CN107098961A - Triptolide artificial antigen and preparation method and polyclonal antibody - Google Patents
Triptolide artificial antigen and preparation method and polyclonal antibody Download PDFInfo
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- CN107098961A CN107098961A CN201710447761.5A CN201710447761A CN107098961A CN 107098961 A CN107098961 A CN 107098961A CN 201710447761 A CN201710447761 A CN 201710447761A CN 107098961 A CN107098961 A CN 107098961A
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- Prior art keywords
- triptolide
- aminobenzoic acid
- preparation
- solution
- artificial antigen
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- DFBIRQPKNDILPW-CIVMWXNOSA-N Triptolide Chemical compound O=C1OCC([C@@H]2C3)=C1CC[C@]2(C)[C@]12O[C@H]1[C@@H]1O[C@]1(C(C)C)[C@@H](O)[C@]21[C@H]3O1 DFBIRQPKNDILPW-CIVMWXNOSA-N 0.000 title claims abstract description 65
- YKUJZZHGTWVWHA-UHFFFAOYSA-N triptolide Natural products COC12CC3OC3(C(C)C)C(O)C14OC4CC5C6=C(CCC25C)C(=O)OC6 YKUJZZHGTWVWHA-UHFFFAOYSA-N 0.000 title claims abstract description 65
- 239000000427 antigen Substances 0.000 title claims abstract description 44
- 102000036639 antigens Human genes 0.000 title claims abstract description 44
- 108091007433 antigens Proteins 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 claims abstract description 90
- 229960004050 aminobenzoic acid Drugs 0.000 claims abstract description 46
- 150000001875 compounds Chemical class 0.000 claims abstract description 23
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 19
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 19
- 241001465754 Metazoa Species 0.000 claims abstract description 11
- 210000002966 serum Anatomy 0.000 claims abstract description 10
- 229940098773 bovine serum albumin Drugs 0.000 claims description 26
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 24
- 238000003756 stirring Methods 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 18
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 15
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 235000015398 thunder god vine Nutrition 0.000 claims description 7
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 claims description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 6
- 108010058846 Ovalbumin Proteins 0.000 claims description 6
- 230000008878 coupling Effects 0.000 claims description 6
- 238000010168 coupling process Methods 0.000 claims description 6
- 238000005859 coupling reaction Methods 0.000 claims description 6
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical compound CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 241000830536 Tripterygium wilfordii Species 0.000 claims description 5
- 230000003197 catalytic effect Effects 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 229940092253 ovalbumin Drugs 0.000 claims description 5
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims description 4
- 229910021529 ammonia Inorganic materials 0.000 claims description 3
- 235000010288 sodium nitrite Nutrition 0.000 claims description 3
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims 2
- 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 claims 1
- 239000005711 Benzoic acid Substances 0.000 claims 1
- 108010000912 Egg Proteins Proteins 0.000 claims 1
- 102000002322 Egg Proteins Human genes 0.000 claims 1
- 235000010233 benzoic acid Nutrition 0.000 claims 1
- 210000004369 blood Anatomy 0.000 claims 1
- 239000008280 blood Substances 0.000 claims 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 claims 1
- 235000014103 egg white Nutrition 0.000 claims 1
- 210000000969 egg white Anatomy 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 8
- 102000004190 Enzymes Human genes 0.000 abstract description 3
- 108090000790 Enzymes Proteins 0.000 abstract description 3
- 239000003547 immunosorbent Substances 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 35
- 230000003053 immunization Effects 0.000 description 12
- 238000000034 method Methods 0.000 description 10
- 238000013019 agitation Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 239000005457 ice water Substances 0.000 description 8
