CN105273060A - Antibody with lysine single methylated derivatization modification and preparation method thereof - Google Patents

Antibody with lysine single methylated derivatization modification and preparation method thereof Download PDF

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CN105273060A
CN105273060A CN201410359860.4A CN201410359860A CN105273060A CN 105273060 A CN105273060 A CN 105273060A CN 201410359860 A CN201410359860 A CN 201410359860A CN 105273060 A CN105273060 A CN 105273060A
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methionin
polypeptide
antibody
modified
monomethyl
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CN105273060B (en
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程仲毅
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PTM Biolabs Inc
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PTM Biolabs Inc
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Priority to CN201710565602.5A priority patent/CN108084047B/en
Priority to PCT/CN2015/084391 priority patent/WO2016011920A1/en
Priority to US15/323,873 priority patent/US10551390B2/en
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P20/00—Technologies relating to chemical industry
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Abstract

The invention relates to artificially synthesized modified single methylated lysine, and modified single methylated lysine derived polypeptides. The invention also relates to production of a new antibody from the modified single methylated lysine and the modified single methylated lysine derived polypeptides as antigen, and the antibody is used for identifying and enriching modified polypeptides produced from external derivatization of the lysine single methylated polypeptide. The antibody is used for detecting whether single methylation modification exists on an amino acid of a polypeptide sequence. The invention also relates to a preparation method of the antibody.

Description

Antibody that a kind of Methionin monomethylation derivatize is modified and preparation method thereof
Technical field
This area relates to ANTIGEN DESIGNThe, antigen is prepared and is utilized antigen to produce the field of antibody, specifically, the present invention relates to a kind of novel modified monomethylation Methionin and the modified monomethylation Methionin derivatize polypeptide of synthetic, and utilize with this modified monomethylation Methionin, and modified monomethylation Methionin derivatize polypeptide produces a kind of brand-new antibody as antigen, this antibody can be used for identifying the modified polypeptide with enrichment Methionin monomethylation polypeptide shedder after outer derivatize, the invention still further relates to the preparation method of described antibody.
Background technology
The modification of albumen or polypeptide is a kind of phenomenon naturally existed in organism, and the amino acid whose residue mainly forming polypeptide or albumen, by some base group modifications, then regulates and controls a series of vital movement in organism.Usual modification can be methylate, acetylize, phosphorylation modification etc.To methylate modification as important Methionin, the type of modification is divided into monomethylation, di-methylation or tri-methylated modification (ChengX, ZhangX.MutatRes (2007))..How effectively whether there is the modification that methylates in detection bodies, particularly monomethylation is modified, and can provide a kind of new method for effectively detecting some important diseases.Such as, such as, because some abiogenous modifications that methylate, monomethylation is modified abnormal, can cause the generation of some serious diseases, cancer etc.(people such as YutakaKondo, the people such as MolCellBiol.23 (1): 206 – 215 (2003), BerdascoM, ProcNatlAcadSci, 106:21830-21835 (2009)).
Methionin methylates and modifies is a kind of normal method of protein modification.Under normal circumstances; the detection of protein modification needs the antibody of this modification of specific recognition; as protein lysine acetylation modification can by lysine acetylation ubiquitin antibody identification (Kouzarides; T.Cell, 128,693 – 705 (2007); Berger; S.L.Nature, 447,407 – 412 (2007).Methionin methylates to modify and comprises monomethylation, di-methylation and tri-methylated three kinds of forms by the combining form of methyl group on Methionin.Correspondingly, the exploitation of highly sensitive, high specific Methionin monomethylation, di-methylation and tri-methylated antibody detects protein lysine to methylate the prerequisite of modifying.(people such as Barski, A., the people such as Cell129,823 – 837 (2007) .Rea, S., Nature406,593 – 599 (2000)).At present, although the Methionin di-methylation and the tri-methylated antibody that have a successfully exploitation are for the detection of Methionin di-methylation or tri-methylated modification, but the antibody that the Methionin monomethylation that success is not developed is modified is used for detection that the Methionin monomethylation modifies (people such as ZiqianLiang, ProteomeScience, 6th volume, (1) 6:2 (2008)).The group modified due to Methionin monomethylation is very little, only has the molecular weight of 15Da, the immune response that pole difficult labour is raw high, and organic attribute (organic) of monomethyl group weakens the immunogenicity of lysine residue further in addition.The attribute of these inherences causes directly there is huge difficulty as immunogen or Methionin monomethylation modified polypeptide as immunogenic antibody exploitation using Methionin monomethylation group.
Therefore, be necessary that a kind of brand-new antibody of exploitation is modified for detecting Methionin monomethylation.
Summary of the invention
The present invention relates to the modified monomethylation Methionin of synthetic, and modified monomethylation Methionin derivatize polypeptide, utilize modified monomethylation Methionin, and modified monomethylation Methionin derivatize polypeptide can be used for identifying the modified polypeptide with enrichment Methionin monomethylation polypeptide shedder after outer derivatize as the antibody that antigen produces, thus play the effect detecting Methionin monomethylation modified polypeptide and modify substrate.
On the one hand, the invention provides a kind of modified monomethylation Methionin, described modified monomethylation Methionin has following structural formula:
In some preferred modes, wherein, R is modification group, 1) R is wherein, R 1for alkyl (modification group carbonatoms is less than 6) or aromatic base (modification group carbonatoms is less than 8); Or, 2) and R is alkylsulfonyl wherein R 2for alkyl (modification group carbonatoms is less than 6) or aromatic base (modification group carbonatoms is less than 8).Preferentially, R is ethanoyl propionyl butyryl radicals more preferentially, R is propionyl
On the other hand, the present invention also provides a kind of modified monomethylation Methionin derivatize polypeptide.The sequence of described modified monomethylation Methionin derivatize polypeptide is CX ngGK*GGX n, wherein X is any monoamino-acid in 19 in common amino acid except halfcystine, and n is 1-20; Wherein, the structure of K* is as follows:
In some preferred modes, wherein, R is modification group, 1) R is wherein, R 1for alkyl (modification group carbonatoms is less than 6) or aromatic base (modification group carbonatoms is less than 8); Or, 2) and R is alkylsulfonyl wherein R 2for alkyl (modification group carbonatoms is less than 6) or aromatic base (modification group carbonatoms is less than 8).Preferentially, R is ethanoyl propionyl butyryl radicals more preferentially, R is propionyl
Preferentially, this peptide sequence is CEGRGDSGGGK*GGSG, and wherein K* is selected from methyl-prop acidylate, methyl vinyl or methylbutyryl modified Methionin, and in some preferred embodiments, K* is methyl-prop acylated lysine.
Another aspect, the invention provides a kind of antigen.Antigen is formed with the carrier protein couplet containing activating group by above-mentioned modified monomethylation Methionin or modified monomethylation Methionin derivatize polypeptide, and described carrier proteins includes but not limited to hemocyanin (KLH), bovine hemoglobin, bovine serum albumin (BSA) or ovalbumin (OVA) etc.
