CN103961694A - 基于p-硝基苯丙氨酸插入法构建的骨质疏松疫苗 - Google Patents
基于p-硝基苯丙氨酸插入法构建的骨质疏松疫苗 Download PDFInfo
- Publication number
- CN103961694A CN103961694A CN201410155986.XA CN201410155986A CN103961694A CN 103961694 A CN103961694 A CN 103961694A CN 201410155986 A CN201410155986 A CN 201410155986A CN 103961694 A CN103961694 A CN 103961694A
- Authority
- CN
- China
- Prior art keywords
- rankl
- osteoporosis
- vaccine
- phenylalanine
- alanine
- 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.)
- Pending
Links
- 208000001132 Osteoporosis Diseases 0.000 title claims abstract description 26
- 229960005486 vaccine Drugs 0.000 title claims abstract description 23
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 title claims abstract description 14
- 235000004279 alanine Nutrition 0.000 title claims abstract description 14
- LQNUZADURLCDLV-UHFFFAOYSA-N nitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC=C1 LQNUZADURLCDLV-UHFFFAOYSA-N 0.000 title claims abstract description 13
- 238000012966 insertion method Methods 0.000 title abstract 2
- 108010025832 RANK Ligand Proteins 0.000 claims abstract description 41
- 102000014128 RANK Ligand Human genes 0.000 claims abstract description 36
- 238000000746 purification Methods 0.000 claims abstract description 20
- 210000002997 osteoclast Anatomy 0.000 claims abstract description 16
- 238000004458 analytical method Methods 0.000 claims abstract description 12
- 210000002966 serum Anatomy 0.000 claims abstract description 12
- 238000003780 insertion Methods 0.000 claims abstract description 11
- 230000037431 insertion Effects 0.000 claims abstract description 11
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 238000003259 recombinant expression Methods 0.000 claims abstract description 8
- 241001465754 Metazoa Species 0.000 claims abstract description 7
- 238000012216 screening Methods 0.000 claims abstract description 7
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 6
- 239000013604 expression vector Substances 0.000 claims abstract description 6
- 235000001014 amino acid Nutrition 0.000 claims abstract description 5
- 150000001413 amino acids Chemical class 0.000 claims abstract description 5
- 238000006396 nitration reaction Methods 0.000 claims description 16
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 12
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 12
- 230000035772 mutation Effects 0.000 claims description 7
- 108020004705 Codon Proteins 0.000 claims description 6
- 241000588724 Escherichia coli Species 0.000 claims description 5
- 238000006073 displacement reaction Methods 0.000 claims description 5
- 238000005516 engineering process Methods 0.000 claims description 5
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 5