- 238000002965 ELISA Methods 0.000 description 7
- 235000012907 honey Nutrition 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 5
- 239000000523 sample 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 4
- 238000000502 dialysis Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000003643 water by type Substances 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- UKJLNMAFNRKWGR-UHFFFAOYSA-N cyclohexatrienamine Chemical group NC1=CC=C=C[CH]1 UKJLNMAFNRKWGR-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 241000256844 Apis mellifera Species 0.000 description 2
- 241000345998 Calamus manan Species 0.000 description 2
- 229920000297 Rayon Polymers 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000012954 diazonium Substances 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-O diazynium Chemical compound [NH+]#N IJGRMHOSHXDMSA-UHFFFAOYSA-O 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- -1 nitrite ions Chemical class 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 235000012950 rattan cane Nutrition 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- OCLUXIQGSXDQNA-WCQYABFASA-N (5ar,9ar)-5a-methyl-4,5,6,8,9,9a-hexahydro-1h-benzo[e][2]benzofuran-3,7-dione Chemical compound C([C@]1([C@H]2CCC(=O)C1)C)CC1=C2COC1=O OCLUXIQGSXDQNA-WCQYABFASA-N 0.000 description 1
- 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 1
- 235000015001 Cucumis melo var inodorus Nutrition 0.000 description 1
- 240000002495 Cucumis melo var. inodorus Species 0.000 description 1
- 240000007849 Macleaya cordata Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 101100203936 Mus musculus Srpra gene Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- OCLUXIQGSXDQNA-UHFFFAOYSA-N Wilforonide Natural products C1C(=O)CCC2C1(C)CCC1=C2COC1=O OCLUXIQGSXDQNA-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- XQDBHSNYTFRCNJ-UDTLMCMUSA-N chembl525628 Chemical compound O([C@H]1[C@@H]2OC(=O)[C@](C)(O)CCC3=NC=CC=C3C(=O)OC[C@]3(C)O[C@]4([C@@]2(C)O)[C@H](OC(C)=O)[C@H]3[C@@H](OC(C)=O)[C@@H](OC(C)=O)[C@@]4([C@H]1OC(C)=O)COC(=O)C)C(=O)C1=CC=CC=C1 XQDBHSNYTFRCNJ-UDTLMCMUSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008236 heating water Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 231100000004 severe toxicity Toxicity 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- IQUAVJOINYOCMR-UHFFFAOYSA-N wilfordine Natural products CC1c2ncccc2C(=O)OCC2(C)OC34C(OC(C)=O)C2C(OC(C)=O)C(OC(C)=O)C3(COC(C)=O)C(OC(C)=O)C(OC(=O)c2ccccc2)C(OC(=O)C1(C)O)C4(C)O IQUAVJOINYOCMR-UHFFFAOYSA-N 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
- 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
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
-
- 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
Abstract
The invention discloses a kind of triptolide artificial antigen and preparation method and polyclonal antibody, it is related to field of immunology.Triptolide artificial antigen is formed by triptolide, p-aminobenzoic acid and carrier protein couplet.Its preparation method it include：By p-aminobenzoic acid diazotising；Diazotizing p-aminobenzoic acid and triptolide are coupled, compound is formed；By the compound and carrier protein couplet, triptolide artificial antigen is produced.Triptolide polyclonal antibody, animal is immunized by triptolide artificial antigen in it, and obtains the serum of animal and obtain.Triptolide artificial antigen and polyclonal antibody constitute enzyme linked immunosorbent detection system, realize the quick detection to triptolide, with it is convenient, sensitivity is high the characteristics of.
Description
Technical field
The present invention relates to field of immunology, especially triptolide artificial antigen and preparation method and polyclonal antibody.
Background technology
Honey has the different physiological roles such as antibacterial, anti-oxidant, raising body immunity, is the agricultural product that dietotherapy develops simultaneously,
It is very popular.Honey quality and safety are all paid high attention to by various circles of society always.