Preferentially, be propionating monomethylation Methionin with the modified monomethylation Methionin of carrier proteins activating group coupling.
Preferably, be the propionating Methionin polypeptide of monomethyl with the modified monomethylation Methionin derivatize polypeptide of carrier proteins activating group coupling, the sequence of polypeptide is CX ngGK*GGX n, wherein X is any monoamino-acid in 19 in common amino acid except halfcystine, and n is 1-20; K* is the propionating Methionin of monomethyl.Preferentially, this peptide sequence is CEGRGDSGGGK*GGSG, and wherein K* is methyl-prop acylated lysine.
Another aspect, the invention provides a kind of antibody.This antibody is prepared from by above-mentioned antigen-immunized animal.Be that antigen immune rabbit can prepare polyclonal antibody with the modified monomethylation Methionin of carrier proteins activating group coupling, preferentially, carrier proteins activating group and the propionating Methionin of monomethyl are that antigen immune rabbit prepares polyclonal antibody.Be that mice immunized with antigen can prepare monoclonal antibody with the modified monomethylation Methionin derivatize polypeptide of carrier proteins activating group coupling.Be CX with the sequence of the polypeptide of carrier proteins activating group coupling ngGK*GGX n, wherein X is any monoamino-acid in 19 in common amino acid except halfcystine, and n is 1-20, is the number of amino-acid residue; K* is the propionating Methionin of monomethyl.Preferentially, the peptide sequence of carrier proteins activating group coupling is CEGRGDSGGGK*GGSG, and wherein K* is methyl-prop acylated lysine.
Can specific combination one peptide species by antibody provided by the invention, this polypeptide comprises one or more modified monomethyl Methionin, and its structure is as follows:
In some preferred modes, wherein, R is modification group, 1) R is wherein, R 1for alkyl (modification group carbonatoms is less than 6) or aromatic base (modification group carbonatoms is less than 8); Or, 2) and R is alkylsulfonyl wherein R 2for alkyl (modification group carbonatoms is less than 6) or aromatic base (modification group carbonatoms is less than 8).Preferentially, R is ethanoyl propionyl butyryl radicals more preferentially, R is propionyl
Possess except the antibody of above-mentioned characteristic except immune animal obtains, according to the principle of antibody exploitation, this antibody can also be screened by modified monomethyl Methionin or modified monomethylation Methionin derivatize polypeptide and be prepared from from phage display library, yeast display storehouse, bacteria display storehouse and ribosomal display storehouse.Preferentially, modified monomethyl Methionin is the propionating Methionin of monomethyl, and modified monomethylation Methionin derivatize peptide sequence is CX ngGK*GGX n, wherein X is any monoamino-acid in 19 in common amino acid except halfcystine, and n is 1-20, is the number of amino-acid residue; K* is the propionating Methionin of monomethyl.Preferentially, this peptide sequence is CEGRGDSGGGK*GGSG, and wherein K* is methyl-prop acylated lysine.
Beneficial effect of the present invention
The invention provides a kind of modified monomethylation Methionin and modified monomethylation Methionin derivatize polypeptide of synthetic, utilize this modified monomethylation Methionin and modified monomethylation Methionin derivatize polypeptide as antigen, a strain specific antibodies can be prepared through immune animal, antibody can be used for identifying the modified polypeptide with enrichment Methionin monomethylation polypeptide shedder after outer derivatize, thus play and detect Methionin monomethylation modified polypeptide and the effect of modifying substrate.
Cell hybridization strain preservation explanation
Preservation center names: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is the PMT-001 cell strain of CGMCCNO.9109, systematic name: mouse source hybridoma, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Accompanying drawing explanation
Figure 1 shows that dot-blot pilot experiments detects Methionin list base propionating rabbit source polyclonal antibody specificity.Applied sample amount corresponding to each Dot blot is 20 nanograms.1: Methionin methyl-prop acyl polypeptide storehouse; 2: Methionin methyl-prop acidylate GG polypeptide; 3: methyl-prop acylated lysine micromolecular compound; The methyl-prop acylated lysine micromolecular compound of 4:KLH coupling; 5: the propionating peptide library of Methionin; 6: the propionating GG polypeptide of Methionin; 7: Methionin methyl vinyl peptide library; 8: Methionin methyl vinyl GG polypeptide; 9: Methionin methylbutyryl peptide library; 10: Methionin methylbutyryl GG polypeptide; 11: the Methionin peptide library of unmodified; 12: the Methionin GG polypeptide of unmodified; 13:KLH.
Figure 2 shows that dot-blot pilot experiments detects Methionin methyl-prop acidylate rabbit source polyclonal antibody specificity.Applied sample amount corresponding to each Dot blot is as schemed shown in left mark.1: Methionin methyl-prop acyl polypeptide storehouse; 2: the propionating peptide library of Methionin; 3: Methionin Butyrylation peptide library; 4: Methionin monomethylation peptide library; 5: Methionin di-methylation peptide library; 6: the tri-methylated peptide library of Methionin.
Figure 3 shows that competitive ELISA experiment detects Methionin methyl-prop acidylate rabbit source polyclonal antibody specificity.In experiment, peptide sequence used is in table 1 and table 2.
Figure 4 shows that dot-blot pilot experiments detects Methionin list base propionating mouse resource monoclonal antibody specificity.Applied sample amount corresponding to each Dot blot is 20 nanograms.1: Methionin methyl-prop acyl polypeptide storehouse; 2: methyl-prop acylated lysine micromolecular compound; 3: Methionin methyl-prop acidylate GG polypeptide; The methyl-prop acylated lysine micromolecular compound of 4:KLH coupling; 5: the propionating peptide library of Methionin; 6: the propionating GG polypeptide of Methionin; 7: Methionin methyl vinyl peptide library; 8: Methionin methyl vinyl GG polypeptide; 9: Methionin methylbutyryl peptide library; 10: Methionin methylbutyryl GG polypeptide; 11: the Methionin peptide library of unmodified; 12: the Methionin GG polypeptide of unmodified; 13:KLH.In experiment, peptide sequence used is in table 3 and table 4.
Figure 5 shows that dot-blot pilot experiments detects Methionin list base propionating mouse resource monoclonal antibody specificity.。Applied sample amount corresponding to each Dot blot is as schemed shown in left mark.1: Methionin methyl-prop acyl polypeptide storehouse; 2: the propionating peptide library of Methionin; 3: Methionin Butyrylation peptide library; 4: Methionin monomethylation peptide library; 5: Methionin di-methylation peptide library; 6: the tri-methylated peptide library of Methionin; In experiment, peptide sequence used is in table 1 and table 2.
Figure 6 shows that competitive ELISA experiment detects Methionin list base propionating mouse resource monoclonal antibody specificity specificity.In experiment, peptide sequence used is in table 1 and table 2.
Describe in detail
definition
Unless otherwise defined, all technology and scientific terminology have identical implication with the term that the those skilled in the art of the technical field of the invention use as used herein.
the modification of Methionin and the design of antigen
Some preferred embodiment in; on the basis that Methionin monomethylation is modified; the modification of ethanoyl, propionyl or butyryl radicals can be carried out further to monomethylation Methionin, then carry out with through the micromolecular compound of dual modification or polypeptide antigen the antibody that immune animal can obtain specific recognition Methionin monomethyl acetylize (Kme-ac), monomethyl propionating (Kme-prop) or monomethyl butyryl radicals (Kme-buty).
Usually, after pancreatin cracking is carried out to the protein sample extracted from organism, external derivative reaction can be carried out to enzymolysis polypeptide, as propionating reaction.Modify as there is Methionin monomethylation in certain polypeptide, then will generate the propionating polypeptide of Methionin monomethylation after external derivatize.And then by through the propionating polypeptide of Methionin monomethylation of derivative reaction and the antibody incubation of above-mentioned exploitation; according to the characteristic of the affine combination of Ag-Ab specificity, the propionating antibody of Methionin monomethyl will specific recognition the propionating polypeptide of enrichment Methionin monomethylation.Take off polypeptide wash-out propionating with the Methionin monomethylation of antibodies through overpickling; peptide sequence and the decorating site information of the propionating modification of Methionin monomethyl can be known by follow-up mass spectroscopy; and these information in fact corresponding be the peptide sequence modified of Methionin monomethylation and decorating site, can obtain finally by further protein data search the protein substrate information that Methionin monomethylation modifies.
It is the technology that persons skilled in the art are known that amino acid carries out base group modification, this modification can carry out abstraction and purification from the natural biology body of the modification had been found that, also manually can carry out external modification and obtain the final amino acid through modifying.Methionin, according to its attribute, is easy to multiple modification occurs in vivo, and as acetylated lysine, methylate Methionin etc.In vitro, under the reaction system optimized and reaction conditions, specific polylysine modification form can also be synthesized, as monomethyl acetylated lysine, the propionating Methionin of monomethyl and monomethyl Butyrylation Methionin.The modified Methionin small molecules of this synthetic, can produce antiserum(antisera) as small molecule antigens and necessary carrier protein couplet immune animal on the one hand, through the process for purification of antibodies of necessity, can produce the antibody of this modification Methionin of specific recognition.On the other hand, using the modified Methionin synthesized as basic raw material (rawmaterial), further artificial synthetic polypeptide antigen.Produce antiserum(antisera) after this polypeptide antigen and necessary carrier protein couplet immune animal, through the process for purification of antibodies of necessity, the antibody of this modification Methionin of specific recognition can be produced.
The length containing modification Methionin polypeptide antigen of synthetic can be within each 30 amino-acid residues in modified Methionin both sides.Preferred sequence length is CX ngGK*GGX n, wherein X is the arbitrary amino acid in 19 kinds of common amino acids except halfcystine, and n is 1-20; K* is modified Methionin, and preferred K* is selected from methyl-prop acidylate, methyl vinyl or methylbutyryl modified Methionin.Some preferred embodiment in, K* is methyl-prop acylated lysine.Some preferred embodiment in, adorned amino acid can be in the mid-way of peptide sequence, also can be in inclined the aminoterminal (-NH of this polypeptide antigen 2) or the position of carboxyl terminal (-COOH).Preferably, polypeptide antigen sequence is CEGRGDSGGGK*GGSG.
the synthesis of antigenic peptide
Modified Methionin polypeptide synthesize existing known technology.Solution method can be adopted to synthesize polypeptide of the present invention, conventional solid-phase synthesis also can be adopted to synthesize polypeptide of the present invention.The chemical synthesising technology of polypeptide is that liquid phase method or solid phase method are all ripe.Some preferred embodiment in, the technology of improvement on synthesis is solid-phase synthesis.1978, the Fmoc (9-fluorenylmethyloxycarbonyl) that the people such as ChangMeienlofer and Atherton adopt Carpino to report was as α amino protecting group, and Fmoc base is very stable to acid, but can use piperidines-CH 2cL 2or piperidines-DMF sloughs.In recent years, Fmoc synthesis method is widely used.In other preferred modes, Fmoc synthesis method is adopted to synthesize peptide sequence of the present invention (Fmoc Solid phase peptide synthssis-a kind of practical approach (FmocSolidPhasePeptideSynthesis) – APracticalApproach. Oxford University Press, 2000).
Employing Fmoc synthesis method is synthesized peptide sequence basic skills of the present invention and is: be first attached to an insoluble carrier on to the amino acid that alpha-amino group is protected by a support arm with Fmoc group by one; subsequently by alpha-amino group deprotection, with solution washing amino acid-support arm-resin.The amino acid of second preactivated alpha-amino group protection is connected by coupled reaction.In addition, also single amino acid can be replaced to carry out coupled reaction by the peptide segment of α-N end and side chain protected, after condensation reaction completes, with solution washing, repeat deprotection, coupling, until obtain object peptide.Finally by peptide-support arm-resin cracking.The solid-phase synthesis of this prolongation peptide chain both can adopt the method for interruption, also can use the method for continuous flow.
the generation of antibody and antibody
Here antibody refers to by utilizing modified Methionin small molecules of the present invention or modified Methionin polypeptide to obtain antibody as antigen-immunized animal.
Some preferred embodiment in, at carbodiimide, as under 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) existent condition, the micromolecular carboxyl terminal (-COOH) of modified Methionin can be reacted with N-hydroxy-succinamide sulfonic acid (Sulfo-NHS), form semistable Sulfo-NHS ester, then with the aminoterminal (-NH of carrier proteins 2) coupling (Taros, J.V., wait people, AnalBiochem, 156:220-2 (1986)).The propionating Methionin of micromolecular compound monomethyl of the present invention (Lys (me-prop)-OH), is mediated by EDC, can with some carrier proteinss-NH 2end coupling, forms activation holoantigen and carrys out immune animal.In the set-up procedure of polypeptide holoantigen, the amino (-NH on carrier proteins 2) first react with 4-(N-maleimidomehyl) hexanaphthene-1-carboxylic acid sulfonic group succinimide ester sodium salt (sulfo-SMCC), form stable peptide bond, react with the halfcystine of antigenic peptide end subsequently, by the mixture that disulfide formation is stable, the holoantigen forming activation thus carrys out immune animal.
Some preferred embodiment in, modified Methionin small molecules of the present invention or antigenic peptide are activated by the coupling with carrier proteins, with the modified Methionin small molecules of carrier proteins or polypeptide antigen, there is stronger immunocompetence, because independent modified Methionin small molecules or peptide sequence itself usually do not have immunocompetence or immunocompetence is very low.The carrier proteins of coupling can be, but be not limited to hemocyanin (Keyholelimpethemocyanin, KLH), bovine serum albumin (BovineSerumAlbumin, BSA), ovalbumin (Ovalbumin, OVA), bovine hemoglobin (Bovinegammaglobulin, BGG), bovine thyroid albumen (BovineThyroglobulin, BTG), Physter macrocephalus myoglobin (SpermWhaleMyoglobin, SWM), Toxoid,tetanus (TetanusToxoid, TT), methylated bovine serum albumin (MethylatedBovineSerumAlbumin, mBSA), human normal immunoglobulin IgG or IgA (HumanimmunoglobulinsIgG, or the immunogen protein of other prior aries etc. IgA).
There are the modified Methionin small molecules of active group or polypeptide can carry out immune animal with above coupling, such as mouse, rabbit, or other Mammalss produce polyclonal antibody, also monoclonal antibody can be produced with hybridoma, these methods are the existing known technology in this area, do not repeat them here, antibody of the present invention or antibody fragment (preparation and use antibody-application manual (MakingANDUsingAntibodies-Apracticalhandbook) .CRCPress, 2007) can be obtained see some textbooks or immune handbook.Certainly, antibody also through naturally producing, also can be through antibody or the antibody fragment of synthetic.These antibody can specific recognition comprise through modification lysine residue, and with its peripheral sequence have nothing to do.Some preferred embodiment in, antibody of the present invention can a certain Methionin polypeptide containing the propionating modification of monomethyl of specific recognition, and can not identify that those contain the polypeptide of other polylysine modification forms.So-called " specificity " refers to that this antibody only can identify or in conjunction with the antigen of a certain particular type, and nonrecognition or the antigen in conjunction with other types.In the present invention; prepared antibody is the peptide sequence of the Methionin that identification form methyl-prop acidylate is modified; and the peptide sequence of other type polylysine modification can not be identified; as including, but not limited to Methionin di-methylation; tri-methylated; acetylize, propionating, Butyrylation modification, or the modified forms of other amino-acid residues, as tyrosine phosphorylation etc.
As combining or identify that the antibody that Methionin methyl-prop acidylate is modified can be antibody of the present invention.Antibody can be the immunoglobulin molecules of immunoglobulin molecules or incomplete antigen specific site, such as those have antigen binding site molecule can special (immune) in conjunction with analyte, the mimicry material of analyte or part.Antibody also comprise synthetic hybrid antibody or through the antibody of modified or Antibody molecule fragments, include, but are not limited to, antibody fragment and Fv fragment.The antibody with conjugated antigen function have some fragments on abiogenous antibody.A binding fragment or antibody fragment include, but are not limited to, (i) Fab fragment, and it comprises VL, VH, CL and CH1 region; (ii) Fd fragment, it comprises VH and CH1 region (iii) Fv fragment, and it comprises VL and the VH region on a strand of antibody; (iv) dAb region (Wardetal., Nature341:544-546 (1989), it comprises VH region; V () be determinant (CDR) independently; (vi) F (ab') 2 fragment, a bivalent fragment comprises two Fab fragments connected in hinge area by disulphide.In addition, although two regions in this Fv fragment are that different genes encodings determined, the connection reagent of synthetic can allow them form single protein chain (known single Fv (scFv) chain) (people such as Bird, Science242:423-426 (1988); With people such as Huston, PNAS85:5879-5883 (1988)).Protein fragments comprises the fragment that those can be cross-linked the target antigen in conjunction with them, such as bivalent fragment, as F (ab') 2 fragment.Optionally, those can not the crosslinked combining target antigen of oneself protein fragments also can together with second antibody combining target antigen.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.。
The following list of the reagent used in the embodiment of the present invention:
Table 1
In the embodiment of the present invention, various solution formula is as follows:
Table 2
The preparation of embodiment 1 polyclonal antibody
The synthesis of 1, the micromolecular synthesis of the propionating Methionin of monomethyl (Lys (me-prop)-OH) namely connects methylpropionyl group on Methionin ε-residue, and NHFMoc is blocking group).
Synthetic route:
1) by 14g (0.029mol) 2-1 (N'-tertbutyloxycarbonyl-N-fluorenylmethyloxycarbonyl-N'-methyl-1B, C 27h 34n 2o 6) dissolve with methylene dichloride (DCM), continue under normal temperature to pass into brand-new HCL gas after 1.5 hours, under HCL strong acid, normal temperature continues reaction 5 hours, and thin-layer chromatography TLC confirms that reaction terminates; 50 DEG C, after being spin-dried under 0.03MPa condition, obtain 14.7g sticky solid, then carry out silica gel column chromatography (eluent ratio: DCM-DCM:MeOH=50:1-DCM:MeOH=20:1) and obtain 10.8g (0.0283mol) 2-2 (N-fluorenylmethyloxycarbonyl-N'-methyl-1B, C 22h 26n 2o 4).
2) 10.8g2-2 adds 30mL acetone solution, then adds 1.1MNaHCO 3after aqueous solution 70mL, stirring at room temperature 2 hours (fully stirring, the HCl combined in removing raw material), slowly adds the acetone soln 50mL being dissolved with 3.93g (0.03mol) propionic anhydride, reacts 1 hour under ice-water bath; Reaction terminates rear 2N hydrochloric acid and adjusts about pH to 3.0, add DCM100mL extracting twice, get organic phase 50 DEG C, 0.03MPa rotary evaporation obtains 12.4g sticky solid, carry out silica gel column chromatography (eluent ratio: DCM-DCM:MeOH=50:1-DCM:MeOH=30:1) and obtain 10g (0.0228mol) 2-3 (N'-propionyl-N-fluorenylmethyloxycarbonyl-N'-methyl-1B, C 25h 30n 2o 5).
3) 10g2-3 adds 70mLN, and add 6mL (0.061mol) piperidines after dinethylformamide (DMF) and 45mL tetrahydrofuran (THF) (THF) dissolve, stirring at normal temperature is spent the night, and TLC confirms that reaction terminates; 8g mix products is obtained after oil pump underpressure distillation evaporate to dryness (0.88kPa) solvent, carry out silica gel column chromatography (eluent ratio: DCM-DCM:MeOH=50:1-DCM:MeOH=30:1), obtain the esterification products that 3.4g (0.0147mol) is pure; Add 0.585g sodium hydroxide, 35mL water, 35mL methyl alcohol is hydrolyzed, and reacts 2 hours under ice-water bath, adjusts pH to 3.0 after esterification products complete hydrolysis with 2NHCl.50 DEG C, 0.03MPa is spin-dried for solvent, then adds 20mL dissolve with ethanol product, and after filtering, filtrate is spin-dried for, and obtains final product 2-4 (Lys (me-prop)-OH, N'-propionyl-N'-methyl-1B C 10h 20n 2o 3) 2.6g.
2, the propionating lysine compound of monomethyl and carrier protein KLH prepare immunogen
(1) 10mgKLH is dissolved in 1mLMES damping fluid;
(2) EDC and the 1.1mgSulfo-NHS of 0.4mg is added respectively; Abundant mixing, room temperature reaction 15 minutes;
(3) add 1.4 μ L beta-mercaptoethanols subsequently, after reaction 10min, with PBS, pH is adjusted to 7.4;
(4), after 2mgLys (me-prop) compound is dissolved in PBS, adds in the KLH solution in step (3) after activation, fully mix, room temperature reaction 2 hours;
(5) add 50mMTris subsequently and react 15min, 4 DEG C of dialysed overnight in PBS solution.
3, animal immune program
1) 8 week age new zealand white rabbit, subcutaneous multi-point injection;
2) first time immunity: getting 500 μ L concentration is that the small molecule immune of the KLH coupling of 0.8mg/ml is former in same volume Split completely mixing and emulsifying, the immunity of dorsal sc multiple spot;
3) after 3 weeks, after getting the small molecules epidemic focus of exempting from KLH coupling and the incomplete freund adjuvant mixing and emulsifying of same volume that 500 μ L concentration are 0.4mg/ml, dorsal sc multi-point injection;
4) every 2 weeks, by the dosage of step 3 and method booster immunization once;
5) the 4th immunity gets 1ml serum in latter 10 days, detects serum titer by ELISA method.If serum titer is greater than 30,000, namely pass through Culling heart blood.If serum titer is defective, then continue repeating step 4, until serum titer is greater than 30,000.
4, polyclonal antibody purification
ProteinA prepurification:
1) titre centrifugal 10 minutes of serum 10mL10000r/min being greater than 30,000, draws supernatant 0.45 μm of filtering with microporous membrane;
2) 3mlProteinA resin equilibrium at room temperature 1 hour, the phosphate buffered saline buffer (PBS, pH7.2) of 4 times of column volumes cleans resin;
3) filter after serum 20ml be loaded in the pillar containing ProteinA resin, and with ProteinA pillar incubated at room 90min after, leave standstill 15min;
4) wash 1 time with the cleaning buffer solution A of 10 times of column volumes respectively, 15 times of column volume PBS wash pillar 1 time;
5) the wash-out Buffer wash-out of 5 times of column volumes, adds neutralization buffer subsequently, 4 DEG C of PBS dialysed overnight;
6) ultrafiltration IgG is 5-10mg/ml to concentration.
Prepared by antigenic peptide coupling pillar
1) equilibrium at room temperature SulfoLinkCouplingResin1 hour, draws 5ml in chromatography void column after mixing, the coupling buffer cleaning of 4 times of column volumes twice;
2) take 5mg antigenic peptide, be dissolved in coupling buffer respectively, add in SulfoLinkCouplingResin chromatography column post and mix, incubated at room 15 minutes;
3) leave standstill 30 minutes, the coupling buffer of 15 times of column volumes washes pillar;
4) add 1 times of column volume Block buffer, incubated at room is after 15 minutes, and 6 times of column volume cleaning buffer solution B clean pillar;
5) obtain respectively containing polypeptide 1 coupling pillar and polypeptide 2 coupling pillar.
Antigenic peptide affinity purification
The IgG obtained after the ultrafiltration of 5mgProteinA prepurification is added in antigenic peptide coupling pillar, incubated at room 2 hours; After giving up effluent liquid, clean pillar with the PBS of the cleaning buffer solution of 5 times of column volumes and 10 times of column volumes respectively, then movable antibody purification according to the following steps:
1) 4 times of column volume elution buffer wash-outs;
2) neutralization buffer is added in elutriant, in 4 DEG C of PBS dialysed overnight in dialysis tubing;
3) ultrafiltration IgG is 5-10mg/ml to concentration;
4) IgG after above-mentioned ultrafiltration is added in polypeptide 2 coupling pillar, incubated at room 30 minutes, collects effluent liquid, be the antibody that purifying is good.
5, Dot blot (DotBlot) detects:
1) polypeptide by listed in table 3 and table 4 is soluble in water, is configured to the polypeptide solution that starting point concentration is 100ng/ μ L;
2) pvdf membrane of the suitable size of cutting, about 40 seconds of anhydrous methanol process, rinsing 3 times in deionized water, seasoning 2 minutes;
3) by starting point concentration be 100ng/ μ L polypeptide solution further respectively dilution for 20ng/ μ L and 4ng/ μ L.Then point sample successively, 1 μ l/ point, puts into six orifice plates after dry 10 minutes;
4) 5% skim-milk room temperature closes 60 minutes, TBST washing lotion rinsing twice, 5 minutes/time;
5) with 5% skim-milk dilution antibody, incubated at room 1 hour, with TBST washing lotion rinsing three times, 10 minutes/time;
6) with 5% skim-milk dilution goat-anti rabbit HRP traget antibody, room temperature 45 minutes, TBST washing lotion rinsing three times, 10 minutes/time;
7) chemoluminescence nitrite ion is evenly laid on film, hatches post-exposure in 5 minutes, the results are shown in Figure 1 and Fig. 2.
Table 3: the sequence (K* represents the Methionin through modifying, and wherein X is any monoamino-acid in 19 in common amino acid except halfcystine) of polypeptide used in Fig. 1 and Fig. 4 dot-blot pilot experiments
Table 4: the sequence (K* represents the Methionin through modifying, and wherein X is any monoamino-acid in 19 in common amino acid except halfcystine) of polypeptide used in Fig. 2 and Fig. 5 dot-blot pilot experiments
Dot blot detects and shows; the polyclonal antibody that the monomethylation designed by the present invention propionating Methionin small molecules is obtained as antigen can specific recognition Methionin methyl-prop acyl polypeptide storehouse, Methionin methyl-prop acidylate GG polypeptide, monomethylation propionating Methionin small molecules and KLH coupling the propionating Methionin small molecules of monomethylation; but also the polypeptide (Fig. 1) of nonrecognition other types polylysine modification, describes the specificity that antibody is good thus.The result of Fig. 2 shows; the propionating Methionin polyclonal antibody of the monomethylation developed can identify the Methionin methyl-prop acyl polypeptide storehouse of 4 nanograms; but nonrecognition similar until other modified Methionin peptide libraries of 100 nanograms; this had both shown the sensitivity of developed antibody, also indicated the specificity of this polyclonal antibody further.
6, ELISA detects:
1) bag quilt: with deionized water dilution Methionin methyl-prop acyl polypeptide to 1X10 -3mg/mL, 50 μ l/ holes, 4 DEG C of coated elisa plates spend the night;
2) close: within second day, wash 1 time with TBST, 5 minutes/time, 200 μ l/ holes, pat dry, 1%BSA/TBS70 μ l closes 45 minutes;
3) compete: the propionating polyclonal antibody PBS1:200 of Methionin monomethylation of purifying dilutes, and adds 0 nanogram respectively subsequently, 10 nanograms, cited polypeptide in 50 nanograms and 100 nanogram tables 1 and table 2,4 DEG C of reactions are spent the night;
4) add the propionating polyclonal antibody of Methionin monomethylation of purifying: second day, 3000g centrifuging and taking supernatant, dilute 10 times, join in enzyme plate, 50 μ l/ holes.Hatch 2 hours for 26 DEG C.
5) goat anti-rabbit igg two adding HRP mark resists: TBST washes 3 times, 5 minutes/time, and 200 μ l/ holes, pat dry, and add two and resist, 1:10000 dilutes (1%BSA/TBS), and 50 μ l/ holes, hatch 45 minutes for 26 DEG C.
6) add substrate: by the method for step 5, TBST washes 3 times, pats dry.50 μ l/ holes add TMB chromogenic substrate, and 26 DEG C are reacted 30 minutes;
7) stop: add 2M sulfuric acid, 50 μ l/ holes.
8) develop the color: microplate reader OD 450measure absorbancy.
As can be seen from the result of Fig. 3; gradually improve and antibody purification compete mutually Methionin methyl-prop acyl polypeptide storehouse, Methionin methyl-prop acidylate GG polypeptide amount; the ELISA signal that antibody and pre-coated Methionin methyl-prop acyl polypeptide react also reduces thereupon gradually; the ELISA signal that after competing with other modified Methionin polypeptide, antibody then reacts with pre-coated Methionin methyl-prop acyl polypeptide remains unchanged substantially, further illustrates the specificity of the developed propionating polyclonal antibody of Methionin monomethylation thus.
the preparation of embodiment 2 monoclonal antibody
1, antigen polypeptide sequence determination and with synthesis
Peptide sequence for immunity: CEGRGDSGGGK*GGSG, wherein, the 11 Methionin (K) residue first line of a couplet order methyl group (me) and propionating (prop) group.
Contrast peptide sequence (control sequence is not used in immunity): CEGRGDSGGGKGGSG, the 11 Methionin (K) residue is without any base group modification.
Antigenic peptide synthesis step is as follows:
A. the synthesis (namely connecting monomethyl propionyl group on Methionin ε-residue) of propionating monomethyl Methionin raw material (Fmoc-Lys (me-prop)-OH))
1) chloroformic acid benzyl-Methionin (trifluoroacetic acid, methyl) preparation of-methyl esters (Z-Lys (TFA, me)-Ome): chloroformic acid benzyl-Methionin (trifluoroacetic acid)-OH (Z-Lys (TFA)-OH), methyl iodide (Me 2i), K 2cO 3react with DMF mono-and reflux, extract reaction solution and send mass spectrometric detection to determine to synthesize successfully;
2) generation of chloroformic acid benzyl-Methionin (methyl)-OH (Z-Lys (me)-OH): the Z-Lys (TFA that step 1 generates, me)-Ome and LiOH saturated solution are greater than 12.0 times at pH and react, and extract reaction solution and send mass spectrometric detection to determine to synthesize successfully;
3) generation of chloroformic acid benzyl-Methionin (methyl-propanoyl)-OH (Z-Lys (me-prop)-OH): Z-Lys (me)-OH, propionic anhydride and triethylamine react under pH is the condition of 9.0, extract reaction solution and send mass spectrometric detection.React completely, acidifying, ethyl acetate extraction.Be condensed into oily matter.
4) generation of H-Lys (me-prop)-OH: Z-Lys (me, the prop)-OH that step 3 obtains dissolves in methyl alcohol, adds palladium carbon Pd/C, logical hydrogen, TLC follows the tracks of reaction.After having reacted, filter, filtrate concentrates to obtain solid, washes 4 times subsequently with ether, dries.
5) generation of Fmoc-Lys (me-prop)-OH: H-Lys (me-prop)-OH, the saturated NaHCO3 solution of fluorenes methoxy carbonyl acyl succinimide (Fmoc-OSu) and acetone are 9.0 reactions at pH, and TLC follows the tracks of reaction.After reacting completely, conventional processing, acidifying, product ethyl acetate extraction, drying is concentrated into oily matter.
B. the synthesis of antigenic peptide
Peptide systhesis carries out on ABI433 Peptide synthesizer.
1) resin swelling: N-fluorenylmethyloxycarbonyl-glycine king resin (Fmoc-Gly-Wangresin) soaks 15 minutes with methylene dichloride, treats that resin expands, pump methylene dichloride;
2) remove amido protecting: add hexahydropyridine/DMF solution that volume ratio is 1:4, use nitrogen to agitate, react 2 times, the time is 5 minutes and 15 minutes, and reaction terminates rear DMF washing resin 9 times.The resin that takes a morsel adds each 2-3 of toner ABC and drips (A liquid: triketohydrindene hydrate/ethanol solution; B liquid: pyridine; C liquid: phenol/ethanol solution) at 100 DEG C, be total to heat 3 minutes, solution and color of resin are blue (amino acid had is red-purple), can judge that amido protecting removes
3) condensation reaction: add Fmoc-Gly-OH and I-hydroxybenzotriazole (HOBT), dissolve with appropriate DMF, add DIEA, nitrogen is agitated, reacts 1 hour, and reaction terminates rear DMF washing resin 6 times.Look tested by the resin that takes a morsel, and method is with step 2, and solution and color of resin should be colourless, can assert that reaction completes.
4) repeating step 2-3, connects successively in advance by the amino acid in the peptide sequence of equimolar ratio example mixing, until completing of sequence, drains after resin methylene dichloride and ether being soaked
5) polypeptide is cut away from resin: add TFA, react 2 hours in constant-temperature table, shaking speed 110 revs/min, temperature 25 degree.
6) separate out crude product: elimination resin, adds anhydrous diethyl ether, obtain solid with after centrifuge in filtrate, add anhydrous diethyl ether washing, more centrifugal, repeat post-drying for several times and can obtain crude product polypeptide.
2, polypeptide holoantigen preparation
1) 20mgKLH is dissolved in 2mL5mMEDTA/H 2in O;
2), after 5mgS μ Lfo-SMCC is dissolved in 40 μ LDMSO completely, adds 160 μ LPBS, mix;
3) add in KLH solution by S μ Lfo-SMCC dropwise, limit edged stirs.Room temperature places 1 hour, then by the 4 DEG C of dialysis 1 hour in the PBS of 1L4 DEG C of pre-temperature of the KLH solution of above-mentioned activation; After changing liquid, dialyse 2 hours, repeat 1 time for 4 DEG C; Take 5mg antigenic peptide, be dissolved in 100 μ LDMSO, then add 400 μ LPBS, mix, add the activation KLH solution that the above-mentioned dialysis of 500 μ L is good subsequently, 4 DEG C of refrigerator overnight;
4) the above-mentioned cross-linked composite being connected with the antigenic peptide of KLH is in 4LPBS solution, 4 DEG C of dialysed overnight;
5) second day takes out KLH-antigenic peptide ,-20 DEG C of storages.
