CN103275222B - 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
- Publication number
- CN103275222B CN103275222B CN201310180792.0A CN201310180792A CN103275222B CN 103275222 B CN103275222 B CN 103275222B CN 201310180792 A CN201310180792 A CN 201310180792A CN 103275222 B CN103275222 B CN 103275222B
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- ab123fr1
- seq
- interleukin
- heavy chain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 17
- 230000000903 blocking effect Effects 0.000 title abstract description 6
- 102000015696 Interleukins Human genes 0.000 title abstract description 3
- 108010063738 Interleukins Proteins 0.000 title abstract description 3
- 108010065805 Interleukin-12 Proteins 0.000 claims abstract description 36
- 102000013462 Interleukin-12 Human genes 0.000 claims abstract description 36
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 14
- 229940117681 interleukin-12 Drugs 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 3
- 101150084750 1 gene Proteins 0.000 claims 1
- 239000000427 antigen Substances 0.000 abstract description 10
- 102000036639 antigens Human genes 0.000 abstract description 10
- 108091007433 antigens Proteins 0.000 abstract description 10
- 201000010099 disease Diseases 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- 201000004681 Psoriasis Diseases 0.000 abstract 1
- 239000002547 new drug Substances 0.000 abstract 1
- 239000002299 complementary DNA Substances 0.000 description 17
- 108091008146 restriction endonucleases Proteins 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 13
- 239000000047 product Substances 0.000 description 10
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000012408 PCR amplification Methods 0.000 description 7
- 208000023275 Autoimmune disease Diseases 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- 102100030698 Interleukin-12 subunit alpha Human genes 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 210000000822 natural killer cell Anatomy 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 101000852992 Homo sapiens Interleukin-12 subunit beta Proteins 0.000 description 5
- 102100037850 Interferon gamma Human genes 0.000 description 5
- 108010074328 Interferon-gamma Proteins 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 101001010600 Homo sapiens Interleukin-12 subunit alpha Proteins 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- 229920002684 Sepharose Polymers 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 208000009386 Experimental Arthritis Diseases 0.000 description 3
- 101100395023 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) his-7 gene Proteins 0.000 description 3
- 230000001363 autoimmune Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 201000002491 encephalomyelitis Diseases 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 102000008299 Nitric Oxide Synthase Human genes 0.000 description 2
- 108010021487 Nitric Oxide Synthase Proteins 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 230000001185 psoriatic effect Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- VEEGZPWAAPPXRB-BJMVGYQFSA-N (3e)-3-(1h-imidazol-5-ylmethylidene)-1h-indol-2-one Chemical compound O=C1NC2=CC=CC=C2\C1=C/C1=CN=CN1 VEEGZPWAAPPXRB-BJMVGYQFSA-N 0.000 description 1
- KISWVXRQTGLFGD-UHFFFAOYSA-N 2-[[2-[[6-amino-2-[[2-[[2-[[5-amino-2-[[2-[[1-[2-[[6-amino-2-[(2,5-diamino-5-oxopentanoyl)amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-(diaminomethylideneamino)p Chemical compound C1CCN(C(=O)C(CCCN=C(N)N)NC(=O)C(CCCCN)NC(=O)C(N)CCC(N)=O)C1C(=O)NC(CO)C(=O)NC(CCC(N)=O)C(=O)NC(CCCN=C(N)N)C(=O)NC(CO)C(=O)NC(CCCCN)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 KISWVXRQTGLFGD-UHFFFAOYSA-N 0.000 description 1
- 208000032116 Autoimmune Experimental Encephalomyelitis Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 206010063827 Extramedullary haemopoiesis Diseases 0.000 description 1
- 102100036701 Interleukin-12 subunit beta Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 208000005777 Lupus Nephritis Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102000047918 Myelin Basic Human genes 0.000 description 1
- 101710107068 Myelin basic protein Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 229940121369 angiogenesis inhibitor Drugs 0.000 description 1
- 239000004037 angiogenesis inhibitor Substances 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 208000012997 experimental autoimmune encephalomyelitis Diseases 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000008807 pathological lesion Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 230000004862 vasculogenesis Effects 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310180792.0A CN103275222B (en) | 2013-05-15 | 2013-05-15 | Monoclonal antibody for blocking 12p40 function of interleukin as well as coding gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310180792.0A CN103275222B (en) | 2013-05-15 | 2013-05-15 | Monoclonal antibody for blocking 12p40 function of interleukin as well as coding gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103275222A CN103275222A (en) | 2013-09-04 |
