CN104211769A - Micromolecule antibody affinity peptide and applications thereof - Google Patents

Micromolecule antibody affinity peptide and applications thereof Download PDF

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Publication number
CN104211769A
CN104211769A CN201310300336.5A CN201310300336A CN104211769A CN 104211769 A CN104211769 A CN 104211769A CN 201310300336 A CN201310300336 A CN 201310300336A CN 104211769 A CN104211769 A CN 104211769A
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affinity peptide
antibody affinity
small molecular
igg
peptide
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CN104211769B (en
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徐霞
韦宇平
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Institute of Process Engineering of CAS
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Institute of Process Engineering of CAS
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Abstract

The invention discloses a micromolecule antibody affinity peptide and applications thereof. The amino acid sequence of the peptide is represented by any one of SEQ ID No. 1-112, and the peptide can combine with the Fc section of human immune globulin IgG, and does not combine with human serum albumin HAS. The provided micromolecule antibody affinity peptide can be taken as the affinity ligand to achieve the high efficient, safe, and economic IgG separation and purification.

Description

A kind of small molecular antibody affinity peptide and application thereof
Technical field
The present invention relates to biology field, particularly, the present invention relates to a kind of small molecular antibody affinity peptide and application thereof.
Background technology
Immunoglobulin IgG, namely antibody is a kind of special protein molecule, by reagent, the medicine of disease therapy, the aglucon etc. of immunoaffinity chromatography as in-vitro diagnosis, has a wide range of applications in life science, biotechnology and medical field.
At present, the natural affinity ligand Protein A/G of separation and purification many employings antibody of IgG, because Protein A/G exists the ability with antibody Fc fragment specific recognition, use Protein A/G affinity chromatography antibody purification, the purity of the product of acquisition is higher.But because Protein A/G also belongs to the activated biological substance of tool, therefore Protein A/G is separated and also there are some problems:
1) expensive: because albumin A aglucon needs to produce purifying by engineered method, therefore production cost is high, and its price reaches $ 10000/ liter of affinity media;
2) lack effective purging method, because albumin A is unstable under alkaline environment, therefore can not use general sodium hydroxide situ cleaning method;
3) because aglucon also belongs to protein, be easily stored in the protease hydrolysis in stock liquid, thus reduce separating effect;
4) compared with general chemical aglucon, the use life cycle of protein A chromatography is shorter;
5) aglucon easily comes off, and the albumin A come off can cause the immune response of people and verified toxic to human body clinically;
6) wash-out pH lower (pH2-3), easily cause some antibody inactivations or occur assembling, usually need limit to collect product, limit neutralizes, and increases the complexity of operation.
For the separation and purification of antibody, also have Many researchers to attempt using micromolecular compound as part, the certain effect also obtained, but also there are some problems in current micromolecular compound part:
1) physiological-toxicity: current micromolecular compound part is many containing heterocycle, has certain toxicity.Such as, 4-Mercapto-Ethyl pyridine (MEP), it is 178mg/kg to mouse 50% lethal quantity.
2) affinity is poor: comparatively Protein A/G is low usually for current micromolecular compound ligand affinity.
3) poor selectivity: current micromolecular compound part also can be combined with main separating impurity HAS while in conjunction with IgG.
So the separation and purification of current IgG lacks a kind of affinity ligand of highly effective and safe economy.
Summary of the invention
The object of the invention is to, provide a kind of small molecular antibody affinity peptide as affinity ligand, realize the IgG separation and purification of highly effective and safe economy.
Another object of the present invention is to the application that above-mentioned small molecular antibody affinity peptide is provided.
Small molecular antibody affinity peptide of the present invention, its aminoacid sequence is as shown in any one in SEQ ID No.1-112.
