CN109865137A - Agarose microbeads through iodoacteyl activation are improving the application in polypeptide or protide immunogen immune originality - Google Patents
Agarose microbeads through iodoacteyl activation are improving the application in polypeptide or protide immunogen immune originality Download PDFInfo
- Publication number
- CN109865137A CN109865137A CN201910065007.4A CN201910065007A CN109865137A CN 109865137 A CN109865137 A CN 109865137A CN 201910065007 A CN201910065007 A CN 201910065007A CN 109865137 A CN109865137 A CN 109865137A
- Authority
- CN
- China
- Prior art keywords
- iodoacteyl
- polypeptide
- agarose microbeads
- activation
- protide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229920000936 Agarose Polymers 0.000 title claims abstract description 74
- 239000011325 microbead Substances 0.000 title claims abstract description 70
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 39
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 38
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 38
- 230000004913 activation Effects 0.000 title claims abstract description 18
- 230000002163 immunogen Effects 0.000 title claims abstract description 5
- 230000005847 immunogenicity Effects 0.000 claims abstract description 25
- 230000003053 immunization Effects 0.000 claims description 35
- 238000002649 immunization Methods 0.000 claims description 35
- 108090000623 proteins and genes Proteins 0.000 claims description 21
- 102000004169 proteins and genes Human genes 0.000 claims description 17
- 150000001875 compounds Chemical class 0.000 abstract description 10
- 230000002427 irreversible effect Effects 0.000 abstract description 2
- 239000011347 resin Substances 0.000 abstract 2
- 229920005989 resin Polymers 0.000 abstract 2
- 239000002671 adjuvant Substances 0.000 description 43
- 238000000034 method Methods 0.000 description 42
- 238000001262 western blot Methods 0.000 description 36
- 241000699666 Mus <mouse, genus> Species 0.000 description 31
- 210000002966 serum Anatomy 0.000 description 24
- 238000012360 testing method Methods 0.000 description 21
- 238000002347 injection Methods 0.000 description 20
- 239000007924 injection Substances 0.000 description 20
- 238000007920 subcutaneous administration Methods 0.000 description 20
- 238000001514 detection method Methods 0.000 description 16
- 241000699670 Mus sp. Species 0.000 description 12
- 238000005119 centrifugation Methods 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 102100028452 Nitric oxide synthase, endothelial Human genes 0.000 description 11
- 101710090055 Nitric oxide synthase, endothelial Proteins 0.000 description 11
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 230000001376 precipitating effect Effects 0.000 description 8
- 210000003462 vein Anatomy 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 238000004132 cross linking Methods 0.000 description 7
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000004945 emulsification Methods 0.000 description 5
- VLEIUWBSEKKKFX-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O VLEIUWBSEKKKFX-UHFFFAOYSA-N 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 101710113864 Heat shock protein 90 Proteins 0.000 description 4
- 102100034051 Heat shock protein HSP 90-alpha Human genes 0.000 description 4
- 210000000683 abdominal cavity Anatomy 0.000 description 4
- 238000004500 asepsis Methods 0.000 description 4
- 210000004556 brain Anatomy 0.000 description 4
- 210000005013 brain tissue Anatomy 0.000 description 4
- 239000003398 denaturant Substances 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 210000003000 inclusion body Anatomy 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000006303 immediate early viral mRNA transcription Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000002107 myocardial effect Effects 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 102100026882 Alpha-synuclein Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 101000834898 Homo sapiens Alpha-synuclein Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101000980463 Treponema pallidum (strain Nichols) Chaperonin GroEL Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005267 amalgamation Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to the new applications of the agarose microbeads activated through iodoacteyl, and in particular to the agarose microbeads through iodoacteyl activation are improving the application in polypeptide or protide immunogen immune originality.Specifically and efficiently with polypeptide or the sulfydryl (- SH) of protide immunogene irreversible react can occur for the iodoacteyl in the agarose microbeads through iodoacteyl activation; generate covalently bound polypeptide-agarose resin compound or albumen-agarose resin compound; compound molecular weight with higher; it is biggish heterogeneous and be difficult to degradability, the immunogenicity of polypeptide or protide immunogene can be greatly improved.
Description
Technical field
The present invention relates to the new applications of the agarose microbeads activated through iodoacteyl, and in particular to living through iodoacteyl
The agarose microbeads of change are improving the application in polypeptide or protide immunogen immune originality.
Background technique
Antibody (antibody) is body under antigenic substance stimulation, caused by the thick liquid cell be divided into as B cell, can
The immunoglobulin that specific binding is reacted occurs with corresponding antigens.The immunogene for being used to prepare antibody can be albumen, polypeptide
Or nucleic acid etc., since small molecular protein or polypeptide antigen are poor, it is single it is immune be difficult to generate good immune response, only and
Carrier and adjuvant appropriate, which combine, can be only achieved ideal effect.The immunogenicity of antigen can be improved in carrier, enhances animal machine
The immune response of body；Adjuvant can also extend the timeliness of antigen other than improving the immunogenicity of antigen.
Currently, more common antigen vectors have: bovine serum albumin(BSA) (BSA), the hemocyanin (KLH), ovum of protide
Pure albumen (OVA) etc.；The poly-D-lysine of poltpeptides；Macromolecule polyalcohol and certain particles, such as polyvinyl pyrrole
Alkanone, active carbon, carboxymethyl cellulose etc..After destination protein or polypeptide and the crosslinking of the carrier of activation, immunogene is obtained.It is another kind of
More common method is that destination protein or polypeptide and certain macromolecule label protein are carried out amalgamation and expression.Two methods
Purpose be all to enhance the immune response of body by increasing the molecular weight of antigen.
Summary of the invention
The purpose of the present invention is to provide the new applications of the agarose microbeads activated through iodoacteyl.
Actually the present invention relates to the agarose microbeads activated through iodoacteyl to improve polypeptide or protide immunogene
Application in immunogenicity.
It further relates to the agarose microbeads activated through iodoacteyl and is improving the application in polypeptide α-syn immunogenicity.
It further relates to the agarose microbeads activated through iodoacteyl and is improving the application in polypeptide Olfr560 immunogenicity.
It further relates to the agarose microbeads activated through iodoacteyl and is improving the application in protein hsp 90 alpha immunization originality.
It further relates to the agarose microbeads activated through iodoacteyl and is improving the application in albumen eNOS immunogenicity.
Iodoacteyl in agarose microbeads of the present invention through iodoacteyl activation can be specifically and efficient
With polypeptide or the sulfydryl (- SH) of protide immunogene occur it is irreversible react, generate covalently bound polypeptide-agarose microbeads
Compound or albumen-agarose microbeads compound, compound molecular weight with higher are biggish heterogeneous and be difficult to degrade
Property, the immunogenicity of polypeptide or protide immunogene can be greatly improved.
In addition, the combination speed of agarose microbeads and polypeptide or protide immunogene through iodoacteyl activation is quickly,
And the combination of polypeptide immunogene, it is just achievable in 2 hours；And the combination of protide immunogene, it is just achievable in 3.5 hours.
Moreover, only polypeptide or protide immunogene need to be mixed with the agarose microbeads activated through iodoacteyl, under stationary state i.e.
It could be incorporated, it is time saving and energy saving；The agarose microbeads binding force also with higher activated through iodoacteyl, reduces immune
Former liquid volume；And the agarose microbeads of the activation and the crosslinking condition of protide immunogene are wide in range, pH7.2-9.0 it
Between can all carry out；Reaction solution can be aqueous solution, organic solvent or denaturant.
The heterologous protein of prokaryotic expression, often exists with inclusion bodies, at this moment needs to dissolve inclusion body with denaturant
After go to be immunized.And the agarose microbeads of the activation and the compatibility of denaturant, make it possible the crosslinking of inclusion body protein.
Detailed description of the invention
Fig. 1 is the embodiment of the present invention one using agarose microbeads as the Western Blot testing result histogram of carrier；
Fig. 2 is the embodiment of the present invention one using KLH as the Western Blot testing result histogram of carrier；
Fig. 3 is the embodiment of the present invention two using agarose microbeads as the Western Blot testing result histogram of carrier；
Fig. 4 is the embodiment of the present invention two using KLH as the Western Blot testing result histogram of carrier；
Fig. 5 is the embodiment of the present invention three using agarose microbeads as the Western Blot testing result histogram of carrier；
Fig. 6 is that the embodiment of the present invention three carries out immune Western Blot testing result histogram in conventional manner；
Fig. 7 is the embodiment of the present invention four using agarose microbeads as the Western Blot testing result histogram of carrier；
Fig. 8 is that the embodiment of the present invention four carries out immune Western Blot testing result histogram in conventional manner.
In figure: 1,2,3 test result for respectively representing three mouse in same group of experiment.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that the described embodiment is only a part of the embodiment of the present invention, instead of all the embodiments.Based on this
Embodiment in invention, every other reality obtained by those of ordinary skill in the art without making creative efforts
Example is applied, shall fall within the protection scope of the present invention.
Embodiment one:
The present embodiment provides the agarose microbeads activated through iodoacteyl to improve polypeptide α-syn (human
60 amino acid of synuclein alpha N-terminal, Gene ID:6622) application in immunogenicity.
It is living that the agarose microbeads activated in the present embodiment through iodoacteyl directly adopt commercially available iodoacteyl
The agarose microbeads (Thermo, prod#20404) changed, hereafter direct abbreviation agarose microbeads；
Application method of the agarose microbeads through iodoacteyl activation in raising polypeptide α-syn immunogenicity is as follows:
(1) microballoon wash: take 1ml agarose microbeads (Thermo, prod#20404) in 2mL centrifuge tube, level from
Scheming is centrifuged (1200rpm) 1min, removes supernatant；Addition 5ml Tris-EDTA (50mM Tris-HCl, 5mM EDTA-Na,
PH8.5), mix well (jog), supernatant is removed in centrifugation；
(2) step (1) is repeated twice；
(3) it is coupled: the agarose microbeads after removal supernatant is centrifuged with 2mL (1mg/mL) polypeptide α-syn solution in 15mL
It is mixed in pipe, is tiltedly placed in ambient temperature overnight and is crosslinked；
(4) mouse (one exempts from) is immunized: 3 mouse of selection are micro- by polypeptide-agarose of overnight crosslinking as experimental subjects
Ball compound moves into 2mL asepsis injector (band needle), and directly subcutaneous or abdominal cavity multi-point injection mouse, 250ug/ is only；
(5) two exempt from: carrying out within the 14th day being immunized for second, immunization method is with for the first time, and 250ug/ is only；
(6) three exempt from: the third time of progress in the 24th day is immune, and immunization method is with for the first time, and 250ug/ is only；
(7) detected whether antibody titer: the 28-29 days, tail vein acquired about 20ul tail blood, 5000 turns of centrifugation 5min,
Haemocyte precipitating is abandoned, serum is drawn, has detected whether antibody generation for Western Blot, detection sample is Hela cell；
(8) four exempt from: carrying out within the 34th day the 4th immune, immunization method same first time, 250ug/；
(9) mouse for having antibody through Western Blot detection in step (7) subsequent monoclonal antibody is carried out to prepare in fact
It tests.
It is following (Normal practice of the prior art) that Mouse Method is immunized after conventional KLH- α-syn polypeptide coupling:
(1) be coupled: taking KLH (Thermo, prod#77600) that 1mL concentration be 2mg/mL and 1mL concentration is 2mg/mL's
Polypeptide α-syn mixing, in being coupled 4h on room temperature shaker.
(2) emulsify: the KLH- α-syn and 1mL Freund's complete adjuvant (sigma, prod#F5881) for taking 1mL to be coupled are in 5mL
It is mixed in syringe, and forms Water-In-Oil mixture by way of injecting repeatedly and can be used to be immunized, it is same to choose 3
Mouse is as experimental subjects.
(3) one exempt from: the 1st day, adjuvant was not formula Freund's complete adjuvant (sigma, prod#F5881), subcutaneous multi-point injection,
250ug/ is only；
(4) two exempt from: the 1st day, adjuvant was not formula Freund's incomplete adjuvant (sigma, prod#F5506), subcutaneous multi-point injection,
250ug/ is only；
(5) three exempt from: the 1st day, adjuvant was not formula Freund's incomplete adjuvant (sigma, prod#F5506), subcutaneous multi-point injection,
250ug/ is only；
(6) detected whether antibody titer: the 28-29 days, tail vein acquired about 20ul tail blood, room temperature 30min, and 5000
Turn centrifugation 5min, abandon haemocyte precipitating, draw serum, Western Blot has detected whether antibody generation, and detection sample is
Hela cell；
(7) four exempt from: the 34th day, adjuvant was not formula Freund's incomplete adjuvant (sigma, prod#F5506), subcutaneous multi-point injection,
250ug/ is only；
(8) mouse for having antibody through Western Blot detection in step (6) subsequent monoclonal antibody is carried out to prepare in fact
It tests.
Two kinds of immunization method results compare:
Western Blot testing result comparison: can using the mice serum that agarose microbeads are obtained as the immunization method of carrier
To detect the α-syn albumen in Hela cell, show that size is the albumen one of 15kDa on Western Blot exposed plate
Band illustrates there is α-syn antibody in serum referring to Fig. 1；It is not detected using the mice serum that KLH is obtained as the immunization method of carrier
α-syn albumen in Hela cell does not have protein band, referring to fig. 2, illustrate in serum on Western Blot exposed plate
Either with or without α-syn antibody.
As it can be seen that the immunogenicity of polypeptide α-syn can be improved using agarose microbeads as carrier, and with KLH commonly used in the art
The immunogenicity of polypeptide α-syn can not be improved for carrier.
Embodiment two:
The present embodiment provides the agarose microbeads activated through iodoacteyl to improve polypeptide Olfr560 (128-162 ammonia
Base acid section, Gene ID:259117) application in immunogenicity.
It is living that the agarose microbeads activated in the present embodiment through iodoacteyl directly adopt commercially available iodoacteyl
The agarose microbeads (Thermo, prod#20404) changed, hereinafter referred to as agarose microbeads；
Agarose microbeads through iodoacteyl activation are improving the application method in polypeptide Olfr560 immunogenicity such as
Under:
(1) microballoon wash: take 1ml agarose microbeads (Thermo, prod#20404) in 2mL centrifuge tube, level from
Scheming is centrifuged (1200rpm) 1min, removes supernatant；Addition 5ml Tris-EDTA (50mM Tris-HCl, 5mM EDTA-Na,
PH8.5), mix well (jog), supernatant is removed in centrifugation；
(2) step (1) is repeated twice；
(3) be coupled: by remove supernatant after agarose microbeads and 2mL (1mg/mL) polypeptide Olfr560 solution in 15mL from
It is mixed in heart pipe, is tiltedly placed in ambient temperature overnight and is crosslinked；
(4) mouse (one exempts from) is immunized: 3 mouse of selection are micro- by polypeptide-agarose of overnight crosslinking as experimental subjects
Ball compound moves into 2mL asepsis injector (band needle), and directly subcutaneous or abdominal cavity multi-point injection mouse, 250ug/ is only；
(5) two exempt from: carrying out within the 14th day being immunized for second, immunization method is with for the first time, and 250ug/ is only；
(6) three exempt from: the third time of progress in the 24th day is immune, and immunization method is with for the first time, and 250ug/ is only；
(7) detected whether antibody titer: the 28-29 days, tail vein acquired about 20ul tail blood, 5000 turns of centrifugation 5min,
Haemocyte precipitating is abandoned, serum is drawn, has detected whether antibody generation for Western Blot, detection sample is mouse brain group
It knits；
(8) four exempt from: carrying out within the 34th day the 4th immune, immunization method same first time, 250ug/；
(9) mouse for having antibody through Western Blot detection in step (7) subsequent monoclonal antibody is carried out to prepare in fact
It tests.
It is following (Normal practice of the prior art) that Mouse Method is immunized after conventional KLH-Olfr560 polypeptide coupling:
(1) be coupled: taking KLH (Thermo, prod#77600) that 1mL concentration be 2mg/mL and 1mL concentration is 2mg/mL's
Polypeptide Olfr560 mixing, in being coupled 4h on room temperature shaker；
(2) emulsify: take 1mL be coupled KLH-Olfr560 and 1mL Freund's complete adjuvant (sigma, prod#F5881) in
It is mixed in 5mL syringe, and forms Water-In-Oil mixture by way of injecting repeatedly and can be used to be immunized, choose 3
Mouse is as experimental subjects；
(3) one exempt from: the 1st day, adjuvant was not formula Freund's complete adjuvant (sigma, prod#F5881), subcutaneous multi-point injection,
250ug/ is only；
(4) two exempt from: the 1st day, adjuvant was not formula Freund's incomplete adjuvant (sigma, prod#F5506), subcutaneous multi-point injection,
250ug/ is only；
(5) three exempt from: the 1st day, adjuvant was not formula Freund's incomplete adjuvant (sigma, prod#F5506), subcutaneous multi-point injection,
250ug/ is only；
(6) detected whether antibody titer: the 28-29 days, tail vein acquired about 20ul tail blood, room temperature 30min, and 5000
Turn centrifugation 5min, abandon haemocyte precipitating, draw serum, Western Blot has detected whether antibody generation, and detection sample is small
Murine brain；
(7) four exempt from: the 34th day, adjuvant was not formula Freund's incomplete adjuvant (sigma, prod#F5506), subcutaneous multi-point injection,
250ug/ is only；
(8) mouse for having antibody through Western Blot detection in step (6) subsequent monoclonal antibody is carried out to prepare in fact
It tests.