| CN103275222B true CN103275222B (en) | 2014-04-16 |
Family
ID=49057860
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310180792.0A Active CN103275222B (en) | 2013-05-15 | 2013-05-15 | Monoclonal antibody for blocking 12p40 function of interleukin as well as coding gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103275222B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20220038775A (en) * | 2019-07-30 | 2022-03-29 | 아케소 바이오파마, 인크. | Anti-human P40 protein domain antibodies and uses thereof |
| CN116355094B (en) * | 2023-02-21 | 2023-10-20 | 武汉爱博泰克生物科技有限公司 | Monoclonal antibody against interleukin 12 of mouse and preparation method |
| CN118620077A (en) * | 2024-08-13 | 2024-09-10 | 苏州百道医疗科技有限公司 | Anti-P40 recombinant rabbit monoclonal antibody and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1351614A (en) * | 1999-03-25 | 2002-05-29 | 克诺尔股份有限公司 | Human antibodies that bind human IL-12 and methods for producing |
| CN101274962A (en) * | 1998-01-23 | 2008-10-01 | 霍夫曼-拉罗奇有限公司 | Antibodies against human IL-12 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU6887096A (en) * | 1996-09-11 | 1998-04-02 | Patrick T. Prendergast | Immune direction therapy |
| CN101490085A (en) * | 2006-06-12 | 2009-07-22 | 特鲁比昂药品公司 | Single-chain multivalent binding proteins with effector function |
| US8715657B2 (en) * | 2009-04-27 | 2014-05-06 | Novartis Ag | Therapeutic antibodies binding IL12Rβ1 |
-
2013
- 2013-05-15 CN CN201310180792.0A patent/CN103275222B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101274962A (en) * | 1998-01-23 | 2008-10-01 | 霍夫曼-拉罗奇有限公司 | Antibodies against human IL-12 |
| CN1351614A (en) * | 1999-03-25 | 2002-05-29 | 克诺尔股份有限公司 | Human antibodies that bind human IL-12 and methods for producing |
Non-Patent Citations (4)
| Title |
|---|
| 抗IL-12/23单克隆抗体Briakinumab;邢爱敏;《药学进展》;20111130;第35卷(第11期);第523-524页 * |
| 新型靶向生物制剂ustekinumab用于自身免疫性疾病治疗;杨清锐等;《世界临床药物》;20100831;第31卷(第8期);第454-459页 * |
| 杨清锐等.新型靶向生物制剂ustekinumab用于自身免疫性疾病治疗.《世界临床药物》.2010,第31卷(第8期),第454-459页. |
| 邢爱敏.抗IL-12/23单克隆抗体Briakinumab.《药学进展》.2011,第35卷(第11期),第523-524页. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103275222A (en) | 2013-09-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| HUP0303171A2 (en) | Artificial proteins with reduced immunogenicity | |
| JPS60222429A (en) | Synthesis of protein by maker peptide | |
| JP4959226B2 (en) | Three finger-like protein library | |
| US20220403012A1 (en) | Peptide-hinge-free flexible antibody-like molecule | |
| CN103275222B (en) | Monoclonal antibody for blocking 12p40 function of interleukin as well as coding gene and application thereof | |
| JP2022511718A (en) | Anti-Taq DNA polymerase antibody and its uses | |
| Todd et al. | The immunogenicity and antigenicity of proteins | |
| US9580479B2 (en) | Peptide tag and uses thereof | |
| ATE208815T1 (en) | GENETIC IGFBP-5 RODING MATERIAL | |
| CN1399646A (en) | Recombinant fusion molecules | |
| CN117866902A (en) | Genetically modified stem cells with anti-IL-17A activity, preparation method thereof and pharmaceutical composition | |
| JP2008520208A (en) | Protein backbone and uses thereof | |
| CN103880953B (en) | One boar P21 protein antibodies and preparation method thereof and application | |
| CN106188279A (en) | A kind of nano antibody of the specific recognition immunoglobulin Fc section in immune library source | |
| US9758571B2 (en) | Antibody for epitope tagging, hybridoma cell line and uses thereof | |
| CN108300725B (en) | Soluble single-chain antibody superantigen fusion gene and protein, and preparation and application thereof | |
| MA | High expression level of human epidermal growth factor (hEGF) using a well-designed fusion protein-tagged construct in E. coli. | |
| Makhzoum et al. | An in silico overview on the usefulness of tags and linkers in plant molecular pharming | |
| JPH06510419A (en) | Interferon-γ-induced monokine-MIG | |
| CY1110308T1 (en) | Molecular Nucleic Acid, which Includes a Code for the Immunocyanic Sequence of a Nucleic Acid and a Tuberculosis | |
| Cooper et al. | Antibodies for immunochemistry | |
| Chin-Fatt et al. | A rationally designed bovine IgA Fc scaffold enhances in planta accumulation of a VHH-Fc fusion without compromising binding to enterohemorrhagic E. coli | |
| RU2801597C1 (en) | Artificial genetic construct for the heterologous expression of the s-protein receptor-binding domain in a fused polypeptide chain with a nucleocapsid protein | |
| WO2022242500A1 (en) | Preparation method for antibody carrying universal fixed-point coupling interface based on genetically modified vertebrate | |
| CN111925447B (en) | Human and mouse egg zona pellucida fusion protein and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |