CN108178793A - It is a kind of to enhance immune carrier protein preparation method for being coupled SNCG, LP-PLA2 and 11- dehydrogenation thromboxane - Google Patents
It is a kind of to enhance immune carrier protein preparation method for being coupled SNCG, LP-PLA2 and 11- dehydrogenation thromboxane Download PDFInfo
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- 101000787273 Homo sapiens Gamma-synuclein Proteins 0.000 title claims abstract description 25
- 102100025615 Gamma-synuclein Human genes 0.000 title claims abstract description 23
- 238000006356 dehydrogenation reaction Methods 0.000 title abstract description 11
- 102000014914 Carrier Proteins Human genes 0.000 title abstract description 9
- 108010078791 Carrier Proteins Proteins 0.000 title abstract description 9
- RZWIIPASKMUIAC-VQTJNVASSA-N thromboxane Chemical compound CCCCCCCC[C@H]1OCCC[C@@H]1CCCCCCC RZWIIPASKMUIAC-VQTJNVASSA-N 0.000 title abstract description 7
- 238000003257 protein preparation method Methods 0.000 title abstract description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 32
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 32
- 108010024976 Asparaginase Proteins 0.000 claims abstract description 20
- 125000002091 cationic group Chemical group 0.000 claims abstract description 15
- PWGJDPKCLMLPJW-UHFFFAOYSA-N 1,8-diaminooctane Chemical class NCCCCCCCCN PWGJDPKCLMLPJW-UHFFFAOYSA-N 0.000 claims abstract description 13
- 102000011931 Nucleoproteins Human genes 0.000 claims abstract description 6
- 108010061100 Nucleoproteins Proteins 0.000 claims abstract description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 6
- 102100031538 Phosphatidylcholine-sterol acyltransferase Human genes 0.000 claims abstract 2
- 239000002253 acid Substances 0.000 claims abstract 2
- 238000005576 amination reaction Methods 0.000 claims abstract 2
- 102000016752 1-Alkyl-2-acetylglycerophosphocholine Esterase Human genes 0.000 claims description 17
- 230000008878 coupling Effects 0.000 claims description 16
- 238000010168 coupling process Methods 0.000 claims description 16
- 238000005859 coupling reaction Methods 0.000 claims description 16
- 230000002163 immunogen Effects 0.000 claims description 16
- 210000004369 blood Anatomy 0.000 claims description 9
- 239000008280 blood Substances 0.000 claims description 9
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 7
- XNRNNGPBEPRNAR-UHFFFAOYSA-N Thromboxane B2 Natural products CCCCCC(O)C=CC1OC(O)CC(O)C1CC=CCCCC(O)=O XNRNNGPBEPRNAR-UHFFFAOYSA-N 0.000 claims description 4
- 125000003277 amino group Chemical group 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 4
- XNRNNGPBEPRNAR-JQBLCGNGSA-N thromboxane B2 Chemical compound CCCCC[C@H](O)\C=C\[C@H]1OC(O)C[C@H](O)[C@@H]1C\C=C/CCCC(O)=O XNRNNGPBEPRNAR-JQBLCGNGSA-N 0.000 claims description 4
- 241000283690 Bos taurus Species 0.000 claims description 2
- 108010088751 Albumins Proteins 0.000 claims 1
- 102000009027 Albumins Human genes 0.000 claims 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
- 239000001257 hydrogen Substances 0.000 claims 1
- 238000005215 recombination Methods 0.000 claims 1
- 230000006798 recombination Effects 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 230000004048 modification Effects 0.000 abstract description 6
- 238000012986 modification Methods 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 4
- 108060003552 hemocyanin Proteins 0.000 abstract description 3
- 230000005847 immunogenicity Effects 0.000 abstract description 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 abstract 2
- 210000000225 synapse Anatomy 0.000 abstract 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 21
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 21
- 230000003053 immunization Effects 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- 239000000427 antigen Substances 0.000 description 12
- 102000036639 antigens Human genes 0.000 description 12
- 108091007433 antigens Proteins 0.000 description 12
- 239000000969 carrier Substances 0.000 description 12
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- 238000002649 immunization Methods 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 239000012460 protein solution Substances 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 6
- 230000000890 antigenic effect Effects 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 101001097889 Homo sapiens Platelet-activating factor acetylhydrolase Proteins 0.000 description 5
- 239000007987 MES buffer Substances 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 102000047182 human PLA2G7 Human genes 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 101001004358 Homo sapiens 1-alkyl-2-acetylglycerophosphocholine esterase Proteins 0.000 description 4
- 101001130226 Homo sapiens Phosphatidylcholine-sterol acyltransferase Proteins 0.000 description 4
- 238000001994 activation Methods 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102100033620 Calponin-1 Human genes 0.000 description 3
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 3
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 3
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 150000001718 carbodiimides Chemical class 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 239000007822 coupling agent Substances 0.000 description 2
- ZQSIJRDFPHDXIC-UHFFFAOYSA-N daidzein Chemical compound C1=CC(O)=CC=C1C1=COC2=CC(O)=CC=C2C1=O ZQSIJRDFPHDXIC-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 235000019800 disodium phosphate Nutrition 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- HKQYGTCOTHHOMP-UHFFFAOYSA-N formononetin Chemical compound C1=CC(OC)=CC=C1C1=COC2=CC(O)=CC=C2C1=O HKQYGTCOTHHOMP-UHFFFAOYSA-N 0.000 description 2
- 102000057593 human F8 Human genes 0.000 description 2
- 102000045642 human SNCG Human genes 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229940047431 recombinate Drugs 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 102100025573 1-alkyl-2-acetylglycerophosphocholine esterase Human genes 0.000 description 1
- KJYIVXDPWBUJBQ-UHHGALCXSA-N 11-dehydro-thromboxane B2 Chemical compound CCCCC[C@H](O)\C=C\[C@H]1OC(=O)C[C@H](O)[C@@H]1C\C=C/CCCC(O)=O KJYIVXDPWBUJBQ-UHHGALCXSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 208000037260 Atherosclerotic Plaque Diseases 0.000 description 1
- 229930185605 Bisphenol Natural products 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 208000025870 aspirin resistance Diseases 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- IISBACLAFKSPIT-UHFFFAOYSA-N bisphenol A Chemical compound C=1C=C(O)C=CC=1C(C)(C)C1=CC=C(O)C=C1 IISBACLAFKSPIT-UHFFFAOYSA-N 0.000 description 1
- 201000001531 bladder carcinoma Diseases 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000007211 cardiovascular event Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000000991 chicken egg Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000007240 daidzein Nutrition 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- RIKPNWPEMPODJD-UHFFFAOYSA-N formononetin Natural products C1=CC(OC)=CC=C1C1=COC2=CC=CC=C2C1=O RIKPNWPEMPODJD-UHFFFAOYSA-N 0.000 description 1
- 229940045109 genistein Drugs 0.000 description 1
- TZBJGXHYKVUXJN-UHFFFAOYSA-N genistein Natural products C1=CC(O)=CC=C1C1=COC2=CC(O)=CC(O)=C2C1=O TZBJGXHYKVUXJN-UHFFFAOYSA-N 0.000 description 1
- 235000006539 genistein Nutrition 0.000 description 1
- ZCOLJUOHXJRHDI-CMWLGVBASA-N genistein 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 ZCOLJUOHXJRHDI-CMWLGVBASA-N 0.000 description 1
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- DSNBHJFQCNUKMA-SCKDECHMSA-N thromboxane A2 Chemical compound OC(=O)CCC\C=C/C[C@@H]1[C@@H](/C=C/[C@@H](O)CCCCC)O[C@@H]2O[C@H]1C2 DSNBHJFQCNUKMA-SCKDECHMSA-N 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 208000010570 urinary bladder carcinoma Diseases 0.000 description 1
- 230000003156 vasculitic effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses the immune carrier protein preparation methods of a kind of Cationic bovine serum albumin, also referred to as amination bovine serum albumin(BSA)；With 1 (3 dimethylamino-propyl) 3 ethyl-carbodiimide hydrochlorides specifically under PH4.75 acid conditions（Abbreviation EDC, No. CAS 25,952 53 8）By 1,8 octamethylenediamines (No. CAS 373 44 4）With bovine serum albumin(BSA)（BSA）It is coupled, positively charged amino is prepared into Cationic bovine serum albumin instead of electronegative carboxylic groups all on bovine serum albumin(BSA) on octamethylenediamine.BSA after the modification as carrier protein, respectively with recombined human γ synapse nucleoproteins（SNCG）, platelet-activating factor acetylhydro-lase（LP‑PLA2）It is coupled with 11 dehydrogenation thromboxane B2s, mouse is immunized, immunogenicity significantly increases, and effect is close compared with hemocyanin or higher, and at low cost.