Further, according to small molecular antibody affinity peptide of the present invention, its constitutional features is as follows to C end from N end:
NH 2-R 1-R 2-R 3-R 4-R 5-COOH，
Wherein, R 1for glutamine (Gln) or L-glutamic acid (Glu) or amino-succinamic acid (Asn) or Aspartic Acid (Asp), R 2for Threonine (Thr) or α-amino-isovaleric acid (Val), R 3for α-amino-isovaleric acid (Val) or leucine (Leu) or nothing, R 4α-amino-isovaleric acid (Val) or leucine (Leu) or nothing, R 5for Histidine (His) or Methionin (Lys).Also can be understood as R 1for acidic amino acid and derivative thereof, R 2for hydroxyl amino acid and leucine (Leu), R 3, R 4for α-amino-isovaleric acid (Val) or leucine (Leu) or nothing, R 5for basic aminoacids.
Small molecular antibody affinity peptide of the present invention, described small molecular antibody affinity peptide is combined with the Fc fragment of human normal immunoglobulin IgG.
Further, the present invention is by being coupled to carrier, for separating of purifying immunoglobulin IgG by described small molecular antibody affinity peptide.
The invention provides a kind of application of small molecular antibody affinity peptide, the isolated or purified of immunoglobulin IgG under different condition can be realized by described small molecular antibody affinity peptide being coupled to different carriers, such as this small molecular antibody affinity peptide is coupled to sepharose, affinity column purifying immunoglobulin IgG can be prepared; And this small molecular antibody affinity peptide is coupled to the hollow-fibre membrane of dialyzer, then can be applicable to the blood purification treatment of autoimmune disorder, optionally remove the IgG that causes a disease.
The present invention compared with prior art, has the following advantages and beneficial effect:
1) efficient: the bonding force of this small molecular antibody affinity peptide is suitable with human normal immunoglobulin native ligand Protein A/G, is not combined with human serum albumin simultaneously.
2) safety: this small molecular antibody affinity peptide molecular weight little (being less than 6 peptides), non-immunogenicity, its residue sequence takes from 20 kinds of indispensable amino acids, safety non-toxic.
3) economical: this small molecular antibody affinity peptide is obtained by solid phase synthesis, cost is lower and be convenient to quality management and control.
Thus adopt this small molecular antibody affinity peptide as affinity ligand, efficient, safe, economic IgG separation and purification can be realized.
Accompanying drawing explanation
Fig. 1 is the SPR kinetic determination curve of small molecular antibody affinity peptide 1 of the present invention and immunoglobulin IgG;
Fig. 2 is the SPR kinetic determination curve of native ligand (Pro G) and immunoglobulin IgG.
Embodiment
Embodiment 1 Peptide systhesis
1) activated resin: take 1000mg FMOC Cys-wang Resin, adds 10 ~ 15mL DMF and soaks 30min(submergence all resins), make it fully swelling.
2) deprotection: the DMF of resin is soaked in press filtration removing; add the DMF solution 10mL containing 20% piperidines; nitrogen blows the reaction 15min that boils; then press filtration removing is containing the DMF solution of 20% piperidines; the FMOC group of deprotection amino; with 10mL washed with isopropyl alcohol resin three times, 10mL DMF washes three times, then detects resin with ninhydrin method and should become black or purple.
3) condensation reaction: connect first amino acid R 5, the consumption taking FMOC-amino acid (His or Lys) is 1.4mmol/g resin, 910mg O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid (TBTU), adds after 10mL DMF and 0.45g I-hydroxybenzotriazole (HOBt) be mixed evenly, add 0.52mL DIEA and be made into reaction solution, ambient temperature under nitrogen blows the reaction 2h that boils, after completion of the reaction, with washed with isopropyl alcohol resin three times, then use DMF washing resin three times, detect amino.
4) repeating step 2) ~ 3): the order of pressing polypeptide extends polypeptide from C end to N end.The process of repetition deprotection, washing, condensation is successively by R 4, R 3, R 2, R 1connect complete, complete the connection of polypeptide.
Wherein, R 1for glutamine (Gln) or L-glutamic acid (Glu) or amino-succinamic acid (Asn) or Aspartic Acid (Asp), R 2for Threonine (Thr) or α-amino-isovaleric acid (Val), R 3for α-amino-isovaleric acid (Val) or leucine (Leu) or nothing, R 4α-amino-isovaleric acid (Val) or leucine (Leu) or nothing, R 5for Histidine (His) or Methionin (Lys).Also can be understood as R 1for acidic amino acid and derivative thereof, R 2for hydroxyl amino acid and leucine (Leu), R 3, R 4for α-amino-isovaleric acid (Val) or leucine (Leu) or nothing, R 5for basic aminoacids.