The honey national standard of China《National food safety standard honey》(GB14963-2011) clear stipulaties honeybee in
Nectar, secretion or the honeydew of herborization answer safety non-toxic, must not be planted from tripterygium wilfordii, macleaya cordata, the poisonous nectar source of the root of langdu
Thing.Tripterygium wilfordii is distributed on the south China the Changjiang river and southwest.In the case that honeybee is less in nectar source, tripterygium wilfordii plant can be made
For its nectar source, the active ingredients of thunder god vine of severe toxicity is caused to enter in honey.As people eat such toxic honey by mistake, poisoning can be caused
It is even dead.Detection method report on active ingredients of thunder god vine in honey is less, in the urgent need to developing new, quick, spirit
Quick active ingredients of thunder god vine detection architecture.
The content of the invention
The goal of the invention of the present invention is：For above-mentioned problem there is provided a kind of triptolide artificial antigen and
Preparation method and polyclonal antibody, wherein triptolide artificial antigen and polyclonal antibody composition enzyme linked immunosorbent detection body
System, triptolide is detected, with it is simple, quick, sensitive the characteristics of.
The technical solution adopted by the present invention is as follows：
A kind of triptolide artificial antigen, it is formed by triptolide, p-aminobenzoic acid and carrier protein couplet.
Because triptolide belongs to micromolecular compound, it is impossible to obtain antibody directly as antigen-immunized animal, and with
After carrier protein is combined, protein macromolecule, thus the function with antigen are formed.The effect of its connecting bridge of p-aminobenzoic acid,
Ensure being stably connected with for triptolide and carrier protein.
In preferred embodiments of the present invention, the carrier protein is bovine serum albumin(BSA) or ovalbumin.
When carrier protein is bovine serum albumin(BSA), compound is immunizing antigen, and triptolide can be prepared with the antigen
Polyclonal antibody.When carrier protein is ovalbumin, because it than bovine serum albumin(BSA) has more preferable immunogenicity, it can use
In screening cell strain of monoclonal antibody.
A kind of preparation method of triptolide artificial antigen, it includes：
By p-aminobenzoic acid diazotising；
Diazotizing p-aminobenzoic acid and triptolide are coupled, compound is formed；
By the compound and carrier protein couplet, triptolide artificial antigen is produced.
Because wilfordine belongs to micromolecular compound, it is impossible to obtain antibody directly as antigen-immunized animal, first will
It is coupled after p-aminobenzoic acid diazotising with triptolide, carboxyl is introduced after coupling（—COOH）, then using mixed acid anhydride
Triptolide-p-aminobenzoic acid and carrier protein are coupled by method, so as to form stable triptolide-to ammonia
Yl benzoic acid-carrier protein complex, i.e. antigen.
In preferred embodiments of the present invention, triptolide-p-aminobenzoic acid diazotising is included：By p-aminophenyl
Formic acid is dissolved in hydrochlorate, obtains p-aminobenzoic acid solution, and ice bath stirring is lower to add NaNO2, and low-temperature dark stirring adds aniline
It is changed into buff to solution.
In preferred embodiments of the present invention, the concentration of the hydrochloric acid is 0.8-1.5mol/L, the p-aminobenzoic acid with
The mass ratio of the NaNO2 is 5-7：1.
Due to taking above-mentioned technical proposal, p-aminobenzoic acid, by diazotising, is that the coupling of its thing triptolide is carried
For architecture basics.
In preferred embodiments of the present invention, diazotizing p-aminobenzoic acid is included with triptolide coupling：By thunder
Public rattan A prime is complete complete soluble in water to obtain triptolide solution, the triptolide solution and diazotizing p-aminophenyl first
Acid mixing, adjusts pH8-9, stirs 1-3h, centrifuging and taking supernatant is separately added into the tri-n-butylamine and isobutyl chlorocarbonate of catalytic amount, low
Temperature stirring 20-30min.
In preferred embodiments of the present invention, the mass ratio of the triptolide and the p-aminobenzoic acid is 3-5:
4-6。
Due to taking above-mentioned technical proposal, diazotizing p-aminobenzoic acid is coupled with triptolide stabilization, and
Carboxyl is introduced in compound, the coupling for compound and carrier protein provides architecture basics.
