CN104804091B - Nano antibody for resisting B cell growth stimulating factor and application - Google Patents
Nano antibody for resisting B cell growth stimulating factor and application Download PDFInfo
- Publication number
- CN104804091B CN104804091B CN201410046088.0A CN201410046088A CN104804091B CN 104804091 B CN104804091 B CN 104804091B CN 201410046088 A CN201410046088 A CN 201410046088A CN 104804091 B CN104804091 B CN 104804091B
- Authority
- CN
- China
- Prior art keywords
- baff
- antibody
- nano antibody
- cell
- nano
- 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
- 210000003719 b-lymphocyte Anatomy 0.000 title claims abstract description 18
- 230000004936 stimulating effect Effects 0.000 title abstract description 7
- 230000010261 cell growth Effects 0.000 title abstract 3
- 230000014509 gene expression Effects 0.000 claims abstract description 22
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 13
- 201000010099 disease Diseases 0.000 claims abstract description 11
- 230000000694 effects Effects 0.000 claims abstract description 8
- 230000002159 abnormal effect Effects 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 43
- 108090000623 proteins and genes Proteins 0.000 claims description 42
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 239000013604 expression vector Substances 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 5
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 claims description 4
- 230000006907 apoptotic process Effects 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 230000001225 therapeutic effect Effects 0.000 claims description 4
- 238000011282 treatment Methods 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 claims description 2
- 101710112752 Cytotoxin Proteins 0.000 claims description 2
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 claims description 2
- 239000004037 angiogenesis inhibitor Substances 0.000 claims description 2
- 230000001028 anti-proliverative effect Effects 0.000 claims description 2
- 230000005784 autoimmunity Effects 0.000 claims description 2
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 claims description 2
- 231100000599 cytotoxic agent Toxicity 0.000 claims description 2
- 239000002619 cytotoxin Substances 0.000 claims description 2
- 208000027866 inflammatory disease Diseases 0.000 claims description 2
- 108020004707 nucleic acids Proteins 0.000 claims description 2
- 102000039446 nucleic acids Human genes 0.000 claims description 2
- 150000007523 nucleic acids Chemical class 0.000 claims description 2
- 101710181056 Tumor necrosis factor ligand superfamily member 13B Proteins 0.000 abstract description 58
- 102100036922 Tumor necrosis factor ligand superfamily member 13B Human genes 0.000 abstract description 55
- 239000000427 antigen Substances 0.000 abstract description 34
- 102000036639 antigens Human genes 0.000 abstract description 34
- 108091007433 antigens Proteins 0.000 abstract description 34
- 230000027455 binding Effects 0.000 abstract description 11
- 238000009739 binding Methods 0.000 abstract description 11
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000035755 proliferation Effects 0.000 abstract description 3
- 230000004071 biological effect Effects 0.000 abstract description 2
- 230000004083 survival effect Effects 0.000 abstract description 2
- 101000851434 Homo sapiens Tumor necrosis factor ligand superfamily member 13B Proteins 0.000 abstract 1
- 102000050326 human TNFSF13B Human genes 0.000 abstract 1
- 239000003446 ligand Substances 0.000 abstract 1
- 238000006386 neutralization reaction Methods 0.000 abstract 1
- 238000000034 method Methods 0.000 description 21
- 241000894006 Bacteria Species 0.000 description 18
- 230000029087 digestion Effects 0.000 description 17
- 239000013612 plasmid Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 10
- 238000005119 centrifugation Methods 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 238000012216 screening Methods 0.000 description 9
- 101000795167 Homo sapiens Tumor necrosis factor receptor superfamily member 13B Proteins 0.000 description 8
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 8
- 102100029675 Tumor necrosis factor receptor superfamily member 13B Human genes 0.000 description 8
- 241001416177 Vicugna pacos Species 0.000 description 8
- 230000002401 inhibitory effect Effects 0.000 description 8
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 description 7
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- BGFTWECWAICPDG-UHFFFAOYSA-N 2-[bis(4-chlorophenyl)methyl]-4-n-[3-[bis(4-chlorophenyl)methyl]-4-(dimethylamino)phenyl]-1-n,1-n-dimethylbenzene-1,4-diamine Chemical compound C1=C(C(C=2C=CC(Cl)=CC=2)C=2C=CC(Cl)=CC=2)C(N(C)C)=CC=C1NC(C=1)=CC=C(N(C)C)C=1C(C=1C=CC(Cl)=CC=1)C1=CC=C(Cl)C=C1 BGFTWECWAICPDG-UHFFFAOYSA-N 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 229960002685 biotin Drugs 0.000 description 5
- 235000020958 biotin Nutrition 0.000 description 5
- 239000011616 biotin Substances 0.000 description 5
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 5
- 238000010494 dissociation reaction Methods 0.000 description 5
- 230000005593 dissociations Effects 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 102100031780 Endonuclease Human genes 0.000 description 4
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 230000002860 competitive effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 108010042407 Endonucleases Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 239000005557 antagonist Substances 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000005611 electricity Effects 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000002823 phage display Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 241001515965 unidentified phage Species 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241000549556 Nanos Species 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 206010042566 Superinfection Diseases 0.000 description 2
- 101710120037 Toxin CcdB Proteins 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000007857 nested PCR Methods 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000000392 somatic effect Effects 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- 101150028074 2 gene Proteins 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 244000303258 Annona diversifolia Species 0.000 description 1
- 235000002198 Annona diversifolia Nutrition 0.000 description 1
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 208000037914 B-cell disorder Diseases 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 102100026596 Bcl-2-like protein 1 Human genes 0.000 description 1
- 241000282836 Camelus dromedarius Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000001493 Cyclophilins Human genes 0.000 description 1
- 108010068682 Cyclophilins Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- -1 Ni+ ion Chemical class 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102100029690 Tumor necrosis factor receptor superfamily member 13C Human genes 0.000 description 1
- 101710178300 Tumor necrosis factor receptor superfamily member 13C Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000037058 blood plasma level Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000016507 interphase Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000003519 mature b lymphocyte Anatomy 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000003716 rejuvenation Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a nanometer antibody for resisting B cell growth stimulating factors and application thereof, belonging to the technical field of biology. A nanometer antibody for resisting B cell growth stimulating factor has an amino acid sequence shown in SEQ ID NO.1 of the sequence table. The invention has the advantages that: according to the invention, a plurality of nano antibodies with high activity and potential neutralization capacity are screened, and finally the anti-BAFF nano antibody 64 is obtained. The nano antibody can specifically bind to a human BAFF antigen, regulate the biological activity related to the BAFF antigen and the ligand thereof, effectively inhibit the binding of the BAFF antigen and the receptor thereof and generate a corresponding signal cascade effect. The anti-BAFF nanobody can block the binding of BAFF to three related receptors thereof and obviously inhibit the proliferation or survival of B cells, and the nanobody can be used for detecting and/or treating various diseases related to the abnormal expression of BAFF.