3, animal immune program
1) 6-8 Balb/c mouse in age in week, subcutaneous multi-point injection;
2) first time immunity is 100 μ L1mg/mL immunogens and same volume Split completely mixing and emulsifying, the immunity of dorsal sc multiple spot;
3) after 3 weeks, after 0.5mg/mL immunogen 100 μ L and the incomplete freund adjuvant mixing and emulsifying of same volume, dorsal sc multi-point injection;
4) later every 2 weeks, by dosage and the method booster immunization of step 3;
5) the 4th immunity got 20 μ L serum after 10 days, detected serum titer by ELISA method.If serum titer defective (serum titer is greater than 30,000), then continue repeating step 4 until serum titer is greater than 30,000.
4, cytogamy
1) after mouse draws neck to put to death, take out spleen, wire netting grinds, collect splenocyte, DMEM substratum cleaning twice, counting;
2) sp2/0 myeloma cell is collected in 50ml centrifuge tube, centrifugal 5 minutes, counting;
3) splenocyte and sp2/0 cell blow even rear collected by centrifugation in the mixing of 1:4 ratio;
4) substratum is clean, beat bottom centrifuge tube, make cell precipitation loose;
5) add 1ml polyoxyethylene glycol (PEG) with pasteur pipet along centrifugal tube wall, stir 60 seconds, add the nutrient solution of 30ml preheating subsequently.Build lid, mixing, centrifugal, evacuation substratum;
6) add fresh HAT medium re-suspended cell to spread into 96 porocyte culture plates.
7) positive cell strain is obtained by ELISA screening, carry out twice subclone, obtain the positive cell model fusion that series is stable, such as be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the PMT-001 cell strain of preserving number 9109, this cell strain can secrete monoclonal antibody.
5, ascites is produced
1) subclone is stable cell expansion is cultivated (be such as deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the PMT-001 cell strain of preserving number 9109);
2) the Balb/c mouse peritoneal injection 0.5ml Freund's incomplete adjuvant more than 8 week age; After 10 days, treat that mouse web portion swells slightly, every mouse immune 1X10 6cell;
3) collect ascites after 7 days, 12000g is centrifugal, removes fat, collects supernatant.
6, ascites purifying
1) centrifugal 10 minutes of 15 milliliters of ascites 12000g, 0.45 μm of membrane filtration;
2) ProteinG of 5 milliliters is loaded in chromatography void column, clean 1 time with 4 times of column volume PBS, equilibrium at room temperature 30 minutes;
3) ascites in step 1 15 milliliters is added in ProteinG pillar, incubated at room 2 hours;
4) leave standstill after 15 minutes, release ascites, the PBS of 15 times of column volumes washs pillar;
5) with the glycine elution liquid wash-out of 6 times of column volumes, collect and add neutralization buffer, 4 DEG C of dialysed overnight in PBS.
7, Dot blot (DotBlot) detects:
1) polypeptide by listed in table 3 and table 4 is soluble in water, is configured to the polypeptide solution that starting point concentration is 100ng/ μ L;
2) pvdf membrane of the suitable size of cutting, about 40 seconds of anhydrous methanol process, rinsing 3 times in deionized water, seasoning 2 minutes;
3) by starting point concentration be 100ng/ μ L polypeptide solution further respectively dilution for 20ng/ μ L and 4ng/ μ L.Then point sample successively, 1 μ l/ point, puts into six orifice plates after dry 10 minutes;
4) 5% skim-milk room temperature closes 60 minutes, TBST washing lotion rinsing twice, 5 minutes/time;
5) with 5% skim-milk dilution antibody, incubated at room 1 hour, with TBST washing lotion rinsing three times, 10 minutes/time;
6) with 5% skim-milk dilution goat-anti rabbit HRP traget antibody, room temperature 45 minutes, TBST washing lotion rinsing three times, 10 minutes/time;
7) chemoluminescence nitrite ion is evenly laid on film, hatches post-exposure in 5 minutes, the results are shown in Figure 3 and Fig. 4.
Dot blot detects and shows; the monoclonal antibody that the CEGRGDSGGGK*GGSG polypeptide designed by the present invention is obtained as antigen can specific recognition Methionin methyl-prop acyl polypeptide storehouse; methyl-prop acylated lysine micromolecular compound; the methyl-prop acylated lysine micromolecular compound of Methionin methyl-prop acidylate GG polypeptide and KLH coupling; but also the polypeptide (Fig. 4) of nonrecognition other types polylysine modification, describes the specificity that antibody is good thus.The result of Fig. 5 shows; the propionating Methionin monoclonal antibody of the monomethylation developed can identify the Methionin methyl-prop acyl polypeptide storehouse of 4 nanograms; but nonrecognition similar until other modified Methionin peptide libraries of 100 nanograms; this had both shown the sensitivity of developed antibody, also indicated the specificity of this polyclonal antibody further.
8, ELISA detects:
1) bag quilt: with deionized water dilution CEGRGDSGGGK*GGSG antigenic peptide to 1X10 -3mg/mL, 50 μ l/ holes, 4 DEG C of coated elisa plates spend the night;
2) close: within second day, wash 1 time with TBST, 5 minutes/time, 200 μ l/ holes, pat dry, 1%BSA/TBS70 μ l closes 45 minutes;
3) compete: the propionating monoclonal antibody PBS1:200 of Methionin monomethylation of purifying dilutes, and adds 0 nanogram respectively subsequently, 10 nanograms, cited polypeptide in 50 nanograms and 100 nanogram tables 1 and table 2,4 DEG C of reactions are spent the night;
4) add the propionating monoclonal antibody of Methionin monomethylation of purifying: second day, 3000g centrifuging and taking supernatant, dilute 10 times, join in enzyme plate, 50 μ l/ holes.Hatch 2 hours for 26 DEG C.
5) goat anti-rabbit igg two adding HRP mark resists: TBST washes 3 times, 5 minutes/time, and 200 μ l/ holes, pat dry, and add two and resist, 1:10000 dilutes (1%BSA/TBS), and 50 μ l/ holes, hatch 45 minutes for 26 DEG C.
6) add substrate: by the method for step 5, TBST washes 3 times, pats dry.50 μ l/ holes add TMB chromogenic substrate, and 26 DEG C are reacted 30 minutes;
7) stop: add 2M sulfuric acid, 50 μ l/ holes.
8) develop the color: microplate reader OD450 measures absorbancy.
