CN107488232A - A kind of synthetic method of His unlabelled antigens - Google Patents
A kind of synthetic method of His unlabelled antigens Download PDFInfo
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- CN107488232A CN107488232A CN201710722610.6A CN201710722610A CN107488232A CN 107488232 A CN107488232 A CN 107488232A CN 201710722610 A CN201710722610 A CN 201710722610A CN 107488232 A CN107488232 A CN 107488232A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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Abstract
A kind of synthetic method of His unlabelled antigens, belongs to technical field of biochemical industry.The present invention includes：The design and synthesis of His label haptens, the synthesis of His label comlete antigens.The present invention is in units of the His tag of 6 His polypeptides, 3 aspartic acids are spaced between two peptide sequences, form the peptide sequence containing three His tag units, and increase a cysteine in sequence aminoterminal, so as to which peptide sequence and carrier protein to be coupled to the sulfydryl of cysteine using SPDP.The antigen of preparation is subjected to mouse immune, has obtained the antibody for the anti-His tag that potency is high, Competitive assays effect is good, therefore shows that this is a kind of synthesis effective ways of His tag antigens, important theoretical foundation is provided for the preparation of His tag antibody.
Description
Technical field
The invention belongs to technical field of biochemical industry, and in particular to a kind of synthetic method of His unlabelled antigens.
Background technology
The small peptide that His-tag is made up of 6 histidines, it is characterized in that molecular weight is small, does not change the life of protein substantially
Thing structure, do not change the dissolubility of protein, be specially designed for the adsorption and purification of recombinant protein, utilize the miaow on histidine
The principle that azoles ring can be combined with bivalent metal ion, people can the purifying of sharp metal ion affinity chromatography technology carry His-
Tag albumen, compared with other labels, His-tag has technical maturity, simple to operate and can be mass-produced and the advantage such as use,
It is the most widely used one kind in the fusion tag currently used for purifying.His-tag antibody can identify His-tag, therefore
It can be used for identifying the protein expression amount with His-tag, cellular localization and other characteristics.
The His-tag antibody types of commercialization in the market are more, but the research to its Antibody preparation is reported but very
It is few, in existing research method, mainly using 6 His peptide sequences as haptens, pass through glutaraldehyde method or carbodiimide
Method is coupled to carrier protein and carries out animal immune as immunogene, to obtain His-tag antibody.Therefore need further to open
The immunogene synthesis for sending out new and the preparation method of highly sensitive antibody with high specificity.
The content of the invention
Technical problems to be solved：The small peptide that His-tag is made up of 6 histidines, molecular weight is small, therefore immunogenicity
It is smaller, and His is a kind of basic amino acid, in acid condition with preferable dissolubility, and in the preparation process of antigen
Synthesize in the basic conditions so that His-tag dissolubility is poor, is unfavorable for the synthesis of antigen.On the other hand, 6 groups
The propylhomoserin composition existing amino of His-tag peptide sequences has carboxyl again, is easy to the coupling method of routine so that between His-tag
Or autoimmunity syndrome is produced between carrier protein.
Technical scheme：The invention discloses a kind of synthetic method of His unlabelled antigens, comprise the following specific steps that：
（1）The design and synthesis of His label haptens
The peptide sequence of His label haptens forms：5 '-CHHHHHHDDDHHHHHHDDDHHHHHH-3 ', according to designing
Peptide sequence, commission Shanghai gill biochemistry Co., Ltd carry out Peptide systhesis.
（2）The synthesis of His label comlete antigens
The synthesis flow of His label comlete antigens is：
Using isodigeranyl functional group crosslinking agent N- succinamides -3- (sulphur of 2- pyridines two)-propionic ester SPDP by keyhole limpet hemocyanin KLH
It is coupled with His tag polypeptides；SPDP is dissolved with DMF first, is configured to the dense of 10mg/mL
Degree, weighs 10mg KLH, it is dissolved with pH7.5 0.01M PBS, 5mg/mL concentration is configured to, is stirring
SPDP solution is added dropwise in the state of mixing so that KLH and SPDP are with 1:10~1:50 mol ratio is reacted, room temperature reaction
After 30 ~ 60min, reaction solution is dialysed 3 times with pH7.40.01M PBS, to remove unnecessary SPDP；More than
To solution in add dithiothreitol (DTT)（DTT）So that its final concentration of 1mM, after reacting 30min under stirring, by solution
Ultrafiltration is carried out with super filter tube, solution is resuspended with PBS again with the DTT for removing unnecessary；To DTT handle solution in
KLH:His tag polypeptides are 1:10~1:50 mol ratio adds His tag polypeptides, at ambient temperature 2 ~ 4h of stirring reaction, instead
After the completion of answering, solution is dialysed with PBS, so as to obtain His label comlete antigens.