- DSSYKIVIOFKYAU-XCBNKYQSSA-N (R)-camphor Chemical compound C1C[C@@]2(C)C(=O)C[C@@H]1C2(C)C DSSYKIVIOFKYAU-XCBNKYQSSA-N 0.000 claims description 4
- 229960000846 camphor Drugs 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 2
- 108020005098 Anticodon Proteins 0.000 claims description 2
- 108091026890 Coding region Proteins 0.000 claims description 2
- 108020004566 Transfer RNA Proteins 0.000 claims description 2
- 238000003757 reverse transcription PCR Methods 0.000 claims description 2
- 210000002536 stromal cell Anatomy 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 7
- 230000003472 neutralizing effect Effects 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 5
- 238000010276 construction Methods 0.000 abstract 1
- 230000006058 immune tolerance Effects 0.000 abstract 1
- 230000003053 immunization Effects 0.000 abstract 1
- 238000002649 immunization Methods 0.000 abstract 1
- 238000006386 neutralization reaction Methods 0.000 abstract 1
- 238000005215 recombination Methods 0.000 abstract 1
- 230000006798 recombination Effects 0.000 abstract 1
- 102000004169 proteins and genes Human genes 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 235000018102 proteins Nutrition 0.000 description 14
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 239000000872 buffer Substances 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 239000007788 liquid Substances 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 230000008859 change Effects 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 6
- 210000000988 bone and bone Anatomy 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 238000011740 C57BL/6 mouse Methods 0.000 description 5
- 102100032236 Tumor necrosis factor receptor superfamily member 11B Human genes 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 239000012531 culture fluid Substances 0.000 description 5
- 230000001900 immune effect Effects 0.000 description 5
- 230000036039 immunity Effects 0.000 description 5
- 230000001009 osteoporotic effect Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 5
- 210000000689 upper leg Anatomy 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 206010010254 Concussion Diseases 0.000 description 4
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 4
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 108010027767 Rank-Fc Proteins 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 238000000137 annealing Methods 0.000 description 4
- 230000009514 concussion Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 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 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 108700020797 Parathyroid Hormone-Related Proteins 0.000 description 3
- 102000043299 Parathyroid hormone-related Human genes 0.000 description 3
- 102100028787 Tumor necrosis factor receptor superfamily member 11A Human genes 0.000 description 3
- 101710178436 Tumor necrosis factor receptor superfamily member 11A Proteins 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000002158 endotoxin Substances 0.000 description 3
- 239000002532 enzyme inhibitor Substances 0.000 description 3
- 229940125532 enzyme inhibitor Drugs 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 229920006008 lipopolysaccharide Polymers 0.000 description 3
- 210000001165 lymph node Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 208000006386 Bone Resorption Diseases 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 238000009007 Diagnostic Kit Methods 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 208000035896 Twin-reversed arterial perfusion sequence Diseases 0.000 description 2
- 230000002146 bilateral effect Effects 0.000 description 2
- 230000036760 body temperature Effects 0.000 description 2
- 230000037182 bone density Effects 0.000 description 2
- 230000024279 bone resorption Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 230000009137 competitive binding Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 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 2
- 239000003446 ligand Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 230000002611 ovarian Effects 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 238000000197 pyrolysis Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 238000002271 resection Methods 0.000 description 2
- 238000005316 response function Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000002303 tibia Anatomy 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 229940122361 Bisphosphonate Drugs 0.000 description 1
- 208000010392 Bone Fractures Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000017897 Carcinoma of esophagus Diseases 0.000 description 1
- 206010010582 Congenital osteodystrophy Diseases 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 208000007652 Dysostoses Diseases 0.000 description 1
- 201000001324 Dysostosis Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010017076 Fracture Diseases 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 102000004889 Interleukin-6 Human genes 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 206010030216 Oesophagitis Diseases 0.000 description 1
- 102000004067 Osteocalcin Human genes 0.000 description 1
- 108090000573 Osteocalcin Proteins 0.000 description 1
- 206010031264 Osteonecrosis Diseases 0.000 description 1
- 208000001164 Osteoporotic Fractures Diseases 0.000 description 1
- 108010035042 Osteoprotegerin Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 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 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 150000004663 bisphosphonates Chemical class 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000004097 bone metabolism Effects 0.000 description 1
- 208000019664 bone resorption disease Diseases 0.000 description 1
- 230000003913 calcium metabolism Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 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
- 229940097572 chloromycetin Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 229960001251 denosumab Drugs 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- XQRLCLUYWUNEEH-UHFFFAOYSA-L diphosphonate(2-) Chemical compound [O-]P(=O)OP([O-])=O XQRLCLUYWUNEEH-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000009547 dual-energy X-ray absorptiometry Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 201000005619 esophageal carcinoma Diseases 0.000 description 1
- 208000006881 esophagitis Diseases 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 229960002348 hepatitis b surface antigen vaccine Drugs 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound 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 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000010603 microCT Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 1
- 230000004719 natural immunity Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- XXUPLYBCNPLTIW-UHFFFAOYSA-N octadec-7-ynoic acid Chemical compound CCCCCCCCCCC#CCCCCCC(O)=O XXUPLYBCNPLTIW-UHFFFAOYSA-N 0.000 description 1
- 238000009806 oophorectomy Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 230000008807 pathological lesion Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229960002275 pentobarbital sodium Drugs 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 208000001685 postmenopausal osteoporosis Diseases 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940023143 protein vaccine Drugs 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 238000000547 structure data Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000028016 temperature homeostasis Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 1
- 235000005282 vitamin D3 Nutrition 0.000 description 1
- 239000011647 vitamin D3 Substances 0.000 description 1
- 229940021056 vitamin d3 Drugs 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明公开了一种基于P-硝基苯丙氨酸插入法构建的骨质疏松疫苗，其特征在于通过如下步骤制备：1)RANKL重组表达载体的构建；2)非天然氨基酸p-硝基化苯丙氨酸编码序列的定点插入/置换；3)p-硝基化苯丙氨酸-RANKL蛋白的重组表达与纯化；4)动物免疫与筛选；5)基于血清中和检测及破骨细胞形成分析后的筛选；6)动物实验，确定所需疫苗。该方法有效突破机体对自体蛋白的免疫耐受，通过主动免疫手段，诱导机体产生针对RANKL的特异性中和抗体，实现对骨质疏松良好的防治；同时克服或绕过工业化生产抗体的高额成本问题。
Description
技术领域
本发明涉及一种治疗骨质疏松症的药物，具体地说是基于P-硝基苯丙氨酸插入法构建的针对破骨细胞分化因子受体配体(RANKL)产生抗体的疫苗，本发明还涉及该药物的制备方法。
背景技术
骨质疏松症主要是一种表现为骨量的减少、骨骼微细结构的破坏，使得骨骼的承受力和强度降低，易于发生脆性骨折一类全身性骨代谢障碍疾病，也是多种骨破坏性疾病共有的病理损害。骨质疏松发病率高，且有逐年增高趋势。骨质疏松性骨折严重影响患者的生活质量，威胁患者的生命安全，同时也带来沉重的经济负担。目前治疗骨质疏松性疾病的药物包括钙剂、维生素D3、双膦酸盐等药物，每种治疗手段都有其局限性。比如，随着双磷酸盐类药物的广泛应用，其长期使用所引起的一些副作用(下颌骨坏死、食道炎以及最近报道的食道癌病例)也不容忽视。因此，仍迫切地需要探索更加有效、安全、简便和经济的治疗手段。