5) polypeptide cutting: polypeptide-resin complexes is dried up, by TFA/phenol/H with nitrogen 2o/thioanisole/EDT/TIS(80/5/5/5/3/2) ratio is made into mixed cutting reagent, polypeptide-resin complexes is placed in round-bottomed flask, add cutting liquid magnetic agitation 2 hours, with 200 order sand core filter removing resins, in the direct suction chilled ethyl ether of filtrate, 3000r/min is centrifugal makes thick peptide precipitate, and namely freeze-drying obtains thick peptide to constant weight, and drying is weighed.
6) thick peptide HPLC is purified to more than 95%, Mass Spectrometric Identification.
Embodiment 2 IgG affinity characteristic measures
1) 10mmol/L Hepes damping fluid is prepared, pH7.4, NaCl content 150mmol/L.
2) with 1) in obtaining liq be moving phase, run BiacoreT200 analyser.
3) detection chip CM5 is inserted, setting flow velocity 30 μ L/min.
4) each 100 μ L of EDC and NHS are injected successively, activation chip.
5) IgG is dissolved in sodium-acetate buffer, pH4.5, IgG100 μ g/mL.
6) 5 are injected) IgG solution 100 μ L, fixing IgG is in chip surface.
7) respectively by small molecular antibody affinity peptide 1(aminoacid sequence as shown in SEQ ID No.11), small molecular antibody affinity peptide 2(aminoacid sequence is as shown in SEQ ID No.34) and native ligand (ProG and ProA), be dissolved in 1) middle obtaining liq, content 100 μ g/mL.
8) inject polypeptide and the native ligand (concentration is followed successively by 500 μm from high to low, 250 μm, 125 μm, 62.5 μm and 31.25 μm) of different concns successively, record the adsorption curve of itself and IgG.Try to achieve its correlation parameter, concrete outcome is shown in that the drafting of the SPR kinetic determination graphic representation of the immunoglobulin IgG of Fig. 1, Fig. 2 and table 1(small molecular antibody affinity peptide 2 is with small molecular antibody affinity peptide 1, and the present embodiment is not repeated and provides).
Table 1 is small molecular antibody affinity peptide of the present invention, Protein A/G and immunoglobulin IgG binding constant ratio
Comparatively show
Ka is binding constant, and Kd is dissociation constant, K dfor reaction equilibrium constant
From Fig. 1, Fig. 2 and table 1, small molecular antibody affinity peptide of the present invention, suitable in conjunction with dissociation effect and native ligand (ProG and ProA) as affinity ligand and IgG, can realize efficient, safe, economic IgG separation and purification.
Embodiment 3 small molecular antibody affinity peptide compares with IgG and HAS characterization of adsorption
1) 10mmol/L Hepes damping fluid is prepared, pH7.4, NaCl content 150mmol/L.
2) with 1) in obtaining liq be moving phase, run BiacoreT200 analyser.
3) detection chip CM5 is inserted, setting flow velocity 30 μ L/min.
4) each 100 μ L of EDC and NHS are injected successively, activation chip.
5) IgG is dissolved in sodium-acetate buffer, pH4.5, IgG100 μ g/mL.Equally HSA is dissolved in sodium-acetate buffer, pH4.5, HSA100 μ g/mL.
6) 5 are injected) IgG solution 100 μ L, fixing IgG is in chip surface.
7) 5 are injected) HSA solution 100 μ L, fixing HAS is in another chip surface.
8) by small molecular antibody affinity peptide 3(aminoacid sequence as shown in SEQ ID No.1), small molecular antibody affinity peptide 4 aminoacid sequence is as shown in SEQ ID No.4) be dissolved in 1) in obtaining liq, content 100 μ g/mL.
9) successively polypeptide is injected to different chip, record the adsorption curve of itself and IgG and HSA.Try to achieve its correlation parameter, concrete outcome is in table 2.
Table 2 compares with IgG and HSA characterization of adsorption for small molecular antibody affinity peptide of the present invention
As shown in Table 2, small molecular antibody affinity peptide of the present invention is to the Fc fragment adsorptive capacity of human normal immunoglobulin IgG much larger than its adsorptive capacity to human serum albumin HSA, and its affinity interaction has high selectivity.
Prepared by embodiment 4 affinity media
1) get 10mg small molecular antibody affinity peptide (aminoacid sequence is as shown in SEQ ID No.5) and be dissolved in 10mL Hepes damping fluid, pH7.4.
2) get sulfhydryl activated after glucose gel 4FF 1mL.
3) both hybrid reactions above-mentioned 2 hours.
4) after completion of the reaction, with Hepes buffer solution resin, affinity media can be obtained.