A kind of synthetic method step of His unlabelled antigens of the present invention（2）Described in KLH and SPDP reaction rub
You are than being 1:20.
A kind of synthetic method step of His unlabelled antigens of the present invention（2）Described in KLH and SPDP reaction when
Between be 40min.
A kind of synthetic method step of His unlabelled antigens of the present invention（2）Described in KLH and His tag polypeptides
The mol ratio of reaction is 1:30.
A kind of synthetic method step of His unlabelled antigens of the present invention（2）Described in His tag polypeptides add after
Reaction time be 3h.
A kind of a kind of synthetic method synthesis of His unlabelled antigens using described in claim 1 of the present invention resists
The method that original prepares His tag antibodies, it is characterised in that comprise the following steps：
（1）Animal immune
The artificial antigen prepared to top method makees immunogene, takes equivalent amount of antigen fully emulsified with Freund's complete adjuvant, by its skin
It is lower injection 3 10 week old BALB/C mice, the immunizing dose of every mouse be 50 μ g, be spaced three weeks after by be subcutaneously injected into
Row booster immunization, it is by equivalent amount of antigen and incomplete Freund's adjuvant mixing and emulsifying, the immunizing dose of every mouse during booster immunization
100μg.Afterbody blood sampling, and centrifuging and taking serum are carried out after immune four times to mouse.
（2）Determine antibody titer
The polypeptide haptens synthesized by the use of above method is carried out as coating antigen with pH9.6 0.01M carbonate buffer solution molten
Solution, concentration is made into as 1 μ g/mL, 0.5 μ g/mL, 0.2 μ g/mL, 0.1 μ g/mL coating concentration, every kind of coating concentration adds 96 holes
One row of ELISA Plate, sample-adding amount are per the μ L of hole 100, are placed in 37 DEG C of baking ovens after incubation 2h with the 0.01M containing 0.5%Tween-20
PH7.4 PBS washing three times, closes 2h at 37 DEG C with 1% gelatin after drying, washs after taking-up and dry three times.Will
Antiserum dilutes 1000,3000,9000,27000 times, and the antibody of every kind of extension rate is added to a row of four ELISA Plates,
Each hole adds 100 μ L, and 37 DEG C are incubated washes clean after 30min, add 1:3000 sheep anti mouse secondary antibody, 37 DEG C of incubation 30min
Washes clean afterwards, terminate liquid is added after adding nitrite ion colour developing 15min, with the light absorption value at ELIASA measure 480nm.
（3）The measure of indirect ELISA
3 μ L/ holes of ELISA Plate 100 are coated with 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL polypeptide haptens, put it into 37 DEG C of bakings
2h is incubated in case, is then washed three times with the PBS of the 0.01M pH7.4 containing 0.5%Tween-20, with 1% after drying
Gelatin as confining liquid 37 DEG C close 2h after, take out washing dry three times.By Amoxicillin standard items be made into 50ng/mL,
40ng/mL, 30ng/mL, 20ng/mL, 10ng/mL, 5ng/mL 6 concentration gradients, the first row of 3 ELISA Plates add 50 μ
The 0.01M pH7.2 in L/ holes PBS, every row sequentially adds 6 groups from low concentration to high concentration since the second row
The μ L/ holes of His tag polypeptides 50 of propylhomoserin composition, then add the 1 of 50 μ L/ holes:The antiserum of 3000 dilutions, 37 DEG C of incubations
Washes clean after 30min, adds 1:The 3000 μ L/ holes of sheep anti mouse secondary antibody 100,37 DEG C are incubated washes clean after 30min, then use
The nitrite ion colour developing 15min in 100 μ L/ holes, the terminate liquid in 50 μ L/ holes is eventually adding, with the extinction at ELIASA measure 480nm
Value.