Two kinds of immunization method results compare:
Western Blot testing result comparison: can using the mice serum that agarose microbeads are obtained as the immunization method of carrier
To detect the Olfr560 albumen in Mice brain tissues, show that size is the egg of 35kDa on Western Blot exposed plate
Informal voucher band illustrates there is Olfr560 antibody in serum referring to Fig. 3；The mice serum obtained using KLH as the immunization method of carrier is not
It detects the Olfr560 albumen in Mice brain tissues, there is no protein band on Western Blot exposed plate, referring to fig. 4,
Illustrate in serum either with or without Olfr560 antibody.As it can be seen that being with agarose microbeads compared to immunization method after standard adjuvant emulsification
The immunogenicity of polypeptide Olfr560 can be improved in the direct immunization method of carrier.
Embodiment one and embodiment two the experimental results showed that, compared with KLH carrier commonly used in the art, agarose microbeads
There is stronger versatility on improving polypeptide immunogenicity as carrier.
Embodiment three:
The present embodiment provides the agarose microbeads activated through iodoacteyl improve protein hsp 90 α (Gene ID:
3320) application in immunogenicity.
It is living that the agarose microbeads activated in the present embodiment through iodoacteyl directly adopt commercially available iodoacteyl
The agarose microbeads (Thermo, prod#20404) changed, hereinafter referred to as agarose microbeads；
Application method of the agarose microbeads through iodoacteyl activation in raising protein hsp 90 alpha immunization originality is as follows:
(1) microballoon wash: take 1ml agarose microbeads (Thermo, prod#20404) in 2mL centrifuge tube, level from
Scheming is centrifuged (1200rpm) 1min, removes supernatant；Addition 5ml Tris-EDTA (50mM Tris-HCl, 5mM EDTA-Na,
PH8.5), mix well (jog), supernatant is removed in centrifugation；
(2) step (1) is repeated twice；
(3) be coupled: by remove supernatant after agarose microbeads and 2mL (1mg/mL) protein hsp 90 α solution in 15mL from
It is mixed in heart pipe, is tiltedly placed in ambient temperature overnight and is crosslinked；
(4) mouse (one exempts from) is immunized: chooses 3 mouse as experimental subjects, by the polypeptide agarose microbeads of overnight crosslinking
Compound moves into 2mL asepsis injector (band needle), and directly subcutaneous or abdominal cavity multi-point injection mouse, 250ug/ is only；
(5) two exempt from: carrying out within the 14th day being immunized for second, immunization method is with for the first time, and 250ug/ is only；
(6) three exempt from: the third time of progress in the 24th day is immune, and immunization method is with for the first time, and 250ug/ is only；
(7) detected whether antibody titer: the 28-29 days, tail vein acquired about 20ul tail blood, 5000 turns of centrifugation 5min,
Haemocyte precipitating is abandoned, serum is drawn, has detected whether antibody generation for Western Blot, detection sample is mouse brain group
It knits；
(8) four exempt from: carrying out within the 34th day the 4th immune, immunization method same first time, 250ug/；
(9) mouse for having antibody through Western Blot detection in step (7) subsequent monoclonal antibody is carried out to prepare in fact
It tests.
