5a behind cDNA insertion carrier pcDNA3.1/myc-his (-) A of p40;
Fig. 2 is the aminoacid sequence after the reading frame of the p40 gene of C end band his6 label is translated, the wherein abbreviation of alphabetical represented amino acid;
Fig. 3 is the SDS-PAGE figure of the antigen p40 albumen of the band his behind the purifying;
Fig. 4 is that cDNA that the cDNA of heavy chain inserts the bacterium colony PCR checking electrophorogram behind the transformed into escherichia coli DH5a behind carrier pcDNA3.1/myc-his (-) A and light chain inserts the bacterium colony PCR checking electrophorogram behind the transformed into escherichia coli DH5a behind carrier pcDNA3.1/myc-his (-) A;
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 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 shown in SEQ ID NO.4;
Fig. 7 is the SDS-PAGE figure of the antibody A b123FR1 behind the purifying;
Fig. 8 is the medium effective concentration EC50 test pattern of P40 antibody A B123FR1.
Embodiment:
Following examples are to further specify of the present invention, rather than limitation of the present invention.
Embodiment 1:
One, the structure of p40 screening antigen presentation carrier
1. buy the cDNA(SC124116 of p40 from Beijing Aureal Dongyuan County company, IL12B (NM_002187) human cD NA clone) as the gene source of antigen p40.
2. be masterplate with SC124116, with F1(5 '-TCTAGAGCCGCCACCATGTGTCACCAGCAGTTG-3 '), R1 (5 '-GTGGTGGTGGTGATGACTGCAGGGCACAGATGC-3 ') be primer, carry out first round pcr amplification, with 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, behind the p40 gene, add the his6 purification tag, and at two ends introducing Xba I, two restriction enzyme sites of Bam H I.Reclaim the amplified production that pcr amplification is taken turns in test kit (TIANGEN DP209-03) recovery second with sepharose.
3. the pcr amplification product of Hui Shouing and carrier pcDNA3.1/myc-his (-) A(are available from Invitrogen) respectively through restriction enzyme Xba I, BamH I (available from NEB company) double digestion, reclaim test kit (TIANGEN DP209-03) recovery enzyme with sepharose and cut product, utilize DNA Ligation Kit Ver.2.0 (TAKARA D6022S) to carry out ligation, pcr amplification product is inserted the Xba I of carrier pcDNA3.1/myc-his (-) A, between the BamH I restriction enzyme site.To connect sky, product transformed into escherichia coli competent cell DH5a(Beijing root biochemistry then, CB101), coat on the LB culture medium flat plate.
4. the mono-clonal on the picking LB culture medium flat plate carries out a small amount of cultivation with the LB liquid nutrient medium, with the primers F 3(5 ' on the carrier-CCAAAAACGAGGAGGATT-3 '), R3(5 '-CATATGCCTTCCGAGTGAGAG-3 ') carries out bacterium colony PCR checking (Fig. 1), contain the clone (clone 4 who inserts fragment, 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, the p40 gene of C end band his6 label has inserted carrier pcDNA3.1/myc-his (-) A(available from Invitrogen) in, aminoacid sequence after the reading frame translation is completely seen Fig. 2, obtain the his fusion expression vector thus, it is that pcr amplification product with step 2 has inserted among carrier pcDNA3.1/myc-his (-) A.
Two, expression and the purifying of p40 screening antigen (p40-his6)
1. will check order and identify correct his fusion expression vector 50ug transfection 5 * 10
7Individual 293f cell (available from invitrogen), 37 degree, 8%CO2,130rpm/min cultivated after 7 days, centrifugal collection supernatant.
With supernatant in the centrifugal 20min of 6000rpm, use the 0.2um membrane filtration again;
3. filtrate is changed liquid to Buffer A(50mM Tris through Millipore labscale ultrafiltration and concentration, 300mM NaCl, 10mM Imidazole, pH8.0) in, obtain concentrating the back sample;
4. concentrate the back sample and after 0.2um filters, go up sample to using PBS (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4,2mM KH2PO4, the 1ml HisTrap HP post that pH7.4) balance is good;
5. treat that intact back is washed with Buffer A on the sample, flow velocity 1ml/min, ultraviolet monitoring are level.
6. use 0-60%Buffer B(50mM Tris, 300mM NaCl, 500mM Imidazole, pH8.0), and 15 column volume linear elutions, flow velocity 1ml/min collects eluting peak, and carries out SDS-PAGE and detect (Fig. 3)
7. merge corresponding elution peak, and change liquid to PBS with the ultrafiltration and concentration pipe is concentrated, obtain antigen p40-his thus.
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 shown in SEQ ID NO.2, wherein the 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 in the cDNA of light chain 5 ' end interpolation EcoR I restriction enzyme site and Kozak sequence, add Hind III restriction enzyme site at 3 ' end, specifically be to connect GAATTCgccgccacc at 5 ' end, connect AAGCTT, the whole EcoR I restriction enzyme site that comprises at 3 ' end, the Kozak sequence, the sequence of the cDNA of light chain and Hind III restriction enzyme site can the direct labor be synthesized.And then its sequence of the cDNA(of synthetic heavy chain is shown in SEQ ID NO.1, wherein the 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 in the cDNA5 ' of heavy chain end interpolation EcoR I restriction enzyme site and Kozak sequence, add Hind III restriction enzyme site at 3 ' end, specifically be to connect GAATTCgccgccacc at 5 ' end, connect AAGCTT, the whole EcoR I restriction enzyme site that comprises at 3 ' end, the Kozak sequence, the sequence of the cDNA of heavy chain and Hind III restriction enzyme site can the direct labor be synthesized.