Note：DNA molecular of the present invention is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd.
Beneficial effect：The His-tag small peptides being made up of 6 histidines, molecular weight is small, and immunogenicity is small, and the present invention is with 6
The His-tag of His polypeptides is unit, and 3 Asp are spaced between two subunit sequences, is formed more containing three His-tag units
Peptide sequence, it can further improve the immunogenicity of antigen.Asp is linear chain structure, and His-tag antigenic determinant will not be produced
It is raw to influence, meanwhile, for Asp as acidic amino acid, the addition in peptide sequence adds new synthetic antigen in alkaline solution
Dissolubility, the basic polypeptide sequence for solving the problems, such as only to be made up of His solubility under alkaline coupling condition is poor, more
Beneficial to the coupling of antigen and carrier protein.Increase a Cys in peptide sequence amino terminal, so as to which mercapto will be contained using SPDP
The peptide sequence and carrier protein of base are coupled, so as to avoid between His-tag caused by conventional method or carrier protein
Between autoimmunity syndrome.
Embodiment
Embodiment 1
A kind of synthetic method of His unlabelled antigens, comprises the following steps：
（1）The design and synthesis of His label haptens
The peptide sequence of His label haptens forms：5 '-CHHHHHHDDDHHHHHHDDDHHHHHH-3 ', according to designing
Peptide sequence, commission Shanghai gill biochemistry Co., Ltd carry out Peptide systhesis.
（2）The synthesis of His label comlete antigens
It is using isodigeranyl functional group crosslinking agent N- succinamides -3- (sulphur of 2- pyridines two)-propionic ester SPDP that KLH and His labels is more
Peptide is coupled；SPDP is dissolved with DMF first, 10mg/mL concentration is configured to, weighs 10mg
KLH, it is dissolved with pH7.5 0.01M PBS, is configured to 5mg/mL concentration, in the state of stirring by
It is added dropwise to SPDP solution so that KLH and SPDP are with 1:20 mol ratio is reacted, after reacting at room temperature 40min, by reaction solution
Dialysed 3 times with pH7.40.01M PBS, to remove unnecessary SPDP；DTT is added in solution derived above to cause
Its final concentration of 1mM, after reacting 30min under stirring, solution is subjected to ultrafiltration with super filter tube, to remove unnecessary DTT
Solution is resuspended with PBS again；With KLH in the solution handled to DTT:His tag polypeptides are 1:30 mol ratio adds
His tag polypeptides, stirring reaction 3h, after the completion of reaction, solution is dialysed with PBS at ambient temperature, so as to
Obtain His label comlete antigens.
The method that His tag antibodies are prepared with the His unlabelled antigens newly synthesized, it is characterised in that comprise the following steps：
（1）Animal immune
The artificial antigen prepared to top method makees immunogene, takes equivalent amount of antigen fully emulsified with Freund's complete adjuvant, by its skin
It is lower injection 3 10 week old BALB/C mice, the immunizing dose of every mouse be 50 μ g, be spaced three weeks after by be subcutaneously injected into
Row booster immunization, it is by equivalent amount of antigen and incomplete Freund's adjuvant mixing and emulsifying, the immunizing dose of every mouse during booster immunization
100μg.Afterbody blood sampling, and centrifuging and taking serum are carried out after immune four times to mouse.
（2）Determine antibody titer
The polypeptide haptens that above method is synthesized is dissolved as coating antigen with pH9.6 0.01M carbonate buffer solution,
Concentration is made into as 1 μ g/mL, 0.5 μ g/mL, 0.2 μ g/mL, 0.1 μ g/mL coating concentration, every kind of coating concentration adds 96 hole enzymes
One row of target, sample-adding amount are per the μ L of hole 100, are placed in 37 DEG C of baking ovens after incubation 2h with the 0.01M containing 0.5%Tween-20
PH7.4 PBS washing three times, closes 2h at 37 DEG C with 1% gelatin after drying, washs after taking-up and dry three times.Will
Antiserum dilutes 1000,3000,9000,27000 times, and the antibody of every kind of extension rate is added to a row of four ELISA Plates,
Each hole adds 100 μ L, and 37 DEG C are incubated washes clean after 30min, add 1:3000 sheep anti mouse secondary antibody, 37 DEG C of incubation 30min
Washes clean afterwards, terminate liquid is added after adding nitrite ion colour developing 15min, with the light absorption value at ELIASA measure 480nm.From following table
The potency of antibody obtained by drawing is higher.