Description
Technical field
The present invention relates to the chemical modification fields of bovine serum albumin(BSA), and in particular to a kind of ox modified using octamethylenediamine
Seralbumin as carrier protein, respectively with recombined human SNCG albumen, recombined human platelet-activating factor acetylhydro-lase and 11- dehydrogenations
Thromboxane B2 is coupled, and prepares immunogene.
Background technology
Research shows that, people γ-synapse nucleoprotein (Synuclein Gamma, SNCG) is high in kinds of tumors tissue herein
Expression, especially content increases in bladder cancer patients tumor tissues and its urine, can become the biology mark of carcinoma of urinary bladder early diagnosis
Will object [1-3].Platelet-activating factor acetylhydro-lase（Lipoprotein-Associated Phospholipase A2, Lp-PLA2)
A kind of vasculitic reaction marker, the generation of activity and its content and cardiovascular disease event in notable positive correlation [4,
5].By detecting the Lp-PLA2 in blood, the degree of inflammation and its stabilization of atherosclerotic plaque can be effectively understood
Property.Thus there can be important clinic to prevention cardiovascular and cerebrovascular accident with the generation of early warning myocardial infarction and cerebral thrombus
It is worth [4,5].Urine 11- dehydrogenation thromboxane B2s are the metabolites that thromboxane A2 is stablized, can hematoblastic activation shape in antimer
State.Aspirin resistance can be differentiated by measuring 11- dehydrogenation thromboxane B2 contents in urine, be conducive to prevent cardiovascular event [6].With
Research deeply, more and more clinical markers are found.
Clinically generally use immunoassay method carries out quantitative analysis to above-mentioned micro biomarker.Wherein, specifically
Property high-titer antibody is the key that various immunoassays, and sensitivity, specificity to immunoassay method etc. play most important
Effect.For this purpose, the preparation of high-affinity antibody is the key that immuno analytical method development.
Recombined human SNCG or LP-PLA2 albumen or micromolecular compound 11- dehydrogenation thromboxane B2s are needed by dividing with big
The carrier protein of son is prepared into the antibody that comlete antigen ability Induction experiments animal generates high-titer after carrying out chemical coupling.Complete
During holoantigen carries out immune response to body, carrier protein can make the immune system of body generate strong T cell table
Position helps target antigen to generate specific b cells, carries out clonal activation, division, proliferation, and secrete for new B cell epitope
Antibody [7].
Only suitable carrier could prepare comlete antigen, and ideal carrier protein molecule should have extremely strong immunogene
Property can simultaneously be combined, and have preferable solubility with hapten conjugation.The protein carrier usually selected has bovine serum albumin(BSA)
(BSA), chicken egg white (0VA), keyhole limpet hemocyanin (KLH) etc..At present, BSA is with the most use, because its property is non-
Often stables, cheap and be easy to get, BSA contains more lysine, thus freely amino is more, and with small point
Still have good soluble [8] after sub- antigen coupling.