Description
Technical field
The present invention relates to a kind of nano antibody and purposes of anti-B cell growth-stimulating factor, belong to biological technical field.
Background technology
Lymthoma category is primary in the malignant tumour of lymph node or lymphoid tissue, there is lymphocyte and (or) histiocytic big
Hyperplasia is measured, accounts for the 8th of China's common cancer, and rejuvenation trend is obvious, the incidence of disease is risen to by 2-4/10 ten thousand in recent years
6.91/10 ten thousand, and risen with annual 5% speed, the annual new hair people of patient about 50,000, death toll is more than 20,000.It is generally acknowledged that suddenly
Strange golden lymthoma is mainly derived from B cell, and most NHL also derives from B cell, therefore thin by removing B
Born of the same parents treat lymthoma turns into a kind of new therapeutic scheme.
Clinical research finds that the malignant B cell such as lymthoma can synthesize and secrete a kind of entitled bone-marrow-derived lymphocyte stimulating factor
The albumen of (B cells-activating fact BAFF), belong to tumor necrosis factor superfamily member, it can be thin in B with expression
The acceptor of cellular surface combines, and then activates NF-kB approach, induces anti-apoptotic gene：Bcl-2, Bcl-xL expression, reduce
The apoptosis of mature B cell.Different subtype B- cell lymphoma patient's serum soluble BAFF contents are obvious compared with normal control
It is elevated, and BAFF blood plasma levels and progress, the order of severity and the therapeutic sensitivity correlation of genetic mutation and disease.
Nano antibody is the heavy chain antibody of the missing light chain by being found in alpaca blood, and molecule is applied through Belgian scientist
The method of biology techniques combination nano-particle science, and the antibody molecule that the newest and molecular weight developed is minimum.It has
Have simple in construction, penetration power is strong, is easy to purify and expresses, and affinity and stability are high, the series of advantages such as no toxicity.
There has been no the report of the nano antibody for BAFF at present.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of anti-BAFF nano antibody and its coding gene sequence.
The invention solves another technical problem be to provide anti-BAFF nano antibody purposes.
To achieve the above object, the present invention uses following technical scheme：
A kind of nano antibody of anti-B cell growth-stimulating factor, its amino acid sequence are sequence table SEQ ID NO.1 institutes
Show.The sequence includes framework region and complementary determining region；Complementary determining region is CDR1-CDR3；Framework region is respectively FR1-FR4；
Complementary determining region has the amino acid sequence being selected from the group, and is specially：
CDR1 amino acid sequence：RRGMG
CDR2 amino acid sequence：LVAGISSDGTTNYAQSVEG
CDR3 amino acid sequence：HARTRWKSY
The FR1-FR4 of framework region amino acid sequence, it is specially：
FR1 amino acid sequence：QVQLVESGGGLVQAGGSLRLSCVASGALIS
FR2 amino acid sequence：WYRQAPGRQRE
FR3 amino acid sequence：RFTISRDNAKRSVYLQMNSLKPEDTAVYYC
FR4 amino acid sequence：WGQGTQVTVSS
The nano antibody of the anti-B cell growth-stimulating factor its amino acid composition order：FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4.（Sequence table SEQ ID NO.1）It is named as anti-BAFF nano antibodies 64.
The nano antibody of described anti-B cell growth-stimulating factor, its amino acid sequence and sequence table SEQ ID NO.1 institutes
The homology of the amino acid sequence shown is more than 80%, preferably greater than 90%, most preferably greater than 95%.
A kind of polypeptide, selected from above-mentioned nano antibody, or single domain antibody fragment, or domain antibody fragment, or " dAb,
VH, VHH ".
Encode the gene order of the nano antibody of above-mentioned anti-B cell growth-stimulating factor.
The gene order is the nucleotide sequence shown in sequence table SEQ ID NO.2.
, can people in vitro according to specific nucle or amino acid sequence in the anti-BAFF nano antibodies variable region of the present invention
Work synthesizes with the nucleotide sequence of this identical antibody weight chain gene or encoded the nucleotide sequence of same amino acid, so as to obtain
Identical antibody gene or the transformation for related gene are obtained, and obtains anti-BAFF nano antibodies or GAP-associated protein GAP or polypeptide production
Thing.
Include the expression vector of the gene order.Carrier can be procaryotic cell expression carrier, eukaryotic cell expression load
Body or insect cell expression vector.
Include the host cell of the expression vector.Described host cell can be prokaryotic expression cell, eukaryotic expression
Cell or insect cell, the preferred Escherichia coli of prokaryotic expression cell.
The method that the nano antibody of anti-B cell growth-stimulating factor is prepared by the host cell.
Pharmaceutical composition, it includes anti-BAFF nano antibodies molecule described in one or more as active ingredient, and wrapped
Containing pharmaceutically acceptable supporting agent.
The pharmaceutical carriers are selected from cytotoxin, radio isotope, immune modulator, anti-angiogenic agent, antiproliferative
Agent, the therapeutic agent for promoting apoptosis agent, chemotherapeutant or therapeutic nucleic acids.