（3）The measure of indirect ELISA
3 μ L/ holes of ELISA Plate 100 are coated with 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL polypeptide haptens, put it into 37 DEG C of bakings
2h is incubated in case, is then washed three times with the PBS of the 0.01M pH7.4 containing 0.5%Tween-20, with 1% after drying
Gelatin as confining liquid 37 DEG C close 2h after, take out washing dry three times.By Amoxicillin standard items be made into 50ng/mL,
40ng/mL, 30ng/mL, 20ng/mL, 10ng/mL, 5ng/mL 6 concentration gradients, the first row of 3 ELISA Plates add 50 μ
The 0.01M pH7.2 in L/ holes PBS, every row sequentially adds 6 groups from low concentration to high concentration since the second row
The μ L/ holes of His-tag polypeptides 50 of propylhomoserin composition, then add the 1 of 50 μ L/ holes:The antiserum of 3000 dilutions, 37 DEG C of incubations
Washes clean after 30min, adds 1:The 3000 μ L/ holes of sheep anti mouse secondary antibody 100,37 DEG C are incubated washes clean after 30min, then use
The nitrite ion colour developing 15min in 100 μ L/ holes, the terminate liquid in 50 μ L/ holes is eventually adding, with the extinction at ELIASA measure 480nm
Value.Find out from following table, obtained antibody is obvious to His-tag inhibition.
。
Claims (6)
1. a kind of synthetic method of His unlabelled antigens, it is characterised in that comprise the following steps：
The design and synthesis of His label haptens
The peptide sequence of His label haptens forms：5’-CHHHHHHDDDHHHHHHDDDHHHHHH-3’；
According to the peptide sequence designed, the synthesis of peptide sequence is carried out using Peptide synthesizer；
The synthesis of His label comlete antigens
The synthesis flow of His label comlete antigens is：
Using isodigeranyl functional group crosslinking agent N- succinamides -3- (sulphur of 2- pyridines two)-propionic ester SPDP by keyhole limpet hemocyanin KLH
It is coupled with His tag polypeptides；SPDP is dissolved with DMF first, is configured to the dense of 10mg/mL
Degree, weighs 10mg KLH, it is dissolved with pH7.5 0.01M PBS, 5mg/mL concentration is configured to, is stirring
SPDP solution is added dropwise in the state of mixing so that KLH and SPDP are with 1:10~1:50 mol ratio is reacted, room temperature reaction
After 30 ~ 60min, reaction solution is dialysed 3 times with pH7.40.01M PBS, to remove unnecessary SPDP；More than
To solution in add dithiothreitol (DTT) and cause its final concentration of 1mM, after reacting 30min under stirring, by solution with super
Chimney filter carries out ultrafiltration, and solution is resuspended with PBS again with the dithiothreitol (DTT) for removing unnecessary；Handled to dithiothreitol (DTT)
With KLH in solution:His tag polypeptides are 1:10~1:50 mol ratio adds His tag polypeptides, and stirring is anti-at ambient temperature
2 ~ 4h is answered, after the completion of reaction, solution is dialysed with PBS, so as to obtain His label comlete antigens.
A kind of 2. synthetic method of His unlabelled antigens according to claim 1, it is characterised in that step（2）Described in
The mol ratio of KLH and SPDP reactions is 1:20.
A kind of 3. synthetic method of His unlabelled antigens according to claim 1, it is characterised in that step（2）Described in
KLH and SPDP reaction time is 40min.
A kind of 4. synthetic method of His unlabelled antigens according to claim 1, it is characterised in that step（2）Described in
The mol ratio of KLH and His tag polypeptides reaction is 1:30.
A kind of 5. synthetic method of His unlabelled antigens according to claim 1, it is characterised in that step（2）Described in
Reaction time after His tag polypeptides add is 3h.