Claims (4)

1. a small molecular antibody affinity peptide, is characterized in that, the aminoacid sequence of described affinity peptide is as shown in any one in SEQ ID No.1-112.
2. small molecular antibody affinity peptide according to claim 1, is characterized in that, described affinity peptide is combined with the Fc fragment of human normal immunoglobulin IgG, is not combined with human serum albumin HAS.
3. the application of small molecular antibody affinity peptide described in claim 1.
4. the application of small molecular antibody affinity peptide according to claim 3, is characterized in that, the described application be applied as in separation, purifying or removing human normal immunoglobulin IgG.
CN201310300336.5A 2013-05-30 2013-07-17 A kind of small molecular antibody affinity peptide and its application Expired - Fee Related CN104211769B (en)

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CN106957352A (en) * 2016-01-08 2017-07-18 中国科学院过程工程研究所 A kind of new small molecule peptide and its alzheimer's disease detection in application
CN109153702A (en) * 2016-03-09 2019-01-04 迈克·安有限责任公司 Class peptide affinity ligand
CN109354603A (en) * 2018-11-06 2019-02-19 南阳师范学院 A kind of tetrapeptide and its application
CN109678932A (en) * 2019-01-07 2019-04-26 中国科学院过程工程研究所 A kind of small-molecular peptides that IgG antibody is affine and its application
CN110818776A (en) * 2019-12-04 2020-02-21 南阳师范学院 Affinity peptide and application thereof
EP3626727A4 (en) * 2017-05-15 2021-02-24 Remer Consultores Assessoria Empresarial Ltda. Compound, synthetic intermediate, use, pharmaceutical composition, and neuromodulatory therapeutic method
CN113906043A (en) * 2019-06-07 2022-01-07 埃斯皮克姆有限责任公司 Biologically active peptides and compositions containing them
CN114853677A (en) * 2022-04-24 2022-08-05 湖北泓肽生物科技有限公司 Preparation method of leucyl histidine

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106957352B (en) * 2016-01-08 2020-10-27 中国科学院过程工程研究所 Novel small molecule peptide and application thereof in Alzheimer's disease detection
CN106957352A (en) * 2016-01-08 2017-07-18 中国科学院过程工程研究所 A kind of new small molecule peptide and its alzheimer's disease detection in application
CN109153702A (en) * 2016-03-09 2019-01-04 迈克·安有限责任公司 Class peptide affinity ligand
CN109153702B (en) * 2016-03-09 2022-05-17 迈克·安有限责任公司 Peptoid affinity ligands
EP3626727A4 (en) * 2017-05-15 2021-02-24 Remer Consultores Assessoria Empresarial Ltda. Compound, synthetic intermediate, use, pharmaceutical composition, and neuromodulatory therapeutic method
CN109354603B (en) * 2018-11-06 2021-12-14 南阳师范学院 Tetrapeptide and application thereof
CN109354603A (en) * 2018-11-06 2019-02-19 南阳师范学院 A kind of tetrapeptide and its application
CN109678932A (en) * 2019-01-07 2019-04-26 中国科学院过程工程研究所 A kind of small-molecular peptides that IgG antibody is affine and its application
CN113906043A (en) * 2019-06-07 2022-01-07 埃斯皮克姆有限责任公司 Biologically active peptides and compositions containing them
CN110818776A (en) * 2019-12-04 2020-02-21 南阳师范学院 Affinity peptide and application thereof
CN110818776B (en) * 2019-12-04 2022-07-12 南阳师范学院 Affinity peptide and application thereof
CN114853677A (en) * 2022-04-24 2022-08-05 湖北泓肽生物科技有限公司 Preparation method of leucyl histidine
CN114853677B (en) * 2022-04-24 2023-11-14 湖北泓肽生物科技有限公司 Preparation method of leucyl histidine

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