骨骼的强度和完整性，依赖于骨骼内骨吸收和骨形成之间的动态平衡，主要体现在对成骨细胞和破骨细胞之间数量和功能状态的精细调节。破骨细胞活动的过度增强是导致骨质疏松的主要病理机制。OPG/RANKL-RANK信号通路(OPG：骨保护素；RANKL：破骨细胞分化因子受体配体；RANK：破骨细胞分化因子受体)是机体控制破骨细胞分化成熟和功能的关键信号通路，通过阻断这一关键信号通路，可以抑制破骨细胞的分化和破骨活性，将实现对骨吸收作用的有效控制，以及对骨质疏松症等骨吸收性疾病的治疗。抗RANKL抗体可以特异性阻断RANKL与其受体的结合，从信号转导途径的上游有效地阻断破骨分化信号的发放，实现对骨质疏松的治疗。目前由美国Amgen公司研制的人源化抗RANKL抗体(商品名denosumab)治疗骨质疏松症的II期临床已经完成，安全性和疗效都得到充分的肯定，但是人源化抗体生产的高额成本使价格问题成为其市场化的一大障碍。
骨质疏松疫苗就是通过主动免疫手段，诱导机体产生针对RANKL的特异性中和抗体，实现对骨质疏松良好的治疗，同时克服或绕过工业化生产抗体的高额成本问题。目前，研制自体蛋白疫苗面临的一个共同技术挑战是如何克服机体对自体蛋白的免疫耐受。目前尚无能够克服机体对自体蛋白RANKL的免疫耐受，从而达到对骨质疏松产生治疗效果的疫苗。
发明内容
为了解决背景技术中所存在的问题，本发明提供了一种基于P-硝基苯丙氨酸插入法构建的骨质疏松疫苗，可以有效地克服自体蛋白RANKL的免疫耐受，从而实现对骨质疏松的治疗。
本发明采用的技术方案是：
(1)RANKL重组表达载体的构建。从骨髓基质细胞中提取RNA，通过RT-PCR扩增RANKL基因编码区，将胞外段克隆入表达载体，构建重组表达载体。
(2)非天然氨基酸p-硝基化苯丙氨酸编码序列的定点插入/置换。通过基于PCR的体外突变技术定点将RANKL基因编码酪氨酸或苯丙氨酸的密码子置换成能被非天然氨基酸p-硝基化苯丙氨酸反密码子特异识别的琥珀突变密码子TAG。也可突变其他位点，或增加突变位点，或不同突变组合的复合突变体。
(3)p-硝基化苯丙氨酸-RANKL蛋白的重组表达与纯化。在共转化表达琥珀突变抑制子tRNA(mutRNACUA)和p-硝基苯丙氨酸-tRNA合成酶的大肠杆菌内表达插入p-硝基化苯丙氨酸RANKL突变体，纯化重组蛋白。
(4)动物免疫与筛选。用纯化的蛋白皮下注射免疫雌性C57BL/6小鼠，通过ELISA检测血清中抗体的产生，并测定滴度。筛选出能够产生高滴度RANKL抗体的突变蛋白。
(5)基于血清中和检测及破骨细胞形成分析后的筛选。建立RANKL和RANK体外结合实验，通过竞争性结合分析检测RANKL突变体免疫小鼠的抗血清对RANKL和RANK结合的中和效应。选择能诱导小鼠产生高滴度、良好中和效应的p-硝基化苯丙氨酸RANKL突变体。大量表达和纯化重组蛋白，即可作为候选疫苗。
(6)动物实验：观察p-硝基苯丙氨酸-RANKL重组蛋白对骨质疏松的防治效果，挑选证实对骨质疏松有良好治疗效果的重组蛋白，大量表达纯化，作为骨质疏松疫苗。
该方法有效突破机体对自体蛋白的免疫耐受，通过主动免疫手段，诱导机体产生针对RANKL的特异性中和抗体，实现对骨质疏松良好的防治；同时克服或绕过工业化生产抗体的高额成本问题。
附图说明
图1是本发明基于P-硝基苯丙氨酸插入法构建骨质疏松疫苗的流程图
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行进一步地描述。
实施例1：p-硝基化苯丙氨酸置换型RANKL疫苗的构建与筛选
(1)RANKL重组表达载体的构建
购取RANKL cDNA克隆，通过PCR扩增RANKL胞外段编码序列，将PCR产物用限制性双酶切，连入同样经过上述双酶切处理的载体(如pET28a等)，构建相应的重组表达载体(如pET28a-RANKL重组表达载体)。通过双向测序确认序列的正确性。
(2)RANKL基因的定点突变(以突变编码酪氨酸的位点为例)
①RANKL蛋白的三维结构分析，确定突变/置换的酪氨酸残基。从蛋白数据库中调取RANKL的晶体结构数据，用Cn3D4.1软件对蛋白结构进行检查分析，并逐个检查蛋白序列中酪氨酸在三维结构中的位置。分析结果表明，RANKL胞外段有8个酪氨酸残基，其中Y187，Y234，Y240，Y272和Y306(Y为酪氨酸)暴露于球蛋白结构表面，可以作为抗原表位的候选位点。
②定点突变：通过基于PCR的体外突变技术将上述编码酪氨酸残基的密码子序列突变成琥珀突变密码子TAG。方法参照【Chiu J，March PE，Lee R，TillettD.Site-directed，Ligase-Independent Mutagenesis(SLIM):a single-tubemethodology approaching100％efficiency in4h.Nucleic Acids Res.2004Dec7；32(21)：e174.】。a.设计突变引物。b.设立突变PCR反应：方法以Y187NO2F突变为例：配制两管50μl高保真PCR反应(含20ng pET28a-RANKL质粒)，按照下列组合添加引物：管1：20pmol Y187NO2F-FL+Y187NO2F-RS，管2：20pmol Y187NO2F-FS+Y187NO2F-RL。加入2U高保真DNA聚合酶(iProof，BIORAD)，进行PCR扩增反应：98℃ 2min→(进入循环)98℃ 20s→55℃ 30s→72℃ 5min(20个循环)→72℃ 10min。(取1μl PCR产物进行琼脂糖电泳分析，判定扩增效果。如果产物条带特异，则进行下述实验；如果无扩增产物或条带不特异，则需要优化PCR反应，如采用热启动、改变退火温度和时间等)。退火反应：从管1和管2各取20μl PCR反应产物，在一个新的PCR管混合，在PCR仪上进行退火反应：98℃ 5min→85℃ 5min→80℃ 5min→75℃ 5min→70℃ 5min→65℃ 5min→60℃ 5min→55℃ 5min→50℃ 10min.用PCR产物纯化试剂盒将退火反应产物纯化，Dpn I酶切8hr(去除野生型)，直接转化大肠杆菌，挑取四个克隆，提取质粒，通过序列测定进行鉴定。对正确突变克隆，再做反向测序进行确认。
(3)野生型与p-硝基苯丙氨酸-RANKL蛋白的重组表达与纯化
①野生型RANKL蛋白的重组表达：将表达载体转化BL-21(DE3)大肠杆菌。挑取单个克隆，接种至5ml含有相应抗生素的LB液体培养基，37℃震荡培养过夜，然后转接入2L含有上述三种抗生素的2×YT液体培养基(30g胰蛋白胨，16g酵母提取物，5g NaCl，1L双蒸水，121℃消毒1hr)，37℃震荡培养8hr，菌液OD600为0.6-1时加入IPTG至终浓度为0.2mM，继续培养7小时。离心收集菌体，取其中少量(约1ml菌液)用于检测蛋白的表达，其余-80℃冻存备用。