Conventional H SP90 α protein immunization Mouse Method is following (Normal practice of the prior art):
(1) it emulsifies: taking 1mLHSP90 α protein solution and 1mL Freund's complete adjuvant (sigma, prod#F5881) in 5mL
It is mixed in syringe, and forms Water-In-Oil mixture by way of injecting repeatedly and can be used to be immunized；Choose 3 mouse
As experimental subjects；
(2) one exempt from: the 1st day, adjuvant was not formula Freund's complete adjuvant (sigma, prod#F5881), subcutaneous multi-point injection,
250ug/ is only；
(3) two exempt from: the 1st day, adjuvant was not formula Freund's incomplete adjuvant (sigma, prod#F5506), subcutaneous multi-point injection,
250ug/ is only；
(4) three exempt from: the 1st day, adjuvant was not formula Freund's incomplete adjuvant (sigma, prod#F5506), subcutaneous multi-point injection,
250ug/ is only；
(5) detected whether antibody titer: the 28-29 days, tail vein acquired about 20ul tail blood, room temperature 30min, and 5000
Turn centrifugation 5min, abandon haemocyte precipitating, draw serum, Western Blot has detected whether antibody generation, and detection sample is small
Murine brain；
(6) four exempt from: the 34th day, adjuvant was not formula Freund's incomplete adjuvant (sigma, prod#F5506), subcutaneous multi-point injection,
250ug/ is only；
(7) mouse for having antibody through Western Blot detection in step (5) subsequent monoclonal antibody is carried out to prepare in fact
It tests.
Two kinds of immunization method results compare:
Western Blot testing result comparison: can using the mice serum that agarose microbeads are obtained as the immunization method of carrier
To detect the HSP90 α albumen in Mice brain tissues, show that size is the egg of 85kDa on Western Blot exposed plate
Informal voucher band illustrates there are HSP90 Alpha antibodies in serum referring to Fig. 5；The mice serum that immunization method obtains after standard adjuvant emulsification is not
It detects the HSP90 α albumen in Mice brain tissues, there is no protein band on Western Blot exposed plate, referring to Fig. 6, say
Either with or without HSP90 Alpha antibodies in bright serum.
It is above-mentioned the experimental results showed that, compared to standard adjuvant emulsification after immunization method, using agarose microbeads as the straight of carrier
Connecing immunization method can be improved the immunogenicity of protein hsp 90 α.
Example IV:
The present embodiment provides the agarose microbeads activated through iodoacteyl to improve albumen eNOS (Gene ID:4846)
Application in immunogenicity.
It is living that the agarose microbeads activated in the present embodiment through iodoacteyl directly adopt commercially available iodoacteyl
The agarose microbeads (Thermo, prod#20404) changed, hereinafter referred to as agarose microbeads；
Application method of the agarose microbeads through iodoacteyl activation in raising albumen eNOS immunogenicity is as follows:
(1) microballoon wash: take 1ml agarose microbeads (Thermo, prod#20404) in 2mL centrifuge tube, level from
Scheming is centrifuged (1200rpm) 1min, removes supernatant；Addition 5ml Tris-EDTA (50mM Tris-HCl, 5mM EDTA-Na,
PH8.5), mix well (jog), supernatant is removed in centrifugation；
(2) step (1) is repeated twice；
(3) it is coupled: by the agarose microbeads after removal supernatant with 2mL (1mg/mL) albumen eNOS solution in 15mL centrifuge tube
Middle mixing is tiltedly placed in ambient temperature overnight and is crosslinked；
(4) mouse (one exempts from) is immunized: chooses 3 mouse as experimental subjects, by the polypeptide agarose microbeads of overnight crosslinking
Compound moves into 2mL asepsis injector (band needle), and directly subcutaneous or abdominal cavity multi-point injection mouse, 250ug/ is only；
(5) two exempt from: carrying out within the 14th day being immunized for second, immunization method is with for the first time, and 250ug/ is only；
(6) three exempt from: the third time of progress in the 24th day is immune, and immunization method is with for the first time, and 250ug/ is only；
(7) detected whether antibody titer: the 28-29 days, tail vein acquired about 20ul tail blood, 5000 turns of centrifugation 5min,
Haemocyte precipitating is abandoned, serum is drawn, has detected whether antibody generation for Western Blot, detection sample is that rat heart muscle is thin
Born of the same parents；
(8) four exempt from: carrying out within the 34th day the 4th immune, immunization method same first time, 250ug/；
(9) mouse for having antibody through Western Blot detection in step (7) subsequent monoclonal antibody is carried out to prepare in fact
It tests.
Conventional eNOS protein immunization Mouse Method is following (Normal practice of the prior art):
(1) it emulsifies: 1mL eNOS protein solution and 1mL Freund's complete adjuvant (sigma, prod#F5881) being taken to infuse in 5mL
It is mixed in emitter, and forms Water-In-Oil mixture by way of injecting repeatedly and can be used to be immunized, in addition, choosing 3
Mouse is as experimental subjects；
(2) one exempt from: the 1st day, adjuvant was not formula Freund's complete adjuvant (sigma, prod#F5881), subcutaneous multi-point injection,
250ug/ is only；
(3) two exempt from: the 1st day, adjuvant was not formula Freund's incomplete adjuvant (sigma, prod#F5506), subcutaneous multi-point injection,
250ug/ is only；
(4) three exempt from: the 1st day, adjuvant was not formula Freund's incomplete adjuvant (sigma, prod#F5506), subcutaneous multi-point injection,
250ug/ is only；
(5) detected whether antibody titer: the 28-29 days, tail vein acquired about 20ul tail blood, room temperature 30min, and 5000
Turn centrifugation 5min, abandon haemocyte precipitating, draw serum, Western Blot has detected whether antibody generation, and detection sample is big
Rat cardiomyocyte；
(6) four exempt from: the 34th day, adjuvant was not formula Freund's incomplete adjuvant (sigma, prod#F5506), subcutaneous multi-point injection,
250ug/ is only；
(7) mouse for having antibody through Western Blot detection in step (5) subsequent monoclonal antibody is carried out to prepare in fact
It tests.
Two kinds of immunization method results compare:
Western Blot testing result comparison: can using the mice serum that agarose microbeads are obtained as the immunization method of carrier
To detect the eNOS albumen in rat myocardial cell, show that size is the egg of 130kDa on Western Blot exposed plate
Informal voucher band illustrates there is eNOS antibody in serum referring to Fig. 7；The mice serum that immunization method obtains after standard adjuvant emulsification is not examined
The eNOS albumen in rat myocardial cell is measured, does not have protein band on Western Blot exposed plate, referring to Fig. 8, explanation
Either with or without eNOS antibody in serum.
It is above-mentioned the experimental results showed that, compared to standard adjuvant emulsification after immunization method, using agarose microbeads as the straight of carrier
Connecing immunization method can be improved the immunogenicity of albumen eNOS.
Above embodiments illustrate through iodoacteyl activate agarose microbeads compared with the prior art in common method,
Have the function of preferably improving polypeptide or protide immunogen immune originality, and is theoretically adapted to various more peptide or proteins
Para-immunity is former, has preferable versatility.
Above embodiments are only exemplary embodiment of the present invention, are not used in the limitation present invention, protection scope of the present invention
It is defined by the claims.Those skilled in the art can within the spirit and scope of the present invention make respectively the present invention
Kind modification or equivalent replacement, this modification or equivalent replacement also should be regarded as being within the scope of the present invention.
Claims (5)
1. the agarose microbeads through iodoacteyl activation are improving the application in polypeptide or protide immunogen immune originality.
2. the agarose microbeads through iodoacteyl activation are improving the application in polypeptide α-syn immunogenicity.
3. the agarose microbeads through iodoacteyl activation are improving the application in polypeptide Olfr560 immunogenicity.
4. the agarose microbeads through iodoacteyl activation are improving the application in protein hsp 90 alpha immunization originality.