Described pharmaceutical composition can also be provided in the form of the pharmaceutical kit comprising packaging, described to be included in
Separate in container or one or more antagonists in single container.
The nano antibody of described anti-B cell growth-stimulating factor is used for the purposes for preparing detection BAFF reagent.Including
But the capture antigen being not limited to use in double-antibody sandwich enzyme-labeled immunity detection BAFF ELISA reagents is coated with nano antibody
The biotin labeling nano antibody of elisa plate and detection antigen.
The nano antibody of described anti-B cell growth-stimulating factor is used to prepare treatment BAFF abnormal expression relevant diseases
The purposes of medicine；The BAFF abnormal expressions relevant disease is B cell non Hodgkin lymphom (non-Hodgkins
Lymphoma), B cell chronic lymphocytic leukemia, Huppert's disease, the autoimmunity disease for being related to B cell or inflammatory disease
Disease.
The present invention constructs the pET30a expression vectors of BAFF extracellular region polypeptides recombination clone, obtains and is marked with His
The BAFF extracellular regions of label（BAFFECD）High Purity albumen, the Ni+ magnetic bead knots with His label proteins are combined with energy high-affinity
Close, the correct space structure of displayed polypeptides, antigen immune alpaca in this format, build specific nano antibody gene pool
（Nano antibody phage display gene pool）, immune nano Antibody geometric mean titer is screened, and obtain the special of B-cell stimulating factor
The nano antibody of property（Or single domain antibody fragment, single domain antibody fragment）, by this gene and expression
Carrier connection restructuring, constructing can be in the nano antibody strain anti-BAFF64 of E. coli.Carried out in laboratory
Shaking flask small-scale production, through Ni+ resin gel affinitive layer purifications, it is pure up to 95%, about 100mg/L to can obtain SDS-PAGE electrophoresis
The nano antibody of bacterial cultures.
The present invention relates to the multi-form nano antibody for being directed to BAFF（Monomer, binary or pentamer）, prepare B cell life
Long stimulating factor（BAFF）Nano antibody all technologies（Technical Reference：Tanha J,Dubuc G,Selection by
phage display of llama conventional V(H)fragments with heavy chain antibody V
(H)H properties.J Immunol Methods.2002,263(1-2):97-109）（Gueorguieva D,Li
S.Identification of single-domain,Bax-specific intrabodies that confer
resistance to mammalian cells against oxidative-stress-induced
apoptosis.FASEB J.2006,20(14):2636-8）And removed based on B cell（B cell depletion) it is immune
Therapy.The antibody can play the effect of antagonist, and can be used for external (in vitro), (in situ) in situ or
The mammalian cell or pathologic condition that (in vivo) is detected in vivo or treatment is related to BAFF presence (or shortage).
The invention provides the side using BAFF antibody blockings or the interphase interaction for neutralizing BAFF and TACI/ or BCMA
Method.The antagonist can also block or neutralize BAFF and TACI/ or BCMA interaction.Anti- BAFF-R antibody can with it is thin
Cellular toxicity agent or enzyme are connected, or are connected with radio isotope, fluorescent chemicals or chemiluminescence compound.
It is an advantage of the invention that：The multiple activity of present invention screening are high and have the nano antibody of potential neutralising capacity, finally
Anti- BAFF nano antibody 64 is obtained.The nano antibody can specifically bind people BAFF antigens, regulation and BAFF antigens and its
The related biological activity of part, effectively suppress BAFF antigens and combine and produce corresponding signal cascade effect with its acceptor.
Anti- BAFF nano antibodies can block the combination of BAFF and its three kinds of associated receptors, significantly inhibit B cell proliferation or survival, and this is received
Meter Kang Ti can be used for detecting and/or treating a variety of and BAFF abnormal expression relevant diseases.
The present invention is described in detail below in conjunction with the drawings and specific embodiments, and the implementation model of the present invention is not limited with this
Enclose.
Brief description of the drawings
Fig. 1：BAFF Extracellular domain proteins are expressed and electrophoresis result after purification
Fig. 2：Protein G affinity chromatographies purify heavy chain antibody
Fig. 3：Alpaca immune serum potency
Fig. 4：The common heavy chain antibodies of first round PCR and single-chain antibody gene
Fig. 5：Second round VHH antibody purpose fragments
Fig. 6：SfiI/Pst digestion pHEN-6 carriers
Fig. 7：SfiI/Pst digestion purpose fragments
Fig. 8：Bacterium colony PCR verifies antibody insertion rate
Fig. 9：Anti- BAFF nano antibodies protein purification result
Figure 10：FPLC methods purify anti-BAFF nano antibodies
Figure 11：Nano antibody and BAFF antigen binding dynamic curves
Figure 12：Competitive inhibitory effect of the nano antibody to acceptor BCMA
Figure 13：Competitive inhibitory effect of the nano antibody to acceptor TACI
Figure 14：Anti- BAFF64 nano antibodies are in vitro to the inhibited proliferation of Raji cells
Embodiment
Embodiment of the present invention is illustrated by the following example.But embodiment of the present invention is not limited to these
The specific detail of embodiment, because for the person of ordinary skill of the art, others change is known, or according to straight
It is obvious to connect disclosure and appended claims.Therefore, all skills realized based on the above of the present invention
Art belongs to the scope of the present invention.
Experimental method described in following embodiments, it is conventional method unless otherwise specified；The reagent and biological material
Material, unless otherwise specified, is commercially obtained.
Embodiment 1：The expression of BAFF Extracellular domain proteins
The fishing of 1.sBAFF genes takes：Normal person's venous blood is extracted, it is thin conventionally to extract the single core of peripheral blood
Born of the same parents (PBMC).RNA extracting is carried out according to the specification of Invitorgen companies.Take 3ug RNA reverse transcriptions
CDNA masterplates are prepared, using it as template, expand BAFF genes.
Primer sequence is：
5’CGGGAATTCCTGGTGCCACGCGGTGCCGTCAGGGTCCAGAAG3’（SEQ ID No.3）
5’CCCAAGCTTTCACAGCAGTTTCAATGC3’（SEQ ID No.4）
PCR reaction conditions：
2. the structure of expression vector：Pcr amplification product is separated with agarose gel electrophoresis, reclaimed, and uses EcoRI（BioLab
Company）、HindIII（BioLab companies）After digestion, the carrier pET-30a with same ferment treatment（Sigma）By 3:1 end ratio
Example carries out cohesive terminus,cohesive termini connection.Connection product presses CaCl2Conversion method transformed competence colibacillus e. coli jm109（Sigma）, utilize card
That chloramphenicol resistance screens recombinant clone.The DNA of positive colony is extracted, EcoR I, HindIII double digestions and agar is carried out and coagulates
Gel electrophoresis are analyzed, and carry out bacterium colony PCR.By the correct positive recombinant of Preliminary Identification, delivering Shanghai, to win sub- biotechnology limited
Company, Shanghai Shen Neng lottery industries Bioisystech Co., Ltd carry out DNA sequence analysis, and by known base in sequencing result and database
The sequence of cause is compared.
3. the expression of target gene：With the expression vector containing the correct BAFF genes of sequencing, by CaCl2Conversion method converts
Competence e. coli bl21（Sigma）；Meanwhile with the empty carrier plasmid transformed competence colibacillus Escherichia coli without BAFF genes
BL21 is as negative control.Picking single bacterium colony, it is inoculated in the LB culture mediums containing kanamycins, in 37 DEG C of cultures to logarithmic phase
Afterwards, add or be added without IPTG to continue to cultivate, induce the expression of target gene.A small amount of induction, the nutrient solution of non-Induction of bacterial are taken,
Centrifugation, precipitation.After removing supernatant, add the bacteria into SDS sample loading buffers and boil, the expression of analysis purpose albumen.
4. great expression and the purifying of destination protein：Single bacterium colony is selected to be inoculated in the LB culture mediums containing kanamycins,
Cultivated in LB to logarithmic phase, add IPTG and continue overnight incubation in 30 DEG C.By bacterial culture fluid in 4 DEG C with 12000r/min from
Heart 15min.After collection bacterium is suspended with bacterial lysate, with ultrasonic disruption thalline.After centrifugation, bacterial supernatant is collected respectively
Precipitated with inclusion body.With nickel chelating sepharose（Beijing Vector Gene Technology Co., Ltd）Fill and be situated between for post
Matter, affinity chromatography is carried out to the extract solution of bacterial supernatant.The elution of destination protein is completed using the concentration of rise imidazoles.Collect not
With the imidazoles eluting peak sample of concentration, with SDS-PAGE analysis purpose albumen.）（Fig. 1）
The amino acid sequence of BAFF Extracellular domain proteins is shown in sequence table SEQ ID No.5：
AVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGH
LIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL
（SEQ ID No.5）
Embodiment 2：The structure of anti-BAFF specific nanos antibody library
1. immune alpaca, enzyme exempts from method analysis nano antibody production.
The subcutaneous multi-point injection antigen of alpaca nape part amounts to 1.5mg, and adds freund adjuvant, point 4 immune, Follow-up observations
Enclosed mass absorbing state is injected, it is immune correct to confirm.Serum antibody titer is determined after immune one month for the first time（Fig. 2）；Thereafter
Immune, each immunization interval time halves, and determines potency.With Protein G posts, affinity chromatography method separates the 4th time and is immunized
Latter all serum, ELISA method detection heavy chain antibody production and antibody titer, after the 4th time immune, blood is immunized in alpaca
Clear potency can reach 1：10000.（Fig. 3）.
2. separate alpaca PBLC
Alpaca PBLC is separated, with RNA extraction agent boxes（QIAGEN）, extracted from obtained lymphocyte
Total serum IgE.
3. nested PCR method obtains camel antibody heavy chain variable region-VHH.
To improve specific amplification, reverse transcriptase primer uses the special primer of heavy chain antibody, the chains of cDNA first is synthesized, with this
Template, enter performing PCR amplification heavy chain antibody VHH genetic fragments with two sets of primers respectively.Using nested PCR method, first time PCR expands
Be more than 800bp in increasing is common heavy chain gene segment, the heavy chain antibody genes for missing light chain between 800~500bp
Fragment（Fig. 4）, gel extraction missing light chain heavy chain antibody gene segments, expanded as template with VHH specific primers through PCR
Obtain VHH target gene（500bp）（Fig. 5）.
The synthesis of diversity primer：
Heavy Chain Fd5’primers：
YTCh-1:CGC CAT CAA GGT ACC AGT TGA（Sequence table SEQ ID NO.6）
YTCh-2:GGG GTA CCT GTC ATC CAC GGA CCA GCT GA（Sequence table SEQ ID NO.7）
Heavy Chain Fd3’primers：
YT1BN:GCC CAG CCG GCC ATG GCC SMK GTR CAG CTG GTG GAK TCT GGG GGA G
（Sequence table SEQ ID NO.8）
YT2BN:GCC CAG CCG GCC ATG GCC CAG GTA AAG CTG GAG GAG TCT GGG GGA G
（Sequence table SEQ ID NO.9）
The connection of 4.VHH fragments and Vector for Phage Display and electricity conversion TG1 competence.
(1) SfI single endonuclease digestions VHH fragments：
Mix, centrifugation of short duration, puts digestion in 50 DEG C of PCR instruments and stayed overnight, and purifies the PCR primer after digestion.
（2）SfI single endonuclease digestion vector plasmids：
Mix, centrifugation of short duration, put digestion in 50 DEG C of PCR instruments and stay overnight, gel reclaims kit（QIA）Purifying.
（3）PstI single endonuclease digestion vector plasmids：
Mix, 37 DEG C of water-bath digestion 1h PCR primer purification kits are put in centrifugation of short duration（QIA）Purify the plasmid after digestion
Carrier, 1% agarose gel electrophoresis, identify plasmid.
（4）Linked system：
Centrifugation of short duration after mixing, put 16 DEG C of PCR instrument connection 28h.
By VHH fragments and pHEN6 carriers (Conrath, KEM other.Antimicrob Agents Chemother
(Antimicrobial Chemotherapy)2001,45:(10) 2807-12., Chinese patent ZL20111028003.1) respectively
Through SfiI/PstI digestions and connect（Fig. 6-7）, electricity conversion is into TG1 competent cells, spread plate, is verified through bacterium colony PCR anti-
Body insertion rate.Recombination cloning efficiency detects：On power taking transformed bacteria solution coating LB/Amp flat boards, 32 DEG C, it is incubated overnight, next day
With the joint efficiency of bacterium colony PCR method validation antibody, the joint efficiency of phage antibody library is more than 90%.（Fig. 8）
The storage capacity of 5.VHH Antibody geometric mean titers and multifarious identification and preservation
The total amount that titre according to being determined after conversion is multiplied by conversion calculates quantity in stock.Screened in addition, selecting at random after electricity turns
20~50 positive colonies of plated growth, sequencing, if not having repetitive sequence, it can be determined that its diversity is good.Will
After remaining bacterium solution adds the culture in 2 hours of appropriate 2YT/Amp culture mediums, glycerol adding packing, frozen in -80 DEG C, so as to obtain BAFF
Immune VHH type Antibody geometric mean titers.
The preparation of 6.VHH phage antibody libraries and primary Function Identification
Antibody library adds helper phage M13K07（Invitrogen）Saved.1 μ l M13 are added to containing
6ml2YT, 0.4ml20%glucose+4 μ l Amp (100mg/ml) culture medium infected cell in, 37 DEG C stand 15min after,
This 6ml culture medium is added to containing 92ml2YT, in 92 μ lAMP culture medium, stands 15min, after 37 DEG C of 250rmp, 1h, is added
100 37 DEG C of μ l Kan (50mg/mL) are overnight.Supernatant takes primary Function Identifications of the 100 μ l for antibody library, remainder after rescue
With PEG8000 precipitating phages, under the conditions of 4 DEG C, precipitation is collected by centrifugation, preserves and determines phage antibody library titre.
Embodiment 3：The acquisition of anti-BAFF nano antibodies
1. the screening of anti-BAFF specific nanos antibody
With biotinylation BAFF antigens, go out anti-BAFF's from phage library screening antibodies in streptomysin avidin magnetic bead method
Nano antibody.The BAFF antigens of biotin labeling are mixed in proportion with phage antibody library, 4 DEG C of reactions are overnight.By antigen-bite
Somatic antibody compound is added in the EP pipes containing Streptavidin MagneSphere, end rotation 0.5h, makes magnetic bead and biotin labeling
Antigen-phage antibody compound fully combine.After PBST (0.05%T20), PBS respectively rinse 10 times, phagocytosis is eluted with TEA
Body, it is stored at room temperature 10min.TEA is closed in 1M Tris-HCl, is preserved on ice.The phage-infect semilog phase is grown into TG1, taken suitable
AMP/LB flat boards are coated with after amount bacterium solution dilution, 32 DEG C of cultures, eluent titre are determined, with M13K07 after the expansion culture of remaining bacterium solution
Simultaneously shaken cultivation is stayed overnight for superinfection, is collected supernatant within the 2nd day, is purified with PEG, is that the secondary for being used for next round screening is bitten after concentration
Somatic antibody storehouse.Screened through 3 wheels, the titre of the specific bacteriophage antibody obtained after measure often wheel screening, and calculate and often take turns
Screening bacteriophage input the index that is enriched with as specific bacteriophage antibody of output ratio (rate of recovery).
Table l：Enrichment effect of the affine screening to phage antibody
2. Phage-ELISA method selects positive colony
From random picking single bacterium colony on the agar plate of the 3rd wheel screening growth bacterium colony, the 2YT liquid containing Amp is seeded in
Cultivated in 96 well culture plates of body culture medium, with helper phage superinfection induced expression phage antibody.Yield table reaches supernatant,
ELISA measure is carried out by antigen of BAFF, picks out BAFF positives hole, through DNA sequencing to identify that anti-BAFF nano antibodies are cloned
Gene order.A series of nano antibody gene orders including sequence table SEQ ID NO.2 gene orders are obtained, are used for
Further expression and screening specificity, the nano antibody of high activity.
Embodiment 4：The structure of specific nano antibody expressing plasmid
The specific nano antibody gene that PCR amplifications embodiment 3 is obtained, and obtain and carry restriction enzyme BbsI
With BamHI sites PCR primer, PCR primer and carrier (pSJF2 carriers) are handled respectively with restriction enzyme BbsI and BamHI
(kim Is.Biosic Biochem.2002,66(5):1148-51, Chinese patent ZL201110280031）, through T4 ligases
Connection restructuring, and acquisition can be in the plasmid BAFF64pSJF2 of E. coli, and gene sequencing is carried out with true
The correctness of its fixed sequence.
1. obtain the PCR amplification conditions of VHH target gene：The composition of 50 μ l PCR systems：
PCR reaction conditions：
Sense primer-TATGAAGACACCAGGCCCAGGTRMAGCTGGWGGAGTCT；（Sequence table SEQ ID NO.10）
Anti-sense primer --- GAAGATCTCCGGATCCTGAGGAGACGGTGACCTGGGT.（Sequence table SEQ ID
NO.11）
2. the digestion of target gene：
3. the digestion of vector plasmid：
Mix, centrifugation of short duration, put digestion 1h in 37 DEG C of water-baths, with glue reclaim kit reclaim digestion after purpose fragment and
Plasmid vector.
4. connect target gene and carrier
Following component is sequentially added in 1.5ml Eppendorf pipes：
Centrifugation of short duration after mixing, 18h is connected in 16 DEG C, obtains specific nano antibody expressing plasmid
BAFF64-pSJF2 plasmid.