Experimental animal immune is carried out using Cationic bovine serum albumin, i.e., it is positively charged using ethylenediamine itself
Amino replaces the carboxyl of negative electrical charge in natural bovine serum albumin(BSA) to form by chemically reacting, with antigen electronegative on cell membrane
Presenting cell has fabulous compatibility, so as to accelerate the speed of immune response, persistently longer time and increases in vivo
The degree of immune response leads to that the antibody [9] of high-titer can be generated in vivo.Document report, since cationization ox blood is pure
Protein surface height is cationized（pI > 11）, the degree of inducing T cell proliferation is apparently higher than natural B SA under same dose
[10]。
When preparing comlete antigen, haptens is by coupling agent and carrier conjugation.Carbodiimide (EDC) makes carboxyl and ammonia
Dehydration forms amido bond between base, and the carboxyl on haptens is first reacted with EDC generates an intermediary, then again with the ammonia on carrier
Base is coupled to form comlete antigen.4- (N- maleimidomethyls) hexamethylene -1- carboxylic acid succinimide esters（SMCC）It is one
Kind bifunctional coupling agent, in Process of Antigen is prepared, the albumen of SMCC activation is often carrier protein（Such as KLH and BSA）, activation
Process is also maleylation, is exactly first to be connect by the amino of carrier protein with SMCC, half conjugate is formed, then with needing to be coupled
Small molecule containing mercapto groups or recombinant protein mixing coupling.
The Cationic bovine serum albumin of document report commercialization is that bovine serum albumin(BSA) is repaiied using ethylenediamine
Decorations（Fig. 1）.By the use of octamethylenediamine as chemical modification molecule in the present invention, advantage is compared to ethylenediamine, can be in bovine serum albumin
The white bridges for extending 6 carbon atoms outward more（Fig. 2）, bovine serum albumin(BSA) is made to make haptens while height is cationized
More stretching, extension is not easy to be shielded, so as to be easier to be identified by B cell.
Bibliography
[1]. Zhao J, Xing N.Identification of γ-synuclein as a stage-specific
marker in bladder cancer by immunohistochemistry.Med Sci Monit. 2014 Dec 5;
20:2550-5.
[2]. Liu C, Shi B, Hao C, et al.,Urine gamma-synuclein as a biomarker for
the diagnosis of bladder cancer.
Oncotarget. 2016 Jul 12;7(28):43432-43441. doi: 10.18632/oncotarget.9468.
[3] . Chen Z, Ji Z, Wang Q, et al.,Expression of γ-Synuclein in Bladder
Carcinoma: A Possible Marker for Prognosis.Anticancer Res. 2016 Mar;36(3):
951-6.
[4]. Packard CJ et al., Lipoprotein-associated phospholipase A2 as an
independent predictor of coronary heart disease. N Engl J Med. 2000;343(16):
1148-55.
[5]. Thompson A et al., Lipoprotein-associated phospholipase A(2) and
risk of coronary disease, stroke, and mortality: collaborative analysis of 32
prospective studies, Lancet. 2010;375(9725):1536-44.
[6]. McCullough PA, Vasudevan A, Sathyamoorthy M,et al.,Urinary 11-
Dehydro-Thromboxane B<sub>2</sub> and Mortality in Patients With Stable
Coronary Artery Disease.Am J Cardiol. 2017 Apr 1;119(7):972-977.
[7] Tam JP.Recent advances in multipe antigen peptides [J] .J.hnmun01.
Meth., 1996,196 (1)：17-32.
[8] .Houerou CL, Lamothe V, Bennetau B, et a1.Syntheses of Novel Hapten-
Protein Conjugates for Production of Highly specific Antibodies to
Formononetin, Daidzein and Genistein [J] .Tetrahedron, 2000,56：295-301.
[9] .Greg TH.Preparation of Hapten-Carrier Immunogen Conjugates [M]
Bioconjugate Techniques, 1996,419-455.
[10] .Yi F, Zhou YX, Zou Q, et a1.Preparation and characterization of
bisphenol A—cationized bovine serum albumin based on Mannich—type reaction
[J] .Immun01.Meth., 2009,340 (2)：138 1 143..
Invention content
In view of the above-mentioned problems, present invention aims at provide a kind of carrier using octamethylenediamine modification bovine serum albumin(BSA)
Albumen is coupled recombined human γ-synapse nucleoprotein respectively（SNCG）, recombined human platelet-activating factor acetylhydro-lase and 11- dehydrogenation thrombus
Alkane B2, it is immune for enhancing.It is characterized in that octamethylenediamine can make bovine serum albumin(BSA) molecular surface be cationized completely, enhancing
Its immunogenicity.Prepare gained Cationic bovine serum albumin can it is freeze-dried after it is steady in a long-term preserve, coupling is reproducible,
Immune-enhancing effect is notable.