5. the agarose microbeads through iodoacteyl activation are improving the application in albumen eNOS immunogenicity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910065007.4A CN109865137A (en) | 2019-01-23 | 2019-01-23 | Agarose microbeads through iodoacteyl activation are improving the application in polypeptide or protide immunogen immune originality |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910065007.4A CN109865137A (en) | 2019-01-23 | 2019-01-23 | Agarose microbeads through iodoacteyl activation are improving the application in polypeptide or protide immunogen immune originality |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109865137A true CN109865137A (en) | 2019-06-11 |
Family
ID=66917885
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910065007.4A Pending CN109865137A (en) | 2019-01-23 | 2019-01-23 | Agarose microbeads through iodoacteyl activation are improving the application in polypeptide or protide immunogen immune originality |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109865137A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1484532A (en) * | 2000-12-06 | 2004-03-24 | Pharmaceutical compositions enhancing the immunogenicity of poorly immunogenic antigens | |
| CN102294024A (en) * | 2011-01-17 | 2011-12-28 | 广东现代农业集团研究院有限公司 | Polypeptide vaccine and preparation method thereof |
| US20130052733A1 (en) * | 2011-08-29 | 2013-02-28 | National Tsing Hua University | Antigen presenting composition and use thereof |
-
2019
- 2019-01-23 CN CN201910065007.4A patent/CN109865137A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1484532A (en) * | 2000-12-06 | 2004-03-24 | Pharmaceutical compositions enhancing the immunogenicity of poorly immunogenic antigens | |
| CN102294024A (en) * | 2011-01-17 | 2011-12-28 | 广东现代农业集团研究院有限公司 | Polypeptide vaccine and preparation method thereof |
| US20130052733A1 (en) * | 2011-08-29 | 2013-02-28 | National Tsing Hua University | Antigen presenting composition and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| 党小军: "《临床免疫学检验技术》", 30 July 2014, 北京：科学技术文献出版社 * |
| 蒋中华，张津辉: "《生物分子固定化技术及应用》", 30 July 1998 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0117934B1 (en) | Antigenic modification of polypeptides | |
| CN104093415B (en) | Companion's diagnostic agent and its using method of anti-hyaluronan agent therapy | |
| JPH03504975A (en) | Heterofunctional cell immunizing agent, vaccine containing the same, and method for using the same | |
| KR870700100A (en) | Polypeptides complementary to proteins or peptides having at least a portion of a known nucleotide sequence or amino acid sequence and methods of designing the same | |
| WO1994026886B1 (en) | Process for the preparation of immunogens or diagnostic reagents, and immunogens or diagnostic reagents thereby obtainable | |
| ALBERICIO et al. | Use of the Npys thiol protection in solid phase peptide synthesis Application to direct peptide‐protein conjugation through cysteine residues | |
| US5191015A (en) | Polymers and polymer-peptide conjugates | |
| US4845042A (en) | Adjuvant for immunization | |
| CN114349855A (en) | Novel coronavirus Delta mutant strain specific antibody and application thereof | |
| CN111505295B (en) | Trichinosis antibody detection kit based on competitive monoclonal antibody and preparation method thereof | |
| GB2226563A (en) | Streptolysin o fragments | |
| CN109865137A (en) | Agarose microbeads through iodoacteyl activation are improving the application in polypeptide or protide immunogen immune originality | |
| Jain et al. | Simple and efficient production and purification of mouse myelin oligodendrocyte glycoprotein for experimental autoimmune encephalomyelitis studies | |
| CN101880316A (en) | Human RBPMS polypeptide and preparation method of antibody thereof | |
| Grant et al. | [17] Edman sequencing as tool for characterization of synthetic peptides | |
| EP2284188A1 (en) | Detection of anti-ribosomal P protein antibodies by means of synthetic peptides | |
| CA1321765C (en) | Immunodiagnostic assays using chimeric antigens | |
| RU2572343C2 (en) | RECOMBINANT DNA pA4, RECOMBINANT DNA pQE 30-pA4, PROVIDING OBTAINING POLYPEPTIDE A4, Esherichia coli STRAIN M 15-A4, TRANSFORMED WITH RECOMBINANT PLASMID DNA pQE 30-pA4, AND EXPRESSING RECOMBINANT POLYPEPTIDE A4, RECOMBINANT POLYPEPTIDE A4, POSSESSING ABILITY TO SELECTIVELY BIND HSA, AFFINE SORBENTS (VERSIONS) AND METHODS FOR HSA AND IgG REMOVAL FROM BLOOD SERUM (VERSIONS) | |
| CN109293779A (en) | A kind of multimeric complexes and preparation method thereof | |
| CN111303276A (en) | B cell epitope polypeptide of trichina intestinal cysteine protease inhibitor, hybridoma cell strain, monoclonal antibody and application | |
| RU2614115C1 (en) | Recombinant somatostatin-containing protein, method for production, injecting drug to increase meat and dairy efficiency of agricultural animals, and method for drug application | |
| CN116606376B (en) | Preparation method and application method of 452 th amino acid phosphorylated PI3Kp85 antibody | |
| EP1072688B1 (en) | Method for in vivo detecting a target protein gene by a drug | |
| CN117801111B (en) | Specific antibody combined with canine red blood cells and application thereof | |
| Boniface et al. | The kinetics of binding of peptide/MHC complexes to T-cell receptors: application of surface plasmon resonance to a low-affinity measurement |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190611 |
|
| RJ01 | Rejection of invention patent application after publication |