6. a kind of a kind of antigen of the synthetic method synthesis of His unlabelled antigens using described in claim 1 prepares His labels
The method of antibody, it is characterised in that comprise the following steps：
（1）Animal immune
The artificial antigen prepared with claim 1 makees immunogene, takes equivalent amount of antigen fully emulsified with Freund's complete adjuvant, by its skin
It is lower injection 3 10 week old BALB/C mice, the immunizing dose of every mouse be 50 μ g, be spaced three weeks after by be subcutaneously injected into
Row booster immunization, it is by equivalent amount of antigen and incomplete Freund's adjuvant mixing and emulsifying, the immunizing dose of every mouse during booster immunization
100 μ g, afterbody blood sampling, and centrifuging and taking serum are carried out after being immunized four times to mouse；
Determine antibody titer
The polypeptide haptens that claim 1 is synthesized is dissolved as coating antigen with pH9.6 0.01M carbonate buffer solution,
Concentration is made into as 1 μ g/mL, 0.5 μ g/mL, 0.2 μ g/mL, 0.1 μ g/mL coating concentration, every kind of coating concentration adds 96 hole enzymes
One row of target, sample-adding amount are per the μ L of hole 100, are placed in 37 DEG C of baking ovens after incubation 2h with the 0.01M containing 0.5%Tween-20
PH7.4 PBS washing three times, closes 2h at 37 DEG C with 1% gelatin after drying, washs after taking-up and dry three times；Will
Antiserum dilutes 1000,3000,9000,27000 times, and the antibody of every kind of extension rate is added to a row of four ELISA Plates,
Each hole adds 100 μ L, and 37 DEG C are incubated washes clean after 30min, add 1:3000 sheep anti mouse secondary antibody, 37 DEG C of incubation 30min
Washes clean afterwards, terminate liquid is added after adding nitrite ion colour developing 15min, with the light absorption value at ELIASA measure 480nm；
The measure of indirect ELISA
3 μ L/ holes of ELISA Plate 100 are coated with 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL polypeptide haptens, put it into 37 DEG C of bakings
2h is incubated in case, is then washed three times with the PBS of the 0.01M pH7.4 containing 0.5%Tween-20, with 1% after drying
Gelatin as confining liquid 37 DEG C close 2h after, take out washing dry three times；By Amoxicillin standard items be made into 50ng/mL,
40ng/mL, 30ng/mL, 20ng/mL, 10ng/mL, 5ng/mL 6 concentration gradients, the first row of 3 ELISA Plates add 50 μ
The 0.01M pH7.2 in L/ holes PBS, every row sequentially adds 6 groups from low concentration to high concentration since the second row
The μ L/ holes of His-tag polypeptides 50 of propylhomoserin composition, then add the 1 of 50 μ L/ holes:The antiserum of 3000 dilutions, 37 DEG C of incubations
Washes clean after 30min, adds 1:The 3000 μ L/ holes of sheep anti mouse secondary antibody 100,37 DEG C are incubated washes clean after 30min, then use
The nitrite ion colour developing 15min in 100 μ L/ holes, the terminate liquid in 50 μ L/ holes is eventually adding, with the extinction at ELIASA measure 480nm
Value.
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| CN201710722610.6A CN107488232A (en) | 2017-08-22 | 2017-08-22 | A kind of synthetic method of His unlabelled antigens |
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| CN201710722610.6A CN107488232A (en) | 2017-08-22 | 2017-08-22 | A kind of synthetic method of His unlabelled antigens |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113150169A (en) * | 2020-08-14 | 2021-07-23 | 河南中泽生物工程有限公司 | High-immunogenicity polypeptide bivalent antigen protein and preparation method and application thereof |
| CN116554338A (en) * | 2023-03-01 | 2023-08-08 | 湖南诺合新生物科技有限公司 | His tag-resistant monoclonal antibody and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113150169A (en) * | 2020-08-14 | 2021-07-23 | 河南中泽生物工程有限公司 | High-immunogenicity polypeptide bivalent antigen protein and preparation method and application thereof |
| CN116554338A (en) * | 2023-03-01 | 2023-08-08 | 湖南诺合新生物科技有限公司 | His tag-resistant monoclonal antibody and application thereof |
| CN116554338B (en) * | 2023-03-01 | 2024-03-08 | 湖南诺合新生物科技有限公司 | His tag-resistant monoclonal antibody and application thereof |
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Application publication date: 20171219 |