CN101293912A - Conjugate for contructing cationised carrier protein and aflatoxin B1 with one-step method - Google Patents
Conjugate for contructing cationised carrier protein and aflatoxin B1 with one-step method Download PDFInfo
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- 102000014914 Carrier Proteins Human genes 0.000 title claims abstract description 29
- 108010078791 Carrier Proteins Proteins 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 15
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 title abstract description 5
- 239000002115 aflatoxin B1 Substances 0.000 title abstract 4
- 229930020125 aflatoxin-B1 Natural products 0.000 title abstract 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 46
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 31
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 31
- 239000007822 coupling agent Substances 0.000 claims abstract description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 5
- 239000001257 hydrogen Substances 0.000 claims abstract description 5
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims abstract description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 36
- 238000002360 preparation method Methods 0.000 claims description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 34
- 239000000243 solution Substances 0.000 claims description 25
- 238000013016 damping Methods 0.000 claims description 23
- 239000008367 deionised water Substances 0.000 claims description 23
- 229910021641 deionized water Inorganic materials 0.000 claims description 23
- 239000012530 fluid Substances 0.000 claims description 23
- 238000002156 mixing Methods 0.000 claims description 22
- 238000006243 chemical reaction Methods 0.000 claims description 20
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical class CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 19
- 238000004108 freeze drying Methods 0.000 claims description 18
- 150000001768 cations Chemical class 0.000 claims description 16
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 14
- 150000002500 ions Chemical class 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 239000003643 water by type Substances 0.000 claims description 10
- 239000000427 antigen Substances 0.000 claims description 7
- 102000036639 antigens Human genes 0.000 claims description 7
- 108091007433 antigens Proteins 0.000 claims description 7
- 125000002091 cationic group Chemical group 0.000 claims description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 229940098773 bovine serum albumin Drugs 0.000 claims description 6
- 230000005611 electricity Effects 0.000 claims description 6
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 5
- 241001465754 Metazoa Species 0.000 claims description 5
- 101100441836 Mus musculus Cyhr1 gene Proteins 0.000 claims description 4
- 210000000612 antigen-presenting cell Anatomy 0.000 claims description 4
- 230000004044 response Effects 0.000 claims description 4
- 108010058846 Ovalbumin Proteins 0.000 claims description 3
- 108010039918 Polylysine Proteins 0.000 claims description 3
- 230000009471 action Effects 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 210000000170 cell membrane Anatomy 0.000 claims description 3
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 3
- 229940092253 ovalbumin Drugs 0.000 claims description 3
- 229920000656 polylysine Polymers 0.000 claims description 3
- 230000000638 stimulation Effects 0.000 claims description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Natural products CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 claims description 2
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 2
- NSOXQYCFHDMMGV-UHFFFAOYSA-N Tetrakis(2-hydroxypropyl)ethylenediamine Chemical compound CC(O)CN(CC(C)O)CCN(CC(C)O)CC(C)O NSOXQYCFHDMMGV-UHFFFAOYSA-N 0.000 claims description 2
- 238000003763 carbonization Methods 0.000 claims 1
- 150000004985 diamines Chemical class 0.000 claims 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 claims 1
- 230000003053 immunization Effects 0.000 claims 1
- 238000002372 labelling Methods 0.000 claims 1
- 102000013415 peroxidase activity proteins Human genes 0.000 claims 1
- 108040007629 peroxidase activity proteins Proteins 0.000 claims 1
- 150000001412 amines Chemical class 0.000 abstract description 2
- 238000006482 condensation reaction Methods 0.000 abstract description 2
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 abstract 1
- 239000013543 active substance Substances 0.000 abstract 1
- 238000007069 methylation reaction Methods 0.000 abstract 1
- 239000007987 MES buffer Substances 0.000 description 14
- 238000010168 coupling process Methods 0.000 description 9
- 238000005859 coupling reaction Methods 0.000 description 9
- 230000008878 coupling Effects 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000010521 absorption reaction Methods 0.000 description 5
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000013459 approach Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 229930195730 Aflatoxin Natural products 0.000 description 2
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 2
- 241000228197 Aspergillus flavus Species 0.000 description 2
- 241000228230 Aspergillus parasiticus Species 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000005409 aflatoxin Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 231100000357 carcinogen Toxicity 0.000 description 2
- 239000003183 carcinogenic agent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005837 enolization reaction Methods 0.000 description 2
- 150000002085 enols Chemical group 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- 102100036227 Cysteine and histidine-rich protein 1 Human genes 0.000 description 1
- 101000947448 Homo sapiens Cysteine and histidine-rich protein 1 Proteins 0.000 description 1
- 101100007427 Manduca sexta COVA gene Proteins 0.000 description 1
- HRNLUBSXIHFDHP-UHFFFAOYSA-N N-(2-aminophenyl)-4-[[[4-(3-pyridinyl)-2-pyrimidinyl]amino]methyl]benzamide Chemical compound NC1=CC=CC=C1NC(=O)C(C=C1)=CC=C1CNC1=NC=CC(C=2C=NC=CC=2)=N1 HRNLUBSXIHFDHP-UHFFFAOYSA-N 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 101710182223 Toxin B Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 150000001728 carbonyl compounds Chemical class 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- -1 imines ion Chemical class 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
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- 231100000614 poison Toxicity 0.000 description 1
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- 150000003141 primary amines Chemical class 0.000 description 1
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Abstract
The invention utilizes amine methylation reaction principle to synthesize a conjugate of aflatoxin B1 and a cationized carrier protein by one step method via condensation reaction between the alpha-reactive hydrogen of the carbonyl group in the structure of the aflatoxin B1 and the aminoethyl group in a cationized protein by using formaldehyde as a coupling agent. Compared with the two-step method in the prior art, which firstly converts the aflatoxin B1 to a carboxyl active substance and then condensates and couples with a carrier protein, the inventive method has the advantages of less steps, simple operation, and high utilization ratio.
Description
Technical field
The present invention relates to proteic preparation of cation carrier and AFB
1The single stage method of-cationic protein conjugate makes up.The conjugate that makes up as the immunogen immune animal, can be improved the immunne response level of body, prepare high-quality aspergillus flavus resisting toxin B
1Antibody.
Background technology
(Aflatoxin is the poisonous secondary metabolite that a class is mainly produced by Aspergillus parasiticus (Aspergillus parasiticus) and the malicious flavus (A.flavus) of product AFT), wherein with AFB to aflatoxin
1(aflatoxin B
1, AFB
1) the harm maximum, be one of carcinogens of present known toxicity maximum.International cancer research institution has classified it as I level carcinogens.Because AFB
1The approach of contaminated food products is a lot, therefore will stop AFB fully
1Pollution be impossible, improve measures for the supervision and adopt the science detection method can reduce AFB to the full extent
1Harm to human health.Present AFB
1Detection method mainly contain based on physics and chemistry and the immunity analytical procedure, the former comprises thin layer chromatography, high performance liquid chromatography, mass spectrum and capillary electrophoresis etc., thin layer chromatography detects AFB at present as China
1National standard method, though easy and simple to handle, with low cost, owing to be semi-quantitative method, sensitivity is low, has been difficult to satisfy the needs of low detectability; Though and highly sensitive, the favorable reproducibility of other instrument analytical methods exists consuming time, shortcomings such as detecting instrument is expensive, complicated operation simultaneously, can not satisfy the demand of field quick detection; And immunological detection methods such as enzyme linked immunological absorption reaction, immune microtrabeculae and immuno-chip because save time, cost is low, easy and simple to handle, be fit to batch detection and on-the-spot the detection, just progressively be subjected to the attention of each quality inspection relevant departments of China.
AFB
1As haptens, need with its with carrier protein couplet after, just can possess the immunogenicity that stimulates animal generation antibody.But since complete antigen preparation process complexity, AFB
1Utilization ratio is low, therefore usually needs to use relatively large AFB
1Just can prepare corresponding antibody; Yet, after the September 11 attacks, AFB
1Biological reagent as a kind of severe toxicity is controlled by the strictness of developed countries such as the U.S..Not only price is high but also be difficult to buy for these toxin standard substance on the market, and this gives research AFB
1Immunological detection method bring very big difficulty, also hindered the application and the popularization of immunological method to a certain extent.
AFB is thought in early stage research
1Character torpescence own does not possess the active group that is connected with carrier proteins, after it need being derived, again with carrier protein couplet, promptly earlier with AFB
1Be converted into AFB
1-activated carboxylic thing (AFB
1-Oxime, AFB
1-O), then, carbodiimide (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide, EDC) wait under the catalysis of coupling agent, with carrier proteins generation condensation reaction, form stable AFB
1-O-carrier protein couplet thing.Wherein, the first step AFB
1Transformation efficiency be 70%～80%, AFB
1The purifying yield of-O is 80%～90%, and only there was 30%～40% AFB in second step
1-O is coupled on the carrier proteins.Therefore, AFB
1Overall utilization about 20%, utilization ratio is lower.
Comprehensive above-mentioned reason, research improves AFB
1The conjugate preparation method of utilization ratio is very important.
The present invention has utilized the cation carrier proteinogenic amines principle that methylates to make up AFB
1The proteic conjugate of-cation carrier.At AFB
1Structure in because the α-reactive hydrogen of its carbonyl under mild acid conditions, exists with enol form, can be by the coupled action of formaldehyde, amine-methylated coupling takes place with the amine ethyl of carrier proteins.Because the proteic carboxyl of cation carrier is almost all replaced by the amine ethyl, thereby whole protein band positive electricity, during as the complete antigen immune animal, than common proteic conjugate, cation carrier albumen is easier to be combined with the surface of cell membrane of the weak negative electricity of band in the body, and then discerned and the intravital immunne response of stimulation machine by antigen presenting cell, simultaneously, also improved body to covalent coupling at cation carrier albumen haptens (AFB
1) identification and reply level, can the stimulator antigen presenting cells to the propagation of offering, promote T lymphocyte and bone-marrow-derived lymphocyte of haptenic group.
AFB among the present invention
1Overall utilization about 50%.Experiment showed, that the conjugate by this method preparation has better immunogenicity, can stimulate body to produce high-quality AFB
1Antibody.Therefore, the present invention is with respect to prior synthesizing method, has that operation steps is few, a toxin utilization ratio height, antibody titer advantages of higher.
This patent inventor has invented with one step of cation carrier albumen structure AFB at the problems that two-step approach exists
1The method of-carrier protein couplet thing.Correlative study does not appear in the newspapers both at home and abroad as yet.
The main reference document
[1]Apple，R.J.，Domen，P.L.，Muckerheide，A.and?Michael，J.G.(1988)Cationization?of?protein?antigens.IV.Increased?antigen?uptake?by?antigen-presenting?cells.J?Immunol?140，3290-5.
[2]Barbiroli，A.，Bonomi，F.，Benedetti，S.，Mannino，S.，Monti，L.，Cattaneo，T.and?Iametti，S.(2007)Bindingof?aflatoxin?M1?to?different?protein?fractions?in?ovine?and?caprine?milk.J?Dairy?Sci?90，532-40.
[3]Chu，F.S.，Lau，H.P.，Fan，T.S.and?Zhang，G.S.(1982)Ethylenediamine?modified?bovine?serum?albumin?asprotein?carrier?in?the?production?of?antibody?against?mycotoxins.J?Immunol?Methods?55，73-8.
[4]Domen，P.L.，Muckerheide，A.and?Michael，J.G.(1987)Cationization?of?protein?antigens.III.Abrogation?oforal?tolerance.J?Immunol?139，3195-8.