②p-硝基化苯丙氨酸mRANKL突变体的表达：将携带p-硝基化苯丙氨酸RANKL突变体的表达质粒与表达琥珀突变抑制子tRNA(mutRNACUA)和p-硝基苯丙氨酸-tRNA合成酶的质粒(mutNO2PheRS)共转化BL-21(DE3)大肠杆菌，在同时含有氨苄青霉素、卡那霉素和氯霉素的琼脂培养基上筛选阳性转化子。挑取单个阳性克隆，接种至5ml含有相应抗生素和1mM p-硝基苯丙氨酸的培养液，37℃震荡培养10hr后，转接入2L上述培养液，继续37℃震荡培养至OD600＝0.5，加入0.02％阿拉伯糖，半小时后加入1mM IPTG，10-37℃持续震荡培养4-16hr。离心收集菌体，取其中少量(约1ml菌液)用于检测蛋白的表达，其余-80℃冻存备用。
③重组蛋白的纯化(野生与突变蛋白纯化方案类同)：A.细胞裂解。1L菌液收集的菌体以50ml T-150(20mM Tris.HCl pH8.0，150mM氯化钠l，10％甘油)洗涤、离心沉淀后，用50ml冰冷的裂解缓冲液(20mM Tris.HCl pH8.0，150mM氯化钠，10％甘油，0.5％TritonX-100，5mMβ-巯基乙醇，5mM咪唑，复合蛋白酶抑制剂(Roche))重悬，放置于冰上超声破菌(20s x5，功率200W，间隔30s)。B.亲和层析初步纯化(常压)将细胞裂解液50000g离心20min，将上清转移至新的50ml塑料离心管，加入3ml镍亲和层析基质，4℃上下颠倒旋转2hr，然后将混悬液加入5ml玻璃层析柱，利用重力除去液体。以10ml细胞裂解液洗涤柱体，再以50ml T-400缓冲液洗涤(20mM Tri s.HCl pH8.0，600mM氯化钠，10％glycerol，80mM咪唑)，然后以10ml含复合蛋白酶抑制剂剂的T-150缓冲液平衡。最后，以10ml洗脱缓冲液(20mM Tris.HCl pH8.0，150mM氯化钠，10％甘油，300mM咪唑，复合蛋白酶抑制剂)将蛋白洗脱，取5μl通过12％SDS-PAGE电泳鉴定纯度和估计产率。C.离子交换层析(FPLC)将洗脱蛋白溶液以10倍20Mm T150缓冲液稀释，同过超级上样环(Super Loop)上样至经过T150缓冲液平衡过的5mi SOURCE-Q阴离子交换柱，以T150作为基本缓冲液，氯化钠梯度洗脱(150-1000mM)，分段收集，从各个洗脱组分中取5μl进行12％SDS-PAGE电泳分析，确定蛋白分离纯化。D.根据蛋白经过B或C步骤纯化的具体情况设计增加后续的纯化方式。备选方案：高分辨率的Mono-Q和S；另外B和C的先后可做不同组合。E.蛋白的浓缩与保存洗脱的蛋白溶液经过凝胶过滤除盐，BCA kit(Sigma)定量，冻干，-20℃保存备用。
(4)确定p-硝基苯丙氨酸的置换与活性分析
将纯化的蛋白(野生型和各种突变体)进行：1)一般的生化分析，如测定大小的变化、蛋白的稳定性；2)同时送样进行质谱分析，确定p-硝基苯丙氨酸的置换以及稳定性；3)受体结合活性分析。将野生型RANKL重组包被在ELISA用的96孔板底(10μg/ml溶于50mM碳酸氢钠缓冲液，pH＝8.5)，将小鼠血清用含有16ng/ml RANK-Fc重组蛋白(向国外实验室获得表达载体，本实验室表达纯化)和0.05％吐温-20的的PBS缓冲液做倍比稀释，然后加入包被有mRANKL蛋白的孔中(100μl/孔)，室温孵育2hr；吸去上清，加入200μl含0.05％吐温-20的的PBS缓冲液洗涤3次。结合的RANK-FC用辣根过氧化物酶标记的抗人IgG抗体检测。
实施例2：p-硝基化苯丙氨酸-RANKL重组疫苗对骨质疏松小鼠的治疗效应
1)小鼠骨质疏松模型的制备根据相关文献，我们采用雌性C57BL/6小鼠行卵巢切除术模拟绝经后骨质疏松症模型。
2)动物免疫与生物学效应观察：选取12周龄的雌性C57BL/6小鼠48只，随机分成四组(每组12只)，
一组：抗原免疫+卵巢切除手术
二组：抗原免疫+假手术
三组：PBS注射+卵巢切除手术
四组：PBS注射+假手术
实验动物均在25±2℃，通风良好条件下分笼饲养，配对摄食、饮水。一、二组小鼠分别在0，14，21，42和63天，靠近淋巴结附近皮下注射10μg/只p-硝基化苯丙氨酸-RANKL重组疫苗；第二、三组注射无菌200μl PBS。第一次免疫后第28天，一、三组(免疫一组和对照组)施以卵巢完全切除术：用戊巴比妥钠腹腔注射麻醉(20mg/kg)，下腹部剪毛消毒后，剪开约1cm的正中切口，模型组行双侧卵巢结扎切除术，逐层缝合，术后肌注青霉素预防感染。假手术组除未行卵巢结扎切除外，其余步骤同模型组。术后12周测量各项指标。
3)各项骨代谢指标检测
①抗体产生与滴度(ELISA)：处死动物，收集血液，分离血清，以野生型RANKL和作为抗原包被在96孔板底，用辣根过氧化物酶偶联的羊抗小鼠IgG抗体进行ELISA实验，检测血清中抗体的产生，并测定滴度。
②钙代谢生物学指标的测定：血钙、磷测定，血中骨钙素的水平
③骨生物学参数的测定：A.骨密度(bone mineral density，BMD)运用DPX L型(美国Lunar)双能X线骨密度检测仪，通过计算机系统软件测量每只小鼠双侧股骨的骨密度，测量数据以SPSS11.5软件处理和分析数据；数据以x±s表示，组间比较采用t检验；B.通过立体结构/密度综合扫描，取小鼠右侧股骨，放入直径为60mm的装满PBS溶液的丙烯管内，用Micro-CT(GEHealthcare)围绕股骨分别进行纵轴旋转扫描和横切扫描(0.5度/层，共200度)，三维像素为33x33x33μm，依据仪器生产商提供的通用优化的阈值，对数据进行分析，获得骨结构和密度综合的评价数据，再以SPSS11.5软件处理和分析数据。
4)血清中和实验(通过竞争性结合分析检测RANKL突变体免疫小鼠的抗血清对RANKL和RANK结合的中和效应)将重组野生型mRANKL蛋白包被在ELISA用的96孔板底(10μg/ml溶于50mM碳酸氢钠缓冲液，pH＝8.5)，将小鼠抗血清用含有16ng/ml RANK-Fc重组蛋白(向国外实验室获得表达载体，本实验室表达纯化)和0.05％吐温-20的的PBS缓冲液做倍比稀释，然后加入包被有野生型mRANKL蛋白的孔中(100μl/孔)，室温孵育2hr；吸去上清，加入200μl含0.05％吐温-20的的PBS缓冲液洗涤3次。结合的RANK-FC用辣根过氧化物酶标记的抗人IgG抗体检测。