In preferred embodiments of the present invention, the compound and carrier protein couplet are included：Bovine serum albumin(BSA) is molten
In PBS, bovine serum albumin solution is obtained, dimethylformamide is added into the bovine serum albumin solution.
In preferred embodiments of the present invention, the mass ratio of the bovine serum albumin(BSA) and the triptolide is 15-
20:1。
Due to taking above-mentioned technical proposal, compound is coupled with bovine serum albumin(BSA), forms stable tripterygium wilfordii first
Element-p-aminobenzoic acid-bovine serum albumin(BSA) immunizing antigen.
A kind of triptolide polyclonal antibody, animal is immunized by above-mentioned triptolide artificial antigen in it, and obtains
The serum of animal and obtain.
It should be noted that the animal can be mouse, and sheep, rabbit, but never only limit and above-mentioned animal.
In summary, by adopting the above-described technical solution, the beneficial effects of the invention are as follows：
By micromolecular compound triptolide and carrier protein couplet, antigen is become, with immunogenicity, and it is anti-with this
The immune animal of original obtains triptolide polyclonal antibody, and antigen-antibody is bonded enzyme linked immunosorbent detection system, realizes to thunder
The quick detection of public rattan A prime, with it is convenient, sensitivity is high the characteristics of.
Brief description of the drawings
Examples of the present invention will be described by way of reference to the accompanying drawings, wherein：
Fig. 1 is the UV spectrophotometer measuring qualification figure of each material.
Fig. 2 is the SDS-PAGE detection figures for being coupled bovine serum albumin(BSA).
Fig. 3 is the Western blot analysis charts of polyclonal antibody.
Embodiment
All features disclosed in this specification, or disclosed all methods or during the step of, except mutually exclusive
Feature and/or step beyond, can combine in any way.
This specification（Including any accessory claim, summary）Disclosed in any feature, unless specifically stated otherwise,
Replaced by other equivalent or with similar purpose alternative features.I.e., unless specifically stated otherwise, each feature is a series of
An example in equivalent or similar characteristics.
Embodiment 1
The present embodiment provides a kind of triptolide artificial antigen and preparation method thereof.Made in this method using bovine serum albumin(BSA)
For carrier protein, prepare in a kind of immunizing antigen.Specifically：
S1：P-aminobenzoic acid 24mg is weighed, is dissolved in 3mL 1mol/L HCl, it is molten to obtain p-aminobenzoic acid after being completely dissolved
Liquid, is placed in ice-water bath and stirs to internal and external temperature balance.
S2：Weigh NaNO2 4mg, adds water and lucifuge stirring at p-aminobenzoic acid solution, 4 DEG C is added after being completely dissolved
10min。
S3：Add the aniline of catalytic amount（2μL）It is changed into buff to solution, obtains diazotizing p-aminobenzoic acid molten
Liquid, is saved backup.
S4：Triptolide 19.2mg is weighed, is dissolved in 3mL ultra-pure waters, ultrasonic 5min is to after being completely dissolved, with diazotising
P-aminobenzoic acid solution mixing, obtain mixed liquor.
S5：Adjust mixed liquor PH to 8.5, magnetic agitation 2h, the supernatant of centrifuging and taking first.
S6：Add 5 μ L tri-n-butylamines into the first supernatant, 3.8 μ L isobutyl chlorocarbonates, magnetic agitation 25min at 4 DEG C,
Obtain compound.
S7：326.4mg BSA are weighed, 5mLPBS is dissolved in（Phosphate buffer）In, it is completely dissolved rear ice-water bath, magnetic force
Stirring is lower to add 5mLDMF, obtains BSA solution.
S8：Compound is added into magnetic stirrer over night, the supernatant of centrifuging and taking second at BSA solution, 4 DEG C.
S9：Second supernatant 0.01mol/L PH7.4 PBSs one week, change medium once on the 1st day, 2-4 per 4h
It does not have 8h to change medium once, then changes medium daily once.