Embodiment 5：The expression and purifying of anti-BAFF nano antibodies
1. nano antibody albumen is in expression in escherichia coli, purifying：
（1）The strain containing plasmid BAFF64-pSJF2 described in embodiment 4 is seeded in the LB containing aminobenzylpenicillin
On culture plate, 37 DEG C overnight.
（2）Select single bacterium colony to be inoculated in the 20ml LB nutrient solutions containing aminobenzylpenicillin, 37 DEG C, shaking table culture mistake
Night.
（3）Transferred species is in 2YT nutrient solutions of the 1L containing aminobenzylpenicillin, 37 DEG C of shaking table cultures, 240 revs/min, and OD is arrived in culture
When value is up to 0.6~1.0,0.1~0.5M IPTG are added, continue overnight incubation.
（4）Centrifugation, receive bacterium.
（5）Add and melt bacterium enzymatic lysis bacterium, centrifuge, receive soluble nano antibody albumen in supernatant.
（6）Purity is obtained up to more than 95% albumen through Ni+ ion affinity chromatography high pressures liquid phase.
The anti-BAFF nano antibodies albumen of expression, through Ni+ resin gel affinitive layer purifications, No. 1-4 is that 250mM imidazoles is washed
Deviating from anti-BAFF nano antibodies, M is standard molecular weight albumen marker, shown in purifying protein SDS-PAGE results, purifying protein
Purity is up to more than 90%.（Fig. 9）
2. fast protein liquid chromatography：
（1）Antigen and buffer solution prepare：With 0.22 μm of filtering with microporous membrane protein solution and PB buffer solutions.
Chromatographic column prepares：Superdex75TM chromatographic columns, flow velocity 0.5ml/min are balanced with PB buffer solutions (PH7.2).
（2）Sample Purification on Single：1ml nano antibodies sample is injected in sample loop with syringe, flow velocity 0.5ml/min, with
UV-detector automatically determines the A280nm values of eluent, collects the nano antibody of required purifying.
Through molecular sieve sephadex（superG75）Shown by the experiment of filtration high pressure liquid phase analysis, anti-BAFF nano antibodies
Eluting peak occur time pass through compared with standard molecular weight albumen Marker, the molecular weight of its peak value should be 15kd.（Figure
10）
Sequencing obtains the amino acid sequence of anti-BAFF nano antibodies 64 as shown in sequence table SEQ ID NO.1.
Experimental example 6：Biological membranous layer interference technique resists BAFF nano antibody affinity determination experiments
1. biotinylated antigen prepares：The BAFF antigens being first dissolved in PBS（It is prepared by the method for embodiment 1）With biotin
By 1:3 mixed in molar ratio is uniform, is stored at room temperature 0.5h, then removes free biotin point by the ultrafiltration of 10KD super filter tubes
Son, biotinylated BAFF antigens are obtained, and determine the concentration of biotinylated antigen.
2. antibody prepares：Broford methods determine antibody concentration, dilute anti-BAFF nano antibodies with PBST (0.005%T20)
For 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM of 5 various concentrations, if 20 μM of anti-CD 20 antibodies (R＆D companies) are unrelated control.
3. envelope antigen：Biotinylated antigen is diluted to 20 μ g/ml with PBST (0.005%T20) buffer solution, then
In OctetQk systems（Fortebio, pull company）, 6 SA sensors are placed in antibody-solutions and reacted, are coupled antigen
On SA sensors.
4. antigen and antibody binding dynamic analysis：In OctetQk systems, the sensor for being coated with antigen is put respectively
In the BAFF nano antibodies and control antibodies solution of 5 various concentrations, 300s is acted on, makes antibody and the antigen knot on sensor
Close, then sensor is placed in PBST solution, makes the antibody dissociation for being incorporated in chip surface, Dissociation time 1500s.This mistake
Machine can record antigen and antibody binding, the real time information of dissociation automatically in journey.
5. affinity costant calculates：The data obtained carries out 1 with analysis software：1Langmuir binding models are fitted, and calculate antigen
The kinetic constant of antibody binding.Binding constant：kon(1/Ms):2.29E+03；Dissociation constant：kdis(1/s)7.29E-04；
Affinity：KD(M)3.19E-07.
Biological membranous layer interference technique method uses various concentrations respectively to the affinity measure of nano antibody and dynamic analysis
（20μM、10μM、5μM、2.5μM、1.25μM）Nano antibody and BAFF antigen bindings, by binding constant and dissociation constant,
The affinity of calculating antibody is 3.19E-07.（Figure 11）
Embodiment 7：The ELISA Competitive assays of anti-BAFF nano antibodies 64 and acceptor BCMA/TACI are tested
1.0.05M NaHCO3（pH9.5）Dilute BAFF antigens（It is prepared by embodiment 1）To 10 μ g/ml, 100 μ l antigen coats
2 piece of 96 orifice plate, 4 DEG C overnight.
2.300 μ l0.5%BSA-PBS close 96 orifice plates, 37 DEG C of 2h.
3. the BCMA that concentration is 50 μ g/ml is first added in the plate hole of the 1st piece of 96 orifice plates（B cell maturation antigen, Sigma）
50 μ l, then anti-BAFF nano antibodies are added, 37 DEG C, 1h with finite concentration dilution proportion by 50 μ l/ holes.
Negative control Anti-CD20（R＆D companies）
Table 2：Competitive inhibitory effect of the nano antibody to acceptor BCMA
4. being loaded method according to the 3rd step, the TACI that concentration is 10 μ g/ml is added in the plate hole of the 2nd piece of 96 orifice plates（Cross-film
Activator and cyclophilin interactant, Sigma）50 μ l, then by anti-BAFF nano antibodies with finite concentration dilution proportion, by 50
μ l/ holes add, 37 DEG C, 1h.
Table 3：Competitive inhibitory effect of the nano antibody to acceptor TACI
5. by HRP-GAH-Ig- antibody（Goat anti human's secondary antibody of horseradish peroxidase-labeled, Abbkine）By 1:5000
Dilute again, after adding 100 μ l, 37 DEG C of 1h per hole, with 0.05%PBST board-washings three times.