As can be seen from the result of Fig. 6; gradually improve and antibody purification compete mutually Methionin methyl-prop acyl polypeptide storehouse, Methionin methyl-prop acidylate antigenic peptide amount; the ELISA signal that antibody and pre-coated Methionin methyl-prop acidylate antigenic peptide react also reduces thereupon gradually; the ELISA signal that after competing with other modified Methionin polypeptide, antibody then reacts with pre-coated Methionin methyl-prop acidylate antigenic peptide remains unchanged substantially, further illustrates the specificity of the developed propionating monoclonal antibody of Methionin monomethylation thus.

Claims (27)

1. a modified monomethyl Methionin, wherein, the structure through the monomethyl Methionin of modified is as follows:
Wherein, R is modification group, and R is wherein, R 1for carbonatoms is less than the aromatic base that the alkyl of 6 or carbonatoms be less than 8.
2. Methionin according to claim 1, wherein, R is alkylsulfonyl wherein R 2for carbonatoms is less than the aromatic base that the alkyl of 6 or carbonatoms be less than 8.
3. Methionin according to claim 1, R is ethanoyl propionyl or butyryl radicals
4. Methionin according to claim 1, R is propionyl
5., according to the Methionin one of claim 1-4 Suo Shu, described Methionin can be coupled to carrier proteins and form immunogen.
6. Methionin according to claim 5, described carrier proteins includes but not limited to, hemocyanin (KLH), bovine hemoglobin (BGG), bovine serum albumin (BSA) or ovalbumin (OVA).
7. a peptide species, this polypeptide comprises one or more modified monomethyl Methionin, and wherein, the structure through the monomethyl Methionin of modified is as follows:
Wherein, R is modification group, and R is wherein, R 1for carbonatoms is less than the aromatic base that the alkyl of 6 or carbonatoms be less than 8; Or R is alkylsulfonyl wherein R 2for carbonatoms is less than the aromatic base that the alkyl of 6 or carbonatoms be less than 8.
8. polypeptide according to claim 7, R is ethanoyl propionyl or butyryl radicals
9. polypeptide according to claim 7, R is propionyl
10., according to the polypeptide one of claim 7-9 Suo Shu, wherein the sequence of polypeptide is CX ngGK*GGX n, or CX nk*X n, wherein X is any monoamino-acid in 19 in common amino acid except halfcystine, and n is 1-20; GGK*GG is a specific motif, and K* is expressed as the described monomethyl Methionin through modified.
11. according to the polypeptide one of claim 7-10 Suo Shu, this polypeptide is a peptide library containing a specific motif (motif) be made up of 2 to 6 amino-acid residues, wherein contains one or more modified ε-amido acyl monomethyl Methionin in this motif.
12. polypeptide according to claim 10, the sequence of polypeptide is that CEGRGDSGGGK*GGSG, K* are expressed as the described monomethyl Methionin through modified.
13. according to the polypeptide one of claim 7-12 Suo Shu, and wherein said polypeptide is coupled to carrier proteins and produces antibody as antigen.
14. polypeptide according to claim 13, described carrier proteins comprises hemocyanin (KLH), bovine hemoglobin (BGG), bovine serum albumin (BSA) or ovalbumin (OVA).
15. 1 kinds of antibody, is characterized in that, this antibody is by the polypeptide one of claim 7-14 Suo Shu, or described the obtaining through the Methionin small molecule immune animal of modifying of one of claim 1-6.
16. antibody according to claim 15, wherein animal is for including but not limited to rabbit, mouse, goat, camel, llama, chicken.
17. antibody according to claim 15, antibody can be polyclonal antibody, also can be monoclonal antibody.
18. antibody according to claim 15, this antibody can specific recognition in conjunction with modified acyl monomethyl Methionin polypeptide, this polypeptide comprises one or more acyl monomethyl Methionin according to claim 1.
19. antibody according to claim 15, this antibody can be screened by the modified monomethyl Methionin one of claim 1-6 Suo Shu and be prepared from from phage display library, yeast display storehouse, bacteria display storehouse and ribosomal display storehouse.
20. antibody according to claim, the wherein antibody that produces of the PMT-001 cell strain of this antibody to be preserving number be CGMCCNO.9109.
The method of the peculiar affinity reagent of acyl monomethylation on 21. 1 kinds of ε-amidos producing protein lysine residues, the method comprises: the 1) ε-amido acyl methylated compound of preparation containing lysine residue, polypeptide or albumen 2) adopt containing the ε-methylated compound of amido acyl of lysine residue, polypeptide or albumen are with the peculiar affinity reagent of acyl monomethylation on the ε-amido crossing immunologic method and go to produce lysine residue.
22. methods according to claim 21, methylated by ε-amido acyl Methionin or the ε-amido acyl lysine analogues that methylates is coupled on carrier proteins and produces antibody as antigen.
23. according to the method for one of claim 21-22, and the methylate structure of Methionin of ε-amido acyl is as follows:
Wherein ,-R is modification group, 1) R is wherein, R 1for carbonatoms is less than the aromatic base that the alkyl of 6 or carbonatoms be less than 8; Or, 2) and R is alkylsulfonyl wherein R 2for the alkyl that carbonatoms is less than 6, or the aromatic base that carbonatoms is less than 8.
24. methods according to claim 23, use bad amino acid monomethylation derivatize polypeptide as antigen, its sequence is CX ngGK*GGX n, or CX nk*X n, wherein X is any monoamino-acid in 19 in common amino acid except halfcystine, and n is 1-20; GGK*GG is a specific motif, and K* is expressed as described ε-amido acyl and methylates Methionin.
25. methods according to claim 24, this polypeptide is a peptide library containing a specific motif (motif) be made up of 2 to 6 amino-acid residues, wherein contains one or more modified ε-amido acyl monomethyl Methionin in this motif.
26. methods according to claim 25, the sequence of polypeptide is that CEGRGDSGGGK*GGSG, K* are selected from the propionating or monomethyl Butyrylation modified Methionin of monomethyl acetylize, monomethyl; Preferentially, K* is the propionating Methionin of monomethyl.
27. methods according to claim 23, on described ε-amido acyl methylates lysine molecule, be connected with carrier proteins, described carrier proteins includes but not limited to hemocyanin (KLH), bovine hemoglobin (BGG), bovine serum albumin (BSA) or ovalbumin (OVA).
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CN106046143A (en) * 2016-07-25 2016-10-26 杭州莱和生物技术有限公司 Method for preparing aniline green artificial antigens
CN115201385A (en) * 2022-06-22 2022-10-18 河北医科大学 Derivatization reagent for electrospray mass spectrometry detection and capable of enabling amino micromolecules to carry two charges, and preparation method and application thereof
CN115201385B (en) * 2022-06-22 2024-01-09 河北医科大学 Derivatization reagent for electrospray mass spectrometry detection for enabling amino small molecules to carry two charges, and preparation method and application thereof

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