[5]Fusheng，C.，Xinchang?Luo，Qi?Zhou.(1999)Immunological?detection?of?afltatoxin?B1?I：preparation?ofaflatoxin?B1?antigen.Mycosystema?18，316～320.(in?Chinese)
[6]Gathumbi，J.K.，Usleber，E.，Ngatia，T.A.，Kangethe，E.K.and?Martlbauer，E.(2003)Application?ofimmunoaffinity?chromatography?and?enzyme?immunoassay?in?rapid?detection?of?aflatoxin?B1?in?chickenliver?tissues.Poult?Sci?82，585-90.
[7]Jaimez，J.，Fente，C.A.，Vazquez，B.I.，Franco，C.M.，Cepeda，A.，Mahuzier，G.and?Prognon，P.(2000)Application?of?the?assay?of?aflatoxins?by?liquid?chromatography?with?fluorescence?detection?in?food?analysis.Journal?of?Chromatography?A?882，1-10.
[8]Muckerheide，A.，Apple，R.J.，Pesce，A.J.and?Michael，J.G.(1987a)Cationization?of?protein?antigens.I.Alteration?of?immunogenic?properties.J?Immunol?138，833-7.
[9]Muckerheide，A.，Domen，P.L.and?Michael，J.G.(1987b)Cationization?of?protein?antigens.II.Alteration?ofregulatory?properties.J?Immunol?138，2800-4.
[10]Ramos?Catharino，R.，de?Azevedo?Marques，L.，Silva?Santos，L.，Baptista，A.S.，Gloria，E.M.，Calori-Domingues，M.A.，Facco，E.M.and?Eberlin，M.N.(2005)Aflatoxin?screening?by?MALDI-TOF?massspectrometry.Anal?Chem?77，8155-7.
[11]Saha，D.，Acharya，D.，Roy，D.，Shrestha，D.and?Dhar，T.K.(2007)Simultaneous?enzyme?immunoassay?forthe?screening?of?aflatoxin?B1?and?ochratoxin?A?in?chili?samples.Anal?Chim?Acta?584，343-9.
[12]Sapsford，K.E.，Taitt，C.R.，Fertig，S.，Moore，M.H.，Lassman，M.E.，Maragos，C.M.and?Shriver-Lake，L.C.(2006)Indirect?competitive?immunoassay?for?detection?of?aflatoxin?B1?in?corn?and?nut?products?using?thearray?biosensor.Biosensors?and?Bioelectronics?21，2298-2305.
Summary of the invention
(1) technical problem that will solve
The purpose of this invention is to provide an a kind of step structure AFB
1The method of-carrier protein couplet thing.
(2) technical scheme
For achieving the above object, the technical solution used in the present invention is:
Enol or contain the carbonyl compound of α-reactive hydrogen under the effect of coupling agent formaldehyde, can carry out coupling with primary amine or secondary amine.
According to this principle, AFB
1α-the reactive hydrogen of last carbonyl just can react with the cationic protein that contains the amine ethyl under the coupled action of formaldehyde, obtains AFB
1The conjugate of-cationic protein.Cationization albumen is for simple albumen, has more amine ethyl, thereby whole protein band positive electricity, during as the complete antigen immune animal, than common proteic conjugate, cation carrier albumen is easier to be combined with the surface of cell membrane of the weak negative electricity of band in the body, and then is discerned and the intravital immunne response of stimulation machine by antigen presenting cell.
Concrete steps:
1. the preparation of cationic protein
Carrier proteins comprises: bovine serum albumin (Bovine serum albumin, BSA), oralbumin (Ovalbumin, OVA), horseradish peroxidase (Horseradish peroxidase, HRP), blood hole spoon albumen (Keyhole limpethemocyanin, KLH) and poly-lysine (Ploy-l-lysine, PLL).
Preparation process:
(1) with quadrol (ethylenediamine, (2-(N-morpholino) ethane sulfonic acid MES) in the damping fluid, and regulates pH to slightly acidic with HCl EDA) to join 2 ethane sulfonic aicd base in the ice bath;
(2) carrier proteins is dissolved in MES after, with above-mentioned EDA solution mixing;
(3) drip coupling agent EDC in (2) described solution, behind the room temperature reaction, use the acetic acid termination reaction;
(4) above-mentioned reactant is fully dialysed with deionized water;
(5) with after its freeze-drying, freeze-drying is standby.