S10：Dialysis is completed, and takes dialyzate, it is freeze-dried after obtain triptolide artificial antigen, -20 DEG C of preservations are standby
With.
Embodiment 2
The present embodiment provides a kind of triptolide artificial antigen and preparation method thereof.Made in this method using bovine serum albumin(BSA)
For carrier protein, prepare in a kind of immunizing antigen.Specifically：
S1：P-aminobenzoic acid 22.5mg is weighed, is dissolved in 3mL 0.8mol/L HCl, p-aminophenyl first is obtained after being completely dissolved
Acid solution, is placed in ice-water bath and stirs to internal and external temperature balance.
S2：Weigh NaNO2 4.5mg, adds water and lucifuge stirring at p-aminobenzoic acid solution, 4 DEG C is added after being completely dissolved
10min。
S3：Add the aniline of catalytic amount（2μL）It is changed into buff to solution, obtains diazotizing p-aminobenzoic acid molten
Liquid, is saved backup.
S4：Triptolide 11.25mg is weighed, is dissolved in 3mL ultra-pure waters, ultrasonic 5min is to after being completely dissolved, with diazonium
The p-aminobenzoic acid solution mixing of change, obtains mixed liquor.
S5：Adjust mixed liquor PH to 8, magnetic agitation 1h, the supernatant of centrifuging and taking first.
S6：Add 5 μ L tri-n-butylamines into the first supernatant, 3.8 μ L isobutyl chlorocarbonates, magnetic agitation 30min at 4 DEG C,
Obtain compound.
S7：225mg BSA are weighed, 5mLPBS is dissolved in（Phosphate buffer）In, it is completely dissolved rear ice-water bath, magnetic force and stirs
Lower addition 5mLDMF is mixed, BSA solution is obtained.
S8：Compound is added into magnetic stirrer over night, the supernatant of centrifuging and taking second at BSA solution, 4 DEG C.
S9：Second supernatant 0.01mol/L PH7.4 PBSs one week, change medium once on the 1st day, 2-4 per 4h
It does not have 8h to change medium once, then changes medium daily once.
S10：Dialysis is completed, and takes dialyzate, it is freeze-dried after obtain triptolide artificial antigen, -20 DEG C of preservations are standby
With.
Embodiment 3
The present embodiment provides a kind of triptolide artificial antigen and preparation method thereof.Made in this method using bovine serum albumin(BSA)
For carrier protein, prepare in a kind of immunizing antigen.Specifically：
S1：P-aminobenzoic acid 25.5mg is weighed, is dissolved in 3mL 1.5mol/L HCl, p-aminophenyl first is obtained after being completely dissolved
Acid solution, is placed in ice-water bath and stirs to internal and external temperature balance.
S2：Weigh NaNO2 3.65mg, adds water and lucifuge stirring at p-aminobenzoic acid solution, 4 DEG C is added after being completely dissolved
10min。
S3：Add the aniline of catalytic amount（2μL）It is changed into buff to solution, obtains diazotizing p-aminobenzoic acid molten
Liquid, is saved backup.
S4：Triptolide 31.88mg is weighed, is dissolved in 3mL ultra-pure waters, ultrasonic 5min is to after being completely dissolved, with diazonium
The p-aminobenzoic acid solution mixing of change, obtains mixed liquor.
S5：Adjust mixed liquor PH to 9, magnetic agitation 3h, the supernatant of centrifuging and taking first.
S6：Add 5 μ L tri-n-butylamines into the first supernatant, 3.8 μ L isobutyl chlorocarbonates, magnetic agitation 25min at 4 DEG C,
Obtain compound.
S7：478mg BSA are weighed, 5mLPBS is dissolved in（Phosphate buffer）In, it is completely dissolved rear ice-water bath, magnetic force and stirs
Lower addition 5mLDMF is mixed, BSA solution is obtained.
S8：Compound is added into magnetic stirrer over night, the supernatant of centrifuging and taking second at BSA solution, 4 DEG C.