The principle of the present invention is to replace natural ox blood by chemically reacting using the positively charged amino of octamethylenediamine itself
The carboxyl of negative electrical charge forms in pure albumen, makes the cationization of its apparent height（pI>11）.
In the present invention, recombined human γ-synapse nucleoprotein（SNCG）From commercialization through Bacillus coli expression purify and
Come；Recombined human platelet-activating factor acetylhydro-lase is purified for commercialization through expressing cho cell；11- dehydrogenation thromboxane B2s are
Commercial reagents, No. CAS is 67910-12-7.
Description of the drawings
Bovine serum albumin(BSA) schematic diagram ethylene diamine-modified Fig. 1
The bovine serum albumin(BSA) schematic diagram of Fig. 2 octamethylenediamines modification
4 immune antiserum titres after Fig. 3 SNCG recombinant proteins are coupled from different immune carriers
Booster immunization antiserum titre after Fig. 4 SNCG recombinant proteins are coupled from different immune carriers
Compared with Fig. 5 SNCG recombinant proteins are coupled immunizing potency with different immune carriers
4 immune antiserum titres after Fig. 6 Lp-PLA2 recombinant proteins are coupled from different immune carriers
Booster immunization antiserum titre after Fig. 7 Lp-PLA2 recombinant proteins are coupled from different immune carriers
Compared with Fig. 8 Lp-PLA2 recombinant proteins are coupled immunizing potency with different immune carriers
4 immune antiserum titres after Fig. 9 11-TXB2 are coupled from different immune carriers
Booster immunization antiserum titre after Figure 10 11-TXB2 are coupled from different immune carriers
Compared with Figure 11 11-TXB2 are coupled immunizing potency with different immune carriers.
Specific embodiment
Embodiment one：Cationic bovine serum albumin（cBSA）Preparation
Natural Bovine Serum Albumin Modified is prepared into Cationic bovine serum albumin with octamethylenediamine, modification specific steps are such as
Under：
1st, by 1,8- octamethylenediamines (the CAS 373-44-4 of 5ml）It adds in 20ml H2O, it is overall with 6N HCl tune PH to 4.75
Product is adjusted to 50ml, balances to room temperature（25℃）；
2nd, in dissolving 500mg BSA to 5ml H2O, above-mentioned solution is then added in；
3rd, 200mg 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides are added in（Hereinafter referred to as EDC, No. CAS
25952-53-8）In above-mentioned solution, after stirring, room temperature 1 hour；
4th, 3ml is added to cause solution reaction stopping above with 4M acetate buffer solutions PH4.75；
5th, it is dialysed with PH7.4 MES；
6th, cationization BSA is prepared by above-mentioned（Abbreviation cBSA）After measured concentration dispense freeze-drying, be stored in 4 DEG C it is spare.
Embodiment two：The coupling synthesis of cBSA-SNCG immunogenes
The amino group of the Cationic bovine serum albumin cBSA of 1,8- octamethylenediamines modification is with commercialization through Bacillus coli expression
Recombined human γ-synapse nucleoprotein of purifying（SNCG）Carboxyl is coupled, and coupling is as follows：
（1）The SNCG antigens of the total 5mg of 1ml is taken to be dissolved in 1ml MES（pH6.0）In buffer solution, totally three groups；
（2）It weighs EDC 4mg and adds in protein solution and react 5min；
（3）With 120 μ l 0.5M Na2HPO4 tune pH=7.2；
（4）Weigh n-hydroxysuccinimide（NHS）6mg, which is added in protein solution, reacts 15min；
（5）Take cBSA, nBSA of 2mg（Unmodified BSA）, KLH is separately added into protein solution and reacts 2 hours；
（6）Albumen coupling mixture is dialysed to PBS（pH7.2）, cBSA- SNCG recombinant proteins immunogene, nBSA- is made
SNCG recombinant proteins immunogene, KLH- SNCG recombinant protein immunogenes measure protein concentration packing freeze-drying.
Embodiment three：The immunological effect of cBSA-SNCG protein immunogen conjugates
1st, prepare immunizing antigen as the cBSA-SNCG recombinant protein immunogenic conjugates prepared in embodiment two, natural cow's serum
Albumin（nBSA）With the conjugate nBSA-PLA2 and hemocyanin of SNCG recombinant proteins（KLH）With SNCG recombinant proteins
Conjugate KLH-SNCG；NBSA- SNCG and KLH-SNCG use the coupling protocols of embodiment two.