Used EDA volume between 20 μ L～100 μ L, used carrier proteins quality between 5mg～100mg, the volume ratio of EDA and MES between 1: 25～1: 5, the pH regulator to 4 of MES damping fluid～6.
Prepare positively charged ion bovine serum albumin (Cationized bovine serum albumin, cBSA), positively charged ion oralbumin (Cationized ovalbumin, cOVA), positively charged ion horseradish peroxidase (Cationized horseradishperoxidase, cHRP), positively charged ion blood hole spoon albumen (Cationized keyhole limpet hemocyanin, cKLH) or the positively charged ion poly-lysine (Cationized ploy-l-lysine, cPLL).
2.AFB
1The preparation of-cation carrier protein conjugate
AFB
1-cation carrier protein conjugate comprises AFB
1-cBSA, AFB
1-cOVA, AFB
1-cHRP, AFB
1-cKLH and AFB
1-cPLL.
Preparation process:
(1) with cation carrier albumen and AFB
1Respectively with after deionized water and the dimethyl formamide dissolving, with AFB
1Solution dropwise joins mixing in the carrier proteins liquid;
(2) in above-mentioned reaction solution, add coupling agent formaldehyde, promptly obtain AFB after the reaction
1-cation carrier protein conjugate;
(3) fully dialyse with deionized water after, freeze-drying is standby.
Wherein, cation carrier albumen and AFB
1Mass ratio between 1: 1～5: 1, the volume ratio of dimethyl formamide and formaldehyde is between 1: 1～5: 1.
(3) beneficial effect
The present invention is with AFB
1Be haptens, utilize the cationic protein single stage method to synthesize AFB
1-carrier protein couplet thing, this method have simple, the AFB of step
1The utilization ratio advantages of higher has been avoided shortcomings such as present used two-step approach step is many, utilization ratio is low.
Description of drawings
Below in conjunction with drawings and Examples patent of the present invention is further specified.
Figure 1A FB
1Structure
The cationization of Fig. 2 carrier proteins
Fig. 3 AFB
1With the proteic coupling mechanism of cation carrier
1. AFB
1Enolization in weak acidic buffer;
2. the imines ionic forms in the cation carrier albumen;
3. the imines ion in the cation carrier albumen is under the formaldehyde effect, with enolization AFB
1Coupling
Embodiment
Embodiment 1AFB
1The preparation of-cBSA conjugate
1.1cBSA preparation:
50～100 μ LEDA are joined in the 500 μ L ice bath MES damping fluids, and with HCl with pH regulator to 5.5 about, again 5～100mg BSA is dissolved in the MES damping fluid of 50 μ L, then, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, use the acetic acid termination reaction, with the deionized water 48～72h that fully dialyses, freeze-drying is standby at last.
1.2AFB
1The preparation of-cBSA conjugate:
CBSA and 2mg AFB with 5～100mg
1Respectively with after 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with above-mentioned AFB
1Solution dropwise joins mixing in the above-mentioned protein soln, adds 50～1000 μ L formaldehyde then rapidly, and 37 ℃ of following jog 24h fully dialyse behind the 48h with deionized water, and freeze-drying is standby.
Embodiment 2AFB
1-cOVA conjugate preparation
2.1cOVA preparation:
50～100 μ L EDA are joined in the 500 μ L ice bath MES damping fluids, and with HCl with pH regulator to 5.5 about, again 5～100mgOVA is dissolved in the above-mentioned damping fluid of 100 μ L, then, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, use the acetic acid termination reaction, with the deionized water 48～72h that fully dialyses, freeze-drying is standby at last.
2.2AFB
1The preparation of-cOVA conjugate:
COVA and 1～50mg AFB with 5～100mg
1Respectively with after 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with above-mentioned AFB
1Solution dropwise joins mixing in the above-mentioned protein soln, adds 50～1000 μ L formaldehyde then rapidly, and 37 ℃ of following jog 24h fully dialyse behind the 48h with deionized water, and freeze-drying is standby.