2. cDNA and carrier pcDNA3.1/myc-his (-) A(Invitrogen that will have the heavy chain of restriction enzyme site) respectively through restriction enzyme EcoR I, Hind III (available from NEB company) double digestion, reclaiming test kit (TIANGE N DP209-03) with sepharose reclaims enzyme respectively and cuts product, utilize DNA Ligation Kit Ver.2.0 (TAKARA D6022S) to carry out ligation, cDNA insertion carrier pcDNA3.1/myc-his (-) A(Invitrogen with heavy chain) EcoR I is between the Hind I II restriction enzyme site.To connect sky, product transformed into escherichia coli competent cell DH5a(Beijing root biochemistry then, CB101), coat on the LB flat board.The mono-clonal of transformed competence colibacillus cell DH5a on the picking LB flat board carries out a small amount of with the LB substratum and cultivates, with primers F 4(5 '-TAATACGACTCACTATAGGG-3 '), R4(5 '-TAGAAGGCACAGTC GAGG-3 ') carries out bacterium colony PCR checking (Fig. 4), to contain the clone that inserts 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 shows that the cDNA of the heavy chain of Ab123FR1 has inserted among carrier pcDNA3.1/myc-his (-) A, inserts correctly, and the aminoacid sequence after the reading frame translation is completely seen Fig. 5, obtains Ab123FR1 heavy chain expression carrier thus.
In like manner, cDNA and pcDNA3.1/myc-his (-) A(Invitrogen that will have the light chain of restriction enzyme site) respectively through restriction enzyme EcoR I, Hind III (available from NEB company) double digestion, reclaiming test kit (TIANGE N DP209-03) with sepharose reclaims enzyme respectively and cuts product, utilize DNA Ligation Kit Ver.2.0 (TAKARA D6022S) to carry out ligation, cDNA insertion carrier pcDNA3.1/myc-his (-) A(Invitrogen with light chain) EcoR I is between the Hind I II restriction enzyme site.To connect sky, product transformed competence colibacillus cell DH5a(Beijing root biochemistry then, CB101), coat on the L B flat board.The mono-clonal of transformed competence colibacillus cell DH5a on the picking LB flat board carries out a small amount of and cultivates, with primers F 4(5 '-TAATACGACTCACTATAGGG-3 '), R4(5 '-TAGAAGGCACAGTCGAGG-3 ') carries out bacterium colony P CR checking (Fig. 4), to contain the clone that inserts 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 shows that the cDNA of the light chain of Ab123FR1 has inserted among carrier pcDNA3.1/myc-his (-) A, and gene inserts correct, and the aminoacid sequence after the reading frame translation is completely seen Fig. 6, obtains the Ab123FR1 light chain expression vector thus.
Four, the expression of p40 antibody A b123FR1 and purifying
1. will check order and identify correct Ab123FR1 heavy chain expression carrier and light chain expression vector (1:1) 50ug cotransfection to 5 * 10
7In the individual 293f cell (available from invitrogen), 37 degree, 8%CO2,130rpm/min cultivated after 7 days, centrifugal collection supernatant.
2. with the centrifugal 20min of supernatant 6000rpm, and use the 0.2um membrane filtration, collect filtrate;
3. filtrate is gone up sample to using PBS (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4,2mM KH2PO4, the 1ml HiTrap protein A HP post that pH7.4) balance is good;
4. treat that intact back is washed with PBS on the sample, flow velocity 1ml/min, ultraviolet monitoring are 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 and detect (Fig. 7)
6. merge corresponding elution peak, and change liquid to PBS with the ultrafiltration and concentration pipe is concentrated, obtain antibody A b123FR1 thus.
Five, measure the medium effective concentration EC50 of P40 antibody A B123FR1 with indirect ELISA (enzyme-linked immunosorbent assay)
1. enzyme plate is hatched 2h for 37 ℃ with antigen p40-his bag quilt.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 is surveyed OD value (seeing Table 1) with microplate reader at the 450nm place.
Data transcription is carried out data analysis to software GraphPad Prism5, and calculating medium effective concentration EC50 is that 0.03992nM(sees Fig. 8).
Table 1:
Claims (5)
1. monoclonal antibody Ab123FR1 who blocks interleukin 12 p40 function, it is characterized in that, comprise heavy chain and light chain, the aminoacid sequence of described heavy chain as the 20th of SEQ ID NO.3 to shown in 468, the aminoacid sequence of described light chain as the 20th of SEQ ID NO.4 to shown in 233.
2. the gene of the monoclonal antibody Ab123FR1 of the blocking-up interleukin 12 p40 function of encoding, it is characterized in that, comprising that coding has as the 20th of SEQ ID NO.3 and has the 20th nucleotide sequence to the light chain of aminoacid sequence shown in 233 as SEQ ID NO.4 to the nucleotide sequence of the heavy chain of aminoacid sequence shown in 468 and coding.
3. the gene of the monoclonal antibody Ab123FR1 of coding according to claim 2 blocking-up interleukin 12 p40 function is characterized in that, the nucleotide sequence of described encoding heavy chain as the 58th of SEQ ID NO.1 to shown in 1407.
4. the gene of the monoclonal antibody Ab123FR1 of coding according to claim 2 blocking-up interleukin 12 p40 function is characterized in that, the nucleotide sequence of described coding light chain as the 58th of SEQ ID NO.2 to shown in 702.
5. the application of the described monoclonal antibody Ab123FR1 of claim 1 in the blocker of preparation IL-12 path.
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