5)破骨细胞形成抑制分析
根据已建立的PTHrP刺激破骨细胞细胞生成系统和M-CSF+RANKL刺激破骨细胞生成系统，在破骨细胞形成分析的基础之上加入免疫小鼠抗血清，观察小鼠抗血清对破骨细胞形成的抑制效应。
①PTHrP刺激法。取C57BL/6小鼠股骨和胫骨，用αMEM冲洗骨髓腔，收集骨髓细胞，在含10％胎牛血清和45ng/mlPTHrP+100100μl免疫小鼠血清的αMEM培养液中培养6-8天(3天更换新鲜培养液以及所有上述添加蛋白)固定细胞，用TRAP染色试剂盒(Sigma诊断试剂盒)检测酒石酸抗性酸性磷酸酶(破骨细胞标志)，显微镜下计破骨细胞数。以OPG作为阳性对照，不加免疫小鼠血清的PTHrP组为阴性对照。
②M-CSF+RANKL刺激法。取C57BL/6小鼠股骨和胫骨，用αMEM冲洗骨髓腔，收集骨髓细胞，在含10％胎牛血清和100ng/ml M-CSF的αMEM培养液中培养3天。0.02％EDTA消化收集悬浮生长的细胞，在10％胎牛血清的αMEM培养液中，每孔7.5×105细胞密度，加入30ng/ml M-CSF+1μg/ml商品化RANKL蛋白+100μl免疫小鼠血清，在48孔培养板内培养6-8天(3天更换新鲜培养液以及所有上述添加蛋白)。固定细胞，用TRAP染色试剂盒(Sigma诊断试剂盒)检测酒石酸抗性酸性磷酸酶(破骨细胞标志)，显微镜下计破骨细胞数。以OPG作为阳性对照，不加免疫小鼠血清的商品化RANKL组为阴性对照。
实施例3：RANKL重组疫苗安全性的观察
主要观察p-硝基化苯丙氨酸-RANKL重组疫苗对免疫系统和体温调节功能的影响
1)全血细胞的组成：血细胞计数仪检测红、白细胞、淋巴细胞和血小板的数量/比例；流式细胞仪检测分析淋巴细胞的组成(B淋巴细胞、T淋巴细胞(CD4+和CD8+)有无改变)。
2)淋巴结和脾组织切片：从每个分组小鼠中抽样选取3个小鼠的肠系膜淋巴结和脾脏以10％福尔马林固定，石蜡包被，切成5μm的组织切片。脱石蜡，分别进行HE染色和用抗CD3抗体作免疫组化染色，观察p-硝基化苯丙氨酸-RANKL重组疫苗免疫小鼠主要淋巴组织结构有无异常改变。
3)免疫功能分析：天然免疫和抗原特异性免疫反应功能的完整性(脂多糖LPS刺激，检测TNF-α，IL-6，IL-1，和IFN-α反映天然免疫的功能；乙肝表面抗原疫苗，检测特异性抗体产生反映抗原特异性免疫反应功能。)
4)测定免疫小鼠的基础体温，以及脂多糖刺激后体温(炎症引起的发热反应)，采用微型红外测温仪测定耳廓温度。
5)停止免疫后1，3，6个月，分别用ELISA检测抗体的滴度变化。
6)其它：骨和肾组织切片，通过免疫组织化学检测有无抗原抗体
对骨质疏松具有良好防治效果、安全性好的p-硝基苯丙氨酸-RANKL重组蛋白即为所需的骨质疏松疫苗。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (4)
1.一种基于P-硝基苯丙氨酸插入法构建的骨质疏松疫苗，其特征在于通过如下步骤制备：1)RANKL重组表达载体的构建；2)非天然氨基酸p-硝基化苯丙氨酸编码序列的定点插入/置换；3)p-硝基化苯丙氨酸-RANKL蛋白的重组表达与纯化；4)动物免疫与筛选；5)基于血清中和检测及破骨细胞形成分析后的筛选；6)动物实验，确定所需疫苗。
2.如权利要求1所述的基于P-硝基苯丙氨酸插入法构建的骨质疏松疫苗，其特征在于：从骨髓基质细胞中提取RNA，通过RT-PCR扩增RANKL基因编码区，将胞外段克隆入表达载体，构建重组表达载体。
3.如权利要求1所述的基于P-硝基苯丙氨酸插入法构建的骨质疏松疫苗，其特征在于：所述定点插入/置换是通过基于PCR的体外突变技术定点将RANKL基因编码酪氨酸或苯丙氨酸的密码子置换成能被非天然氨基酸p-硝基化苯丙氨酸反密码子特异识别的琥珀突变密码子TAG。
4.如权利要求1所述的基于P-硝基苯丙氨酸插入法构建的骨质疏松疫苗，其特征在于：在共转化表达琥珀突变抑制子tRNA(mutRNACUA)和p-硝基苯丙氨酸-tRNA合成酶的大肠杆菌内表达插入p-硝基化苯丙氨酸RANKL突变体，纯化重组蛋白。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410155986.XA CN103961694A (zh) | 2014-04-18 | 2014-04-18 | 基于p-硝基苯丙氨酸插入法构建的骨质疏松疫苗 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410155986.XA CN103961694A (zh) | 2014-04-18 | 2014-04-18 | 基于p-硝基苯丙氨酸插入法构建的骨质疏松疫苗 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103961694A true CN103961694A (zh) | 2014-08-06 |
Family
ID=51232012
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410155986.XA Pending CN103961694A (zh) | 2014-04-18 | 2014-04-18 | 基于p-硝基苯丙氨酸插入法构建的骨质疏松疫苗 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103961694A (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105597927A (zh) * | 2016-03-04 | 2016-05-25 | 合肥工业大学 | 一种工地道路扬尘处理系统 |
| CN112375774A (zh) * | 2020-10-26 | 2021-02-19 | 浙江新码生物医药有限公司 | 一种重组蛋白表达用工程菌株的构建方法 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102755646A (zh) * | 2002-07-19 | 2012-10-31 | 艾博特生物技术有限公司 | TNF α相关疾病的治疗 |
-
2014