Embodiment 3AFB
1The preparation of-cHRP conjugate
3.1cHRP preparation:
50～100 μ L EDA are joined in the 500 μ L MES damping fluids of ice bath, and with HCl with pH regulator to 5.5 about, again 5～100mgHRP is dissolved in the above-mentioned MES damping fluid of 500 μ L, then, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, use the acetic acid termination reaction, with the deionized water 48～72h that fully dialyses, freeze-drying is standby at last.
3.2AFB
1The preparation of-cHRP conjugate:
CHRP and 1～50mg AFB with 5～100mg
1Respectively with after 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with above-mentioned AFB
1Solution dropwise joins in the above-mentioned protein soln behind the mixing, adds 50～1000 μ L formaldehyde rapidly, and 37 ℃ of following jog 24h fully dialyse behind the 48h with deionized water, and freeze-drying is standby.
Embodiment 4AFB
1The preparation of-cKLH conjugate
4.1cKLH preparation:
50～100 μ L EDA are joined in the 500 μ L MES damping fluids of ice bath, and with HCl with pH regulator to 5.5 about, again 5～100mg KLH is dissolved in the above-mentioned MES damping fluid of 800 μ L, then, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, use the acetic acid termination reaction, with the deionized water 48～72h that fully dialyses, freeze-drying is standby at last.
4.2AFB
1The preparation of-cKLH conjugate:
CKLH and 1～50mg AFB with 5～100mg
1Respectively with after 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with above-mentioned AFB
1Solution dropwise joins mixing in the above-mentioned protein soln, adds 50～1000 μ L formaldehyde then rapidly, and 37 ℃ of following jog 24h fully dialyse behind the 48h with deionized water, and freeze-drying is standby.
Embodiment 5AFB
1The preparation of-cPLL conjugate
5.1cPLL preparation:
50～100 μ L EDA are joined in the 500 μ L MES damping fluids of ice bath, and with HCl with pH regulator to 5.5 about, again 5～100mg PLL is dissolved in the above-mentioned MES damping fluid of 1000 μ L, then, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, use the acetic acid termination reaction, with the deionized water 48～72h that fully dialyses, freeze-drying is standby at last.
5.2AFB
1The preparation of-cPLL conjugate:
CPLL and 1～50mg AFB with 5～100mg
1Respectively with after 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with above-mentioned AFB
1Solution dropwise joins mixing in the above-mentioned protein soln, adds 50～1000 μ L formaldehyde then rapidly, and 37 ℃ of following jog 24h fully dialyse behind the 48h with deionized water, and freeze-drying is standby.
Above-mentioned used MES buffer concentration is 0.1mol/L, and HCl is 1mol/L, and acetic acid is 4mol/L.
AFB
1The evaluation of-cationic protein conjugate:
Adopt ultraviolet spectrophotometer to cation carrier albumen, AFB
1Mark product and conjugate scan, and determine according to its charateristic avsorption band whether coupling is successful.
The result shows, AFB
1-cation carrier protein conjugate has two main absorption peaks at 270nm and 366nm, and previous absorption peak is carrier proteins (280nm) and AFB
1Stack peak (266nm), and the latter is AFB
1Another feature absorption peak (363nm).According to the coupling ratio formula,, can calculate AFB in conjunction with absorption peak wavelength and absorbancy on the uv-spectrogram
1Coupling ratio with cationic protein.
Formula:
Wherein:
AFB in the conjugate
1Molar mass;
M
Carrier proteins: the molar mass of carrier proteins in the conjugate;
A
Conjugate 278nm: conjugate is in the absorbancy at 278nm place;
A
Conjugate 366nm: conjugate is in the absorbancy at 366nm place;
ε
Carrier proteins 278nm: carrier proteins is at the molar absorptivity at 278nm place
Through calculating AFB
1With the coupling ratio of cationic protein about 6.1, proved successfully to prepare AFB
1-cationic protein conjugate.