S9：Second supernatant 0.01mol/L PH7.4 PBSs one week, change medium once on the 1st day, 2-4 per 4h
It does not have 8h to change medium once, then changes medium daily once.
S10：Dialysis is completed, and takes dialyzate, it is freeze-dried after obtain triptolide artificial antigen, -20 DEG C of preservations are standby
With.
Embodiment 4
The present embodiment provides a kind of triptolide artificial antigen and preparation method thereof.Ovalbumin is used in this method as load
Body protein, is prepared in a kind of envelope antigen.Specifically：
S1：P-aminobenzoic acid 24mg is weighed, is dissolved in 3mL 1.2mol/L HCl, p-aminobenzoic acid is obtained after being completely dissolved
Solution, is placed in ice-water bath and stirs to internal and external temperature balance.
S2：Weigh NaNO2 4mg, adds water and lucifuge stirring at p-aminobenzoic acid solution, 4 DEG C is added after being completely dissolved
10min。
S3：Add a small amount of aniline（2μL）It is changed into buff to solution, obtains diazotizing p-aminobenzoic acid solution, protects
Deposit standby.
S4：Triptolide 24mg is weighed, is dissolved in 3mL ultra-pure waters, ultrasonic 5min is and diazotizing to after being completely dissolved
P-aminobenzoic acid solution is mixed, and obtains mixed liquor.
S5：Adjust mixed liquor PH to 8.5, magnetic agitation 2h, the supernatant of centrifuging and taking first.
S6：Add 5 μ L tri-n-butylamines into the first supernatant, 3.8 μ L isobutyl chlorocarbonates, magnetic agitation 27min at 4 DEG C,
Obtain compound.
S7：432mg ovalbumins are weighed, 5mL PBS are dissolved in（Phosphate buffer）In, be completely dissolved rear ice-water bath,
5mLDMF is added under magnetic agitation, ovalbumin solution is obtained.
S8：Compound is added into magnetic stirrer over night, the supernatant of centrifuging and taking second at BSA solution, 4 DEG C.
S9：Second supernatant 0.01mol/L PH7.4 PBSs one week, change medium once on the 1st day, 2-4 per 4h
It does not have 8h to change medium once, then changes medium daily once.
S10：Dialysis is completed, and takes dialyzate, it is freeze-dried after obtain triptolide artificial antigen, -20 DEG C of preservations are standby
With.
Embodiment 5
The present embodiment provides the identification of immunizing antigen, and the purpose is to determine triptolide to be successfully coupled with bovine serum albumin(BSA).
Test antigen is the immunizing antigen that embodiment 1 is provided.It includes：
Experimental method：
1. UV scanning is identified
The immunizing antigen that triptolide, p-aminobenzoic acid, bovine serum albumin(BSA) and embodiment 1 are provided is weighed respectively, is matched somebody with somebody
5mg/mL solution is made, is scanned in 200-600nm wave-length coverages, its characteristic absorption peak is determined respectively.
Experimental result is as shown in Figure 1.In Fig. 1, P represents wilforonide, and BSA represents bovine serum albumin(BSA), and PABA is represented
P-aminobenzoic acid.As shown in Figure 1, triptolide has individual absworption peak respectively in 270nm and 345nm or so, and BSA exists
285nm or so has an absworption peak, and p-aminobenzoic acid has absworption peak in 270 your left and right, and triptolide-p-aminobenzoic acid-
Bovine serum albumin(BSA) only has the absworption peak of absworption peak, i.e. bovine serum albumin(BSA) in 285nm or so, illustrates that three is successfully coupled, only
There is the absworption peak of macromolecular.
2.SDS-PAGE gel electrophoresises are identified
Experimental method：
S1：Prepare and liquid is outwelled after fluid-tight after 12% separation gel, encapsulating, solidification, and blotted with filter paper.
S2：Prepare 5% and concentrate insertion glue comb, static 30min after glue, encapsulating.