6. adding TMB100 μ l, lucifuge is stored at room temperature 20min.
7.1mol/LHCl100 μ l terminating reactions.
8. determine the sample OD values under 450nm wavelength with ELIASA.
Figure 12 and Figure 13 are shown, in the acceptor TACI of nano antibody and BAFF antigens and the competition of BAFF antigen bindings
In ELISA experiments, it is found that nano antibody acts on TACI inhibiting rate up to 33%, and act on special（The anti-CD20 nanometers of unrelated control resist
Body result is negative）.
Embodiment 8：Anti- BAFF nano antibodies stimulate BAFF the inhibitory action of Raji cells propagation
1. cell culture：Raji cells（Human B lymphocyte knurl, is purchased from ATCC）In 10%FBS 1640 cultures（Gibico
Company）Based on cellar culture under the conditions of 37 DEG C, 5%CO2.
2. cell count：Culture cell is counted, cell is configured to 106After/ml cell suspension, added with every μ l of hole 100
Enter into 96 porocyte culture plates, per hole about 105Individual cell.
3.BAFF stimulates Raji cells propagation：By BAFF antigens（It is prepared by embodiment 1）By final concentration 0ng/ml, 125ng/
Ml, 250ng/ml, 500ng/ml, 750ng/ml, 1 μ g/ml, control group are cultivated for 100 μ l serum-free modified forms RPMI-1640
Base, every group sets 3 repeating holes, after cultivating 24h, 48h, 72h respectively, adds 10 μ l CCK-8（Doj indo
Laboratories）, continue cellar culture 5h under the conditions of 37 DEG C, 5%CO2.Under 450nm wavelength, the absorbance in each hole is detected
Value, observation BAFF stimulate Raji cel l proliferations.
Table 4：BAFF stimulates Raji cells propagation
4. increment of the nano antibody to cell suppresses：After BAFF antigenic stimulus Raji cells breed 24h and 48h, add
Entering anti-BAFF nano antibodies 64 makes its final concentration of 0 μ g/ml, 25 μ g/ml, 50 μ g/ml, 75 μ g/ml, 100 μ g/ml, every group 3
Multiple holes, then continue to cultivate cell 48h, 24h respectively, BAFF antigenic stimulus Raji cells propagation is reached 72h.
Figure 14 shows that for 750ng/ml BAFF antigens to stimulating Raji cell 72h, cell is visible substantially to see propagation.
Nano antibody is added after BAFF antigenic stimulus 48h, the inhibitory action of cell proliferation is more obvious, and anti-BAFF nano antibodies
64 inhibiting rate is 50%.
Claims (9)
1. a kind of nano antibody of anti-B cell growth-stimulating factor (B cells-activating factor, BAFF), it is special
Sign is：Its amino acid sequence is shown in sequence table SEQ ID NO.1.
2. encode the gene order of the nano antibody of the anti-B cell growth-stimulating factor described in claim 1.
3. the gene order of the nano antibody of the anti-B cell growth-stimulating factor of coding according to claim 2, its feature exist
In：The gene order is the nucleotide sequence shown in sequence table SEQ ID NO.2.
4. include the expression vector of gene order described in Claims 2 or 3.
5. include the host cell of gene order described in Claims 2 or 3.
6. the host cell of claim 5 is used for the nano antibody for preparing the anti-B cell growth-stimulating factor described in claim 1
Purposes.
7. Pharmaceutical composition, it is characterised in that：Its include claim 1 described in anti-BAFF nano antibodies molecule as activity into
Part, and include pharmaceutical carriers；The pharmaceutical carriers are selected from cytotoxin, radio isotope, immune modulator, anti-angiogenesis
Agent, antiproliferative, promote apoptosis agent, therapeutic nucleic acids or chemotherapeutant.
8. the nano antibody of the anti-B cell growth-stimulating factor described in claim 1 is used for the use for preparing detection BAFF reagent
On the way.
9. the nano antibody of the anti-B cell growth-stimulating factor described in claim 1 is used to prepare treatment BAFF abnormal expression phases
The purposes of the medicine of related disorders；The BAFF abnormal expressions relevant disease is B cell non Hodgkin lymphom, B cell is chronic
Lymphocytic leukemia, Huppert's disease, the autoimmunity disease or inflammatory disease for being related to B cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410046088.0A CN104804091B (en) | 2014-01-29 | 2014-01-29 | Nano antibody for resisting B cell growth stimulating factor and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410046088.0A CN104804091B (en) | 2014-01-29 | 2014-01-29 | Nano antibody for resisting B cell growth stimulating factor and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104804091A CN104804091A (en) | 2015-07-29 |
| CN104804091B true CN104804091B (en) | 2018-03-27 |
Family
ID=53689314
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410046088.0A Active CN104804091B (en) | 2014-01-29 | 2014-01-29 | Nano antibody for resisting B cell growth stimulating factor and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104804091B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1492878A (en) * | 2000-06-16 | 2004-04-28 | �Ϻ���ͨ��ѧ | Antibody that immunospecifically bind to BLYS |
| CN103421113A (en) * | 2012-05-22 | 2013-12-04 | 武汉华鑫康源生物医药有限公司 | Anti-BLyS antibody |
| CN103497253A (en) * | 2013-09-26 | 2014-01-08 | 东南大学 | Nanometer antibody, encoding sequence and application of H2A.Z variant |
-
2014
- 2014-01-29 CN CN201410046088.0A patent/CN104804091B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1492878A (en) * | 2000-06-16 | 2004-04-28 | �Ϻ���ͨ��ѧ | Antibody that immunospecifically bind to BLYS |
| CN103421113A (en) * | 2012-05-22 | 2013-12-04 | 武汉华鑫康源生物医药有限公司 | Anti-BLyS antibody |
| CN103497253A (en) * | 2013-09-26 | 2014-01-08 | 东南大学 | Nanometer antibody, encoding sequence and application of H2A.Z variant |
Non-Patent Citations (1)
| Title |
|---|