Claims (6)
1. cationic protein is characterized in that structural formula:
Wherein, the carboxyl of carrier proteins is almost all replaced by the amine ethyl, whole protein band positive electricity, thereby during as the complete antigen immune animal, cation carrier albumen is easier to be combined with the surface of cell membrane of the weak negative electricity of band in the body, and then is discerned and the intravital immunne response of stimulation machine by antigen presenting cell.
2.AFB
1-cationic protein conjugate is characterized in that its molecular structural formula is:
Wherein, carrier proteins can be positively charged ion bovine serum albumin (Cationized bovine serum albumin, cBSA), positively charged ion oralbumin (Cationized ovalbumin, cOVA), positively charged ion horseradish peroxidase (Cationizedhorseradish peroxidase, cHRP), positively charged ion blood hole spoon albumen (Cationized keyhole limpet hemocyanin, cKLH) or positively charged ion poly-lysine (Cationized ploy-L-lysine, cPLL), respectively with AFB
1In conjunction with obtaining AFB
1-cBSA, AFB
1-cOVA, AFB
1-cHRP, AFB
1-cKLH and AFB
1-cPLL.
3. prepare the proteic method of the described a kind of cation carrier of claim 1, it is characterized in that comprising step:
(1) preparation cBSA, step is as follows:
With 20～100 μ L quadrol (ethylenediamine, EDA) join 500 μ L 0.1mol/L 2 ethane sulfonic aicds (2-(N-morpholino) the ethane sulfonic acid of ice bath, MES) in the damping fluid, and with 1mol/L HCl with pH regulator to 4～6, then, after 5～100mg BSA being dissolved in the above-mentioned MES damping fluid of 50 μ L, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add again 2～50mg the inferior diamines of carbonization (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide, EDC), stirring at room 1～12h is with the acetic acid termination reaction of 4mol/L, fully dialyse behind 48～72h with deionized water, its cold storage is standby.
(2) preparation cOVA, step is as follows:
20～100 μ L EDA are joined in the 500 μ L 0.1mol/L MES damping fluids of ice bath, and with 1mol/L HCl with pH regulator to 4～6, then, after 5～100mg OVA being dissolved in the above-mentioned MES damping fluid of 100 μ L,, in above-mentioned mixed solution, add the EDC of 2～50mg again with above-mentioned EDA solution mixing, stirring at room 1～12h, with the acetic acid termination reaction of 4mol/L, fully dialyse behind 48～72h with deionized water, its cold storage is standby.
(3) preparation cHRP, step is as follows:
20～100 μ L EDA are joined in the 500 μ L 0.1mol/L MES damping fluids of ice bath, and with 1mol/L HCl with pH regulator to 4～6, then, after 5～100mg HRP being dissolved in the above-mentioned MES damping fluid of 500 μ L,, in above-mentioned mixed solution, add the EDC of 2～50mg again with above-mentioned EDA solution mixing, stirring at room 1～12h, with the acetic acid termination reaction of 4mol/L, fully to dialyse behind 48～72h with deionized water, cold storage is standby.
(4) preparation cKLH, step is as follows:
20～100 μ L EDA are joined in the 500 μ L 0.1mol/L MES damping fluids of ice bath, and with 1mol/L HCl with pH regulator to 4～6, after 5～100mg KLH being dissolved in the above-mentioned MES damping fluid of 800 μ L again, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, with the acetic acid termination reaction of 4mol/L, fully dialyse behind 48～72h with deionized water, cold storage is standby.
(5) preparation cPLL, step is as follows:
20～100 μ L EDA are joined in the 500 μ L 0.1mol/L MES damping fluids of ice bath, and with 1mol/L HCl with pH regulator to 4～6, again 5～100mg PLL is dissolved in the above-mentioned MES damping fluid of 1000 μ L, then, with above-mentioned EDA solution mixing, in above-mentioned mixed solution, add the EDC of 2～50mg again, behind stirring at room 1～12h, with the acetic acid termination reaction of 4mol/L, with the deionized water 48～72h that fully dialyses, cold storage is standby at last.