S3：The immunizing antigen that bovine serum albumin(BSA) and embodiment 1 are provided, is configured to 5mg/mL solution, respectively with 2 ×
SDS- sample buffers are mixed in equal volume, and 100 DEG C of heating water baths obtain sample in 3-5 minutes.
S4：The gel flat made is put into vertical panel electrophoresis apparatus, lower groove puts SDS- electrode buffers into.Extract glue
Comb, takes each sample and each 20 μ l of albumen maker to be added in sample cell, adds SDS- electrode buffers, starts electrophoresis.Start
When electric current be 10mA or so；After sample enters separation gel, 20 ~ 30mA is changed to；When fuel forward position is away from silica gel frame base 1.5cm
When, stop electrophoresis, close power supply.
S5：After electrophoresis terminates, gel is taken out.By soak in dyeing liquor, 4h is dyed, destainer is then carried out and takes off
Color, a few hours change a destainer, until background is colourless.
Experimental result is as shown in Figure 2.In Fig. 2, BSA represents bovine serum albumin(BSA), and P+P+B represents triptolide-to ammonia
Yl benzoic acid-bovine serum albumin(BSA) immunizing antigen.There is the band close with BSA columns band in P+P+B columns, further illustrate Thunder God
Rattan A prime is successfully coupled with bovine serum albumin(BSA).
Embodiment 6
The present embodiment provides a kind of triptolide polyclonal antibody, and the triptolide polyclonal antibody has embodiment 1 to provide
Immunizing antigen be immunized mouse and obtain.And its potency is detected with Elisa, Western blot have detected its specificity.Specifically：
8 week old BALB/C systems mouse are immunized with immunizing antigen.After three exempt from, by mouse docking blood sampling separation serum, indirect ELISA is used
The positive mouse of method and indirect competitive ELISA method screening serum titer height and high specificity, obtains polyclonal antibody.
Elisa is detected：
S1：Appropriate recombinant protein after purification is added in ELISA Plate, substrate is used as.
S2：100-150 μ L coating buffer is separately added into the every hole of ELISA Plate, 4 DEG C of coatings is placed in and stays overnight（Or 37 DEG C of 2-3
Hour）.
S3：Cleaned 3-5 times with PBST liquid.
S4：Add and be incubated 1-2 hours at 150-200uL 3-5% BSA dilutions, 37 DEG C into the every hole of ELISA Plate.
S5：PBST is cleaned 3-5 times.
S6：Add the polyclonal antiserum of different dilution factors（1:500、1:1000、1:2000、1:5000、1:10000、1:
20000、1:24000、1:36000）, per hole 150-200 μ L.
S7：150-200 μ L negative serum is added in control.
S8：It is placed at 37 DEG C and is incubated 2-3 hours.
S9：PBST is cleaned 3-5 times.
S10：By 1:1 adds the sheep anti mouse of HRP marks into ELISA Plate（1:5000/1：10000）Solution, 37 DEG C of incubation 1-
2 hours.
S11：PBST is cleaned 3-5 times.
S12：100-150 μ L TMB nitrite ions are added per hole, 37 DEG C of lucifuges are reacted 30-45 minutes.
S13：100-150 μ L, 2mol/L H are added per hole2SO4, terminating reaction.
S14：ELIASA determines D（490nm）Value.
Elisa testing results are as shown in table 1：
The ELISA testing results of the polyclonal antibody potency of table 1
| Positive serum | 1:500 | 1:1000 | 1:2000 | 1:5000 | 1:10000 | 1:20000 | Negative serum |
| D（490nm） | 0.979 | 0.852 | 0.730 | 0.651 | 0.339 | 0.115 | 0.054 |
As shown in Table 1,2 times with the OD values of positive serum more than negative control OD value is standards, the antibody effect of this serum sample
Valency is 1:20000.
Western blot are detected：
Experimental method：
S1：Recombinant protein after purification is subjected to SDS-PAGE, electrophoresis takes out blob of viscose and is dipped in film transfer buffer solution after terminating.