2nd, cBSA-SNCG, nBSA-PLA2 and KLH-SNCG immunogenes made from embodiment two use conventional method
Inoculation experiments animal mouse respectively（BALB/c）, mouse resisting anteserum is taken after booster immunization, is as follows：
CBSA-SNCG, nBSA-PLA2 and KLH-SNCG immunogenes of above-mentioned synthesis are diluted to 1mg/ml with sterile water, are obtained
To antigenic solution, experiment mice is immunized after then being mixed with the antigenic solution of 1.0ml with the Freund's complete adjuvant of equivalent,
Every mouse subcutaneous injection 100ug；
After 1 week, then experiment mice is immunized after being mixed with the antigenic solution of 1.0ml with the incomplete Freund's adjuvant of equivalent,
Next primary, inoculation 4 times altogether are immunized week about；
By it is above-mentioned it is immune after experiment mice take the specific antibody of the isolated anti-SNCG of blood, and with recombinate SNCG albumen into
Row enzyme-linked immunosorbent assay（ELISA）Carry out comparison antibody titer.
3rd, Fig. 3 and Fig. 4 after being coupled from different immune carriers for SNCG recombinant proteins haptens in embodiment three exempt from for 4 times
It detects its serum titer after epidemic disease and booster immunization to compare, using cBSA as immune carrier than using natural nBSA or KLH as exempting from
Epidemic disease carrier makes immune mouse generate higher SNCG specific antibodies（Fig. 5）.
Example IV：The coupling synthesis of cBSA-Lp-PLA2 immunogenes
The amino group of the Cationic bovine serum albumin cBSA of 1,8- octamethylenediamines modification and commercialization are pure through expressing cho cell
The recombined human platelet-activating factor acetylhydro-lase of change（Lp-PLA2）Carboxyl is coupled, and coupling is as follows：
（1）The Lp-PLA2 antigens of the total 5mg of 1ml is taken to be dissolved in 1ml MES（pH6.0）In buffer solution, totally three groups；
（2）It weighs EDC 4mg and adds in protein solution and react 5min；
（3）With 120 μ l 0.5M Na2HPO4 tune pH=7.2；
（4）Weigh n-hydroxysuccinimide（NHS）6mg, which is added in protein solution, reacts 15min；
（5）Take cBSA, nBSA of 2mg（Unmodified BSA）, KLH is separately added into protein solution and reacts 2 hours；
（6）Albumen coupling mixture is dialysed to PBS（pH7.2）, cBSA-Lp-PLA2 recombinant proteins immunogene, nBSA- is made
Lp-PLA2 recombinant proteins immunogene and KLH-Lp-PLA2 recombinant protein immunogenes measure protein concentration packing freeze-drying.
Embodiment five：The immunological effect of cBSA-Lp-PLA2 protein immunogen conjugates
1st, prepare immunizing antigen as the cBSA-Lp-PLA2 recombinant protein immunogenic conjugates prepared in embodiment two, natural ox blood
Pure albumen（nBSA）With the conjugate nBSA-PLA2 and hemocyanin of Lp-PLA2 recombinant proteins（KLH）With Lp-PLA2 weights
Conjugate KLH-the Lp-PLA2 of histone；NBSA- Lp-PLA2 and KLH-Lp-PLA2 use the coupling side of embodiment two
Case.
2nd, cBSA-Lp-PLA2, nBSA-PLA2 and KLH-Lp-PLA2 immunogenes made from embodiment two are using normal
Rule method distinguishes inoculation experiments animal mouse（BALB/c）, mouse resisting anteserum is taken after booster immunization, is as follows：
CBSA-Lp-PLA2, nBSA-PLA2 and KLH-Lp-PLA2 immunogenes of above-mentioned synthesis are diluted to sterile water
1mg/ml obtains antigenic solution, to experiment mice after then being mixed with the antigenic solution of 1.0ml with the Freund's complete adjuvant of equivalent
It is immunized, every mouse subcutaneous injection 100ug；
After 1 week, then experiment mice is immunized after being mixed with the antigenic solution of 1.0ml with the incomplete Freund's adjuvant of equivalent,
Next primary, inoculation 4 times altogether are immunized week about；
Above-mentioned immune rear experiment mice take to the specific antibody of the isolated anti-Lp-PLA2 of blood, and to recombinate Lp-PLA2
Albumen carries out enzyme-linked immunosorbent assay（ELISA）Carry out comparison antibody titer.