- 2014-04-18 CN CN201410155986.XA patent/CN103961694A/zh active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102755646A (zh) * | 2002-07-19 | 2012-10-31 | 艾博特生物技术有限公司 | TNF α相关疾病的治疗 |
Non-Patent Citations (2)
| Title |
|---|
| ADMIN: "2010年国家自然科学基金资助清单-医学科学部", 《HTTP://D.DXY.CN/PREVIEW/2091181》 * |
| JAN ET AL: "Immunochemical termination of self-tolerance", 《PNAS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105597927A (zh) * | 2016-03-04 | 2016-05-25 | 合肥工业大学 | 一种工地道路扬尘处理系统 |
| CN105597927B (zh) * | 2016-03-04 | 2017-08-25 | 合肥工业大学 | 一种工地道路扬尘处理系统 |
| CN112375774A (zh) * | 2020-10-26 | 2021-02-19 | 浙江新码生物医药有限公司 | 一种重组蛋白表达用工程菌株的构建方法 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Schmitz et al. | IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines | |
| Roškar et al. | The role of inflammasomes in osteoarthritis and secondary joint degeneration diseases | |
| Murata et al. | CD69‐null mice protected from arthritis induced with anti‐type II collagen antibodies | |
| US20080241130A1 (en) | Methods and compositions for modulating il-17f/il-17a biological activity | |
| KR20150020734A (ko) | Ｉｌ-１７ 길항제를 사용한 류마티스성 관절염의 치료 방법 | |
| KR20160125409A (ko) | 인간 페리오스틴을 검출하기 위한 신규 검정법 | |
| Wang et al. | Evaluation of the immune response afforded by combined immunization with orf virus DNA and subunit vaccine in mice | |
| Sazinsky et al. | Antibodies targeting human or mouse VSIG4 repolarize Tumor-Associated macrophages providing the potential of potent and specific clinical Anti-Tumor response induced across multiple Cancer types | |
| JP2022545312A (ja) | 関節リウマチの診断及び処置の方法 | |
| CN103961694A (zh) | 基于p-硝基苯丙氨酸插入法构建的骨质疏松疫苗 | |
| Warren et al. | Manipulation of receptor oligomerization as a strategy to inhibit signaling by TNF superfamily members | |
| CN112345769A (zh) | 一种基于多克隆抗体的骨钙素胶乳增强比浊法检测试剂盒及其制备方法 | |
| Liu et al. | Identification, Characterization, and Structure of Tm16 from Trichuris muris | |
| EP1687330A1 (en) | Peptides with anti-obesity activity and other related uses | |
| WO2010105446A1 (zh) | 抗人肿瘤坏死因子受体α单抗及其应用 | |
| AU2019427773B2 (en) | mRNA display antibody library and methods | |
| Vitale et al. | From Bench to Bedside in Rheumatoid Arthritis from the “2022 GISEA International Symposium” | |
| CN116106548A (zh) | 一种用于检测人pcsk9蛋白水平的免疫学试剂 | |
| CN115304670A (zh) | 一种猫冠状病毒核衣壳蛋白的鼠源单克隆抗体及其编码基因和应用 | |
| US20230287390A1 (en) | Compositions and methods for identifying epitopes | |
| Zhang et al. | Integrative analysis of TNFRSF6B as a potential therapeutic target for pancreatic cancer | |
| CN103204939A (zh) | CD147-HAb18MAb复合物晶体结构及应用 | |
| Printsev et al. | The Opposite Functions of CD30 Ligand Isoforms | |
| CN104415328B (zh) | 异源动物细胞因子突变体疫苗及其应用 | |
| CA2540006A1 (en) | Treating inflammation using a biological agent that causes cells to release cytokine receptors |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140806 |