S2：Clip and the PVDE films of the similar size of blob of viscose, are cleaned with ultra-pure water after soaking 1-2 minutes in methyl alcohol, put
In film transfer buffer solution.
S3：Film transfer buffer solution immersion sponge, pvdf membrane, PAGE films, sponge are overlayed turn successively from top to bottom
In film instrument.
S4：Switch on power, constant pressure transferring film 30-35 minutes.
S5：The pvdf membrane transferred is placed in 4 DEG C of shaking table closings in 4-6% skimmed milk power confining liquid to stay overnight.
S6：Confining liquid is abandoned, pvdf membrane is cleaned 3-5 times with TBST liquid room temperature shakers, each 10-15 minutes.
S7：Pvdf membrane is incubated with the polyclonal antiserum room temperature shaker of preparation 3-3.5 hours.
S8：TBST is cleaned.
S9：Incubation at room temperature pvdf membrane about 1-2 hours after being diluted with alkali phosphatase enzyme mark sheep anti-mouse antibody.
S10：TBST is cleaned.
S11：It is put into TMB nitrite ions and develops the color, observes.
Western blot testing results are as shown in figure 3, M swimming lanes are marker in Fig. 3, and 1 swimming lane is polyclonal antibody, by
Fig. 3 understands that polyclonal antibody specificity is good.
The invention is not limited in foregoing embodiment.The present invention, which is expanded to, any in this manual to be disclosed
New feature or any new combination, and disclose any new method or process the step of or any new combination.
Claims (10)
1. a kind of triptolide artificial antigen, it is characterised in that it is by triptolide, p-aminobenzoic acid and carrier egg
White coupling is formed.
2. triptolide artificial antigen according to claim 1, it is characterised in that the carrier protein is that ox blood is pure
Albumen or ovalbumin.
3. the preparation method of triptolide artificial antigen according to claim 1 or 2, it is characterised in that it includes：
By p-aminobenzoic acid diazotising；
Diazotizing p-aminobenzoic acid and triptolide are coupled, compound is formed；
By the compound and carrier protein couplet, triptolide artificial antigen is produced.
4. preparation method according to claim 3, it is characterised in that include p-aminobenzoic acid diazotising：Will be to ammonia
Yl benzoic acid is dissolved in hydrochloric acid, obtains p-aminobenzoic acid solution, and ice bath stirring is lower to add NaNO2, and low-temperature dark stirring is added
Aniline to solution is changed into buff.
5. preparation method according to claim 4, it is characterised in that the concentration of the hydrochloric acid is 0.8-1.5mol/L, institute
State p-aminobenzoic acid and the NaNO2Mass ratio be 5-7：1.
6. preparation method according to claim 3, it is characterised in that by diazotizing p-aminobenzoic acid and tripterygium wilfordii first
Element coupling includes：By triptolide it is complete it is complete it is soluble in water obtain triptolide solution, the triptolide solution with again
Nitridation p-aminobenzoic acid mixing, adjust pH8-9, stir 1-3h, centrifuging and taking supernatant, be separately added into catalytic amount tri-n-butylamine and
Isobutyl chlorocarbonate, low temperature stirring 20-30min.
7. preparation method according to claim 6, it is characterised in that the triptolide and the p-aminobenzoic acid
Mass ratio be 3-5:4-6.
8. preparation method according to claim 3, it is characterised in that include the compound and carrier protein couplet：
Bovine serum albumin(BSA) is dissolved in PBS, bovine serum albumin solution is obtained, dimethyl is added into the bovine serum albumin solution
Formamide.
9. preparation method according to claim 8, it is characterised in that the bovine serum albumin(BSA) and the triptolide
Mass ratio be 15-20:1.
10. a kind of triptolide polyclonal antibody, it is characterised in that it is as the triptolide people described in claim 1 or 2
Work antigen-immunized animal, and obtain the serum of animal and obtain.
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