3rd, Fig. 6 and Fig. 7 carries out 4 after being coupled for Lp-PLA2 recombinant proteins haptens in embodiment three from different immune carriers
Its serum titer is detected after secondary immune and booster immunization to compare, using cBSA as immune carrier than with natural nBSA or KLH works
Immune mouse is made to generate higher Lp-PLA2 specific antibodies for immune carrier（Fig. 8）.
Embodiment six：The coupling synthesis of cBSA-11-DH-TXB2 small molecule immunes original
CBSA-11-DH-TXB2 immunogenes are by the amino group of Cationic bovine serum albumin cBSA and 11 dehydrogenation thromboxane of people
B2（11- TXB2）The carboxylic group of recombinant protein is coupled, and coupling is as follows：
（1）The cBSA of 11 dehydrogenation thromboxane B2 of people and 10mg for weighing 10mg is dissolved separately in 1ml MES buffer solutions；
（2）EDC 2mg are weighed to be dissolved in the MES buffer solutions of 200 μ l, after adding in above-mentioned three kinds of solution mixings immediately, magnetic at room temperature
Power blender stirs 2 hours；
（3）The protein solution for reacting end is used into PBS at 4 DEG C（pH7.2）Dialysis, obtained cBSA-11- TXB2 are immunized anti-
Original measures protein concentration packing freeze-drying.
Embodiment seven：The immunological effect of cBSA-11- TXB2 small molecule immune original conjugates
CBSA-11- TXB2, nBSA-11- TXB2 made from embodiment two and KLH 11- TXB2 are to experimental animal mouse
Be immunized, blood, detection taken to be carried out with reference to the step in embodiment three, after immune the 4th and booster immunization is laggard
Row takes blood, and former using 11- TXB2 and ovalbumin conjugate OVA-11- TXB2 as detection（Its coupling protocols and embodiment
Four is consistent）It carries out ELISA and carries out comparison antibody titer.
Fig. 9 and Figure 10 is that 11- TXB2 small haptens carry out 4 times after being coupled from different immune carriers in embodiment three
After immune and booster immunization its serum titer is detected to compare, using cBSA as immune carrier than using natural nBSA or KLH as
Immune carrier makes immune mouse generate higher 11- TXB2 specific antibodies（Figure 11）.
Claims (4)
1. a kind of modify natural bovine serum albumin(BSA) with octamethylenediamine, Cationic bovine serum albumin or amination cow's serum are prepared
The method of albumin uses 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide hydrochlorides specifically under PH4.75 acid conditions
Salt（Abbreviation EDC）By 1,8-octamethylenediamine is coupled with bovine serum albumin(BSA), and positively charged amino is pure instead of ox blood on octamethylenediamine
All electronegative carboxylic groups, are prepared into Cationic bovine serum albumin on albumen.
2. Cationic bovine serum albumin according to claim 1 is through EDC and recombined human γ-synapse nucleoprotein（SNCG）
CBSA- SNCG immunogenes are made, for mouse to be immunized in coupling.
3. according to Cationic bovine serum albumin prepared by claim 1 through through EDC and recombination platelet-activating factor acetylhydro-lase
（LP-PLA2）CBSA- Lp-PLA2 immunogenes are made, for mouse to be immunized in coupling.
4. it is taken off according to 11- of the Cationic bovine serum albumin amino group through EDC and carboxyl group prepared by claim 1
Hydrogen thromboxane B2 is coupled, cBSA-11-dehydro TXB2 obtained, for mouse to be immunized.
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| CN110713519A (en) * | 2019-11-02 | 2020-01-21 | 深圳市安帝宝科技有限公司 | 11-dehydrothromboxane B2 antigen and preparation method thereof |
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