| 骆驼纳米抗体在医疗领域研究进展;刘微等;《动物医学进展》;20130508;第34卷(第3期);101-105 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104804091A (en) | 2015-07-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107686520B (en) | anti-PD-L1 nano antibody and application thereof | |
| US11912770B2 (en) | Blocking type PD-L1 single-domain camel antibody and application thereof | |
| WO2016188449A1 (en) | Single-domain antibody targeting cd47 | |
| JP7289562B2 (en) | Anti-BCMA single domain antibody and its application | |
| CN112500480B (en) | Nanobodies against novel coronaviruses and uses thereof | |
| CN104926941B (en) | Ox source property anti-Staphylococcus aureus phage single chain antibody, preparation method and its usage | |
| CN104804092B (en) | A kind of nano antibody of anti-B cell growth-stimulating factor and application thereof | |
| CN110003335A (en) | VHH chain of CD47 single domain antibody, CD47 single domain antibody, nucleotide sequence and kit | |
| CN108299561A (en) | A kind of PD-1 nano antibodies and its cloning expression method and application | |
| CN105949316A (en) | Anti-EGFRvIII chimeric antigen receptor, encoding gene, recombinant expression vector, construction method of recombinant expression vector, and application | |
| CN114262377B (en) | Preparation method of anti-human CD70 nano antibody for blocking binding of CD70 and ligand CD27 thereof and coding sequence thereof | |
| CN108164600A (en) | A kind of resisting GPC 3 antibody and its preparation method and application | |
| CN109897110A (en) | Nano antibody and preparation method thereof | |
| CN104804090B (en) | Nano antibody for resisting B cell growth stimulating factor and application | |
| CN103724413B (en) | Trichina paramyosin B cell antigen epi-position 8A1 and application thereof | |
| CN102174106B (en) | Anti-Met humanized Fab, anti-Met humanized Fab and doxorubicin conjugate and preparation method and application of anti-Met humanized Fab and doxorubicin conjugate | |
| CN106854244A (en) | A kind of nano antibody and its clinical practice for HER3 | |
| WO2023125842A1 (en) | Development of novel upar single-domain antibody | |
| CN104804091B (en) | Nano antibody for resisting B cell growth stimulating factor and application | |
| CN108276475B (en) | Chicken bone marrow-derived dendritic cell targeting peptide AH and application thereof | |
| CN106928358B (en) | CD105 nano antibody Nb168 | |
| CN106928355B (en) | CD105 nano antibody Nb184 | |
| CN111333732B (en) | Preparation and application of bispecific antibody targeting human BCMA and activating NK cells | |
| CN106928360B (en) | CD105 nano antibody Nb68 | |
| CN106928359B (en) | CD105 nano antibody Nb59 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20170607 Address after: Zi street Jinhui road Shenzhen 518118 Guangdong province Pingshan pit No. 14 Shenzhen Biomedical Innovation Industrial Park, building 1, 605A Applicant after: PREGENE (SHENZHEN) BIOTECHNOLOGY CO.,LTD. Address before: 300457 No. 220, Dongting Road, Tianjin economic and Technological Development Zone, Binhai New Area, Tianjin, thirteen floor, experimental building, Tianjin international biological medicine Joint Research Institute, S1305 Applicant before: TIANJIN SHENGFA NABIO TECH Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190428 Address after: 518000 Building 402, Shenzhen Biomedical Innovation Industrial Park, No. 14 Jinhui Road, Kengzi Street, Pingshan District, Shenzhen City, Guangdong Province Co-patentee after: PREGENE (SHENZHEN) BIOTECHNOLOGY CO.,LTD. Patentee after: SHENZHEN PREGENE BIOPHARMA Co.,Ltd. Address before: 518118 Building 605A, Shenzhen Biomedical Innovation Industrial Park, 14 Jinhui Road, Kengzi Street, Pingshan New District, Shenzhen City, Guangdong Province Patentee before: PREGENE (SHENZHEN) BIOTECHNOLOGY CO.,LTD. |
|
| EE01 | Entry into force of recordation of patent licensing contract | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20150729 Assignee: Shenzhen south intellectual property operation center Co.,Ltd. Assignor: SHENZHEN PREGENE BIOPHARMA Co.,Ltd. Contract record no.: X2020980008793 Denomination of invention: A nano antibody against B cell growth stimulating factor and its application Granted publication date: 20180327 License type: Exclusive License Record date: 20201203 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A nano antibody against B cell growth stimulating factor and its application Effective date of registration: 20201207 Granted publication date: 20180327 Pledgee: Shenzhen south intellectual property operation center Co.,Ltd. Pledgor: SHENZHEN PREGENE BIOPHARMA Co.,Ltd. Registration number: Y2020980008886 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20220401 Granted publication date: 20180327 Pledgee: Shenzhen south intellectual property operation center Co.,Ltd. Pledgor: SHENZHEN PREGENE BIOPHARMA Co.,Ltd. Registration number: Y2020980008886 |
|
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: Room 605, building 1, Shenzhen Biomedical Innovation Industrial Park, 14 Jinhui Road, Jinsha community, Kengzi street, Pingshan District, Shenzhen, Guangdong 518000 Patentee after: Shenzhen prijin biopharmaceutical Co.,Ltd. Patentee after: PREGENE (SHENZHEN) BIOTECHNOLOGY CO.,LTD. Address before: 518000 Building 402, Shenzhen Biomedical Innovation Industrial Park, No. 14 Jinhui Road, Kengzi Street, Pingshan District, Shenzhen City, Guangdong Province Patentee before: SHENZHEN PREGENE BIOPHARMA Co.,Ltd. Patentee before: PREGENE (SHENZHEN) BIOTECHNOLOGY CO.,LTD. |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A nano antibody against B cell growth stimulating factor and its application Effective date of registration: 20230216 Granted publication date: 20180327 Pledgee: Shenzhen hi tech investment small loan Co.,Ltd. Pledgor: Shenzhen prijin biopharmaceutical Co.,Ltd. Registration number: Y2023980032758 |