Used EDA volume between 20 μ L～100 μ L, used carrier proteins quality between 5mg～100mg, the volume ratio of EDA and MES between 1: 25～1: 5, the pH regulator to 4 of MES damping fluid～6.
4. as a kind of AFB as described in the claim 2
1The preparation method of-cation carrier protein conjugate is characterized in that AFB
1α-the reactive hydrogen of last carbonyl can be under the coupled action of formaldehyde and the amine ethyl reaction of cationic protein, obtains AFB
1The proteic conjugate of-cation carrier can prepare the AFB that comprises immunizing antigen, envelope antigen and enzyme labelling thing
1-cationic protein conjugate obtains AFB
1Complete antigen.
5. as a kind of AFB as described in the claim 2
1The preparation method of-cation carrier protein conjugate is characterized in that coupling agent formaldehyde.
6. method according to claim 4 is characterized in that comprising step:
(1) AFB
1The preparation of-cBSA, step is as follows: with 5～100mg cBSA and 1～50mg AFB
1Respectively with 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with AFB
1Solution dropwise joins in the above-mentioned protein soln, behind the mixing, adds 50～1000 μ L formaldehyde rapidly, and 37 ℃ of following jog 24h fully promptly obtain AFB after the reaction
1-cBSA fully dialyses behind the 48h with deionized water, and freeze-drying is standby.
(2) AFB
1The preparation of-cOVA, step is as follows: with cOVA and the 1～50mg AFB of 5～100mg
1Respectively with 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with AFB
1Solution dropwise joins in the above-mentioned protein soln, behind the mixing, adds 50～1000 μ L formaldehyde rapidly, and 37 ℃ of following jog 24h fully promptly obtain AFB after the reaction
1-cOVA then fully dialyses behind the 48h with deionized water, and freeze-drying is standby.
(3) preparation of AFB1-cHRP, step is as follows: with cHRP and the 1～50mg AFB of 5～100mg
1Respectively with 500～10000 μ L deionized waters and the dissolving of 500 μ L dimethyl formamides, again with AFB
1Solution dropwise joins in the above-mentioned protein soln, behind the mixing, adds 50～1000 μ L formaldehyde rapidly, and 37 ℃ of following jog 24h fully promptly obtain AFB after the reaction
1-cHRP then fully dialyses behind the 48h with deionized water, and freeze-drying is standby.
(4) AFB
1The preparation of-cKLH, step is as follows: with cKLH and the 1～50mg AFB of 5～100mg
1Respectively with 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with AFB
1Solution dropwise joins in the above-mentioned protein soln, behind the mixing, adds 50～1000 μ L formaldehyde rapidly, and 37 ℃ of following jog 24h fully promptly obtain AFB after the reaction
1-cKLH then fully dialyses behind the 48h with deionized water, and freeze-drying is standby.
(5) AFB
1The preparation of-cPLL, step is as follows: with cPLL and the 1～50mg AFB of 5～100mg
1Respectively with 500～10000 μ L deionized waters and the dissolving of 50～1000 μ L dimethyl formamides, again with above-mentioned AFB
1Solution dropwise joins in the above-mentioned protein soln, behind the mixing, adds 50～1000 μ L formaldehyde rapidly, and 37 ℃ of following jog 24h fully promptly obtain AFB after the reaction
1-cPLL then fully dialyses behind the 48h with deionized water, and freeze-drying is standby.
Wherein, cation carrier albumen and AFB
1Mass ratio between 1: 1～5: 1, the volume ratio of dimethyl formamide and formaldehyde is between 1: 1～5: 1.
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| CNA2007100519367A CN101293912A (en) | 2007-04-23 | 2007-04-23 | Conjugate for contructing cationised carrier protein and aflatoxin B1 with one-step method |
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| CN109725143A (en) * | 2018-12-27 | 2019-05-07 | 国家食品安全风险评估中心 | Aflatoxin ELISA kit and detection method |
| CN114252591A (en) * | 2021-12-27 | 2022-03-29 | 深圳市亚辉龙生物科技股份有限公司 | Magnetic bead coating and preparation method thereof and detection kit |
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