CN109265520A - A kind of efficient identification Protein Serine heptan glycosylated polyclonal antibody, preparation method and applications - Google Patents
A kind of efficient identification Protein Serine heptan glycosylated polyclonal antibody, preparation method and applications Download PDFInfo
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Abstract
The present invention relates to a kind of glycosylated polyclonal antibody in efficient identification Protein Serine heptan, preparation method and applications, the antibody be used to detect include glycosylation modified native protein and new antibiotic in various configuration serine heptan screening and discovery.The preparation process of antibody includes: to construct seven carbon sugar skeletons first with Wittig reaction, the heptose of 6 two kinds of absolute configurations is obtained using Sharpless asymmetric oxidation reaction afterwards, with the help of the glycosylation that sulphur glycosides and three chlorimide esters mediate, it obtains and the glycosylation modified consistent serine heptose of configuration in organism, corresponding glycopeptide, sequence are as follows: Ac-GS (Hep) GL-OH are obtained using Solid-phase synthesis peptides method afterwards；By on obtained hapten conjugation to carrier protein BSA, antigen is obtained, with antigen-immunized animal, immune new zealand rabbit blood is collected and prepares antiserum, and IgG is obtained by affinity purification.Antibody specificity prepared by the present invention is good, and potency is high.
Description
Technical field
The present invention relates to technical field of medical chemistry, specifically, being a kind of glycosylation in efficient identification Protein Serine heptan
Polyclonal antibody, preparation method and applications.
Background technique
Host cell can be adhered to or tissue is that most pathogenic bacterias realize the prerequisite effectively infected.It causes a disease thin
It is thin to efficiently be adhered to that bacterium would generally specifically identify the receptor of host cell surface using a kind of albumen for being known as adhesin
Cellular surface.Spread adhesion Escherichia coli (Diffuse-adheringE.coli, DAEC) and enterotoxigenic escherichia coil
(EnterotoxigenicE.coli, ETEC) is a kind of important pathogen for causing people and cub diarrhea, they are separately encoded
Two kinds of adhesins of AIDA-I and TibA.Before research shows that both adhesins are directly related with Pathogenicity of Bacteria, and have report
Road claims AIDA-I and TibA is glycoprotein, but it glycosylates mechanism and concrete function is unknown.In a newest research, Shao
Peak team identifies a seven novel carbon sugar transferase (BAHT:bacterialautotransporterheptosyl-
Transferase) family, the Heptose for being catalyzed AIDA-I and TibA is glycosylation modified, to realize bacterium to host cell
Adherency and effective field planting (Fig. 1) in mouse intestinal.
BAHT family and known glycosyl transferase do not have the homology in sequence, so it is with unique biochemical
Matter, but BAHT catalysis and substrate recognition mechanism are unknown.TibC monomer molecule in the parsing discovery BAHT family of crystal structure can
To be divided into N-terminal β-pleated sheet barrel structure domain and C-terminal catalytic core domain.12 TibC monomer molecules are by " hand in hand " and " lean against
Back " two ways aggregates into a kind of 12 dimeric structures of novel garland shape；It is formed among 12 dimeric complexes one very big
Cylindrical body channel, the catalytic active center of all monomer molecules all towards this channel interior, prompts the glycosylation of substrate
Journey may be by substrate across this channel to realize.The compound of further ADP-D, D-Heptose and TibC are brilliant
Body does not only disclose the Key residues and catalytic mechanism for participating in glycosyl transfer, and also illustrates BAHT family different members to matching
The structural principle of body spatial configuration selectivity.The glycosylation transfer of the catalysis of BAHT first is that configuration inversion reacts: ADP-D, D-
The end Heptose position glycosidic bond is beta comfiguration in Heptose substrate, after Heptose is transferred to substrate, obtained Ser-O-Hep
The end position glycosidic bond of modified glucoprotein is changed into α configuration (Fig. 2).Secondly, research shows that AAH can shift D, D-Heptose and
On L, D-Heptose to substrate protein, and TibC is merely able to D, and D-Heptose shows that different BAHT are mediated not as ligand
The Ser-O-Hep of isomorphism type is modified.
But still it has the following problems:
Whether modified containing others Ser-O-Hep in including but not limited to above-mentioned pathogenic bacteria? it is different in various pathogenic bacteria
How is the ligand stereoselectivity for the Ser-O-Hep modification that BAHT is mediated? how targeted bacteria surface Ser-O-Hep modification
Is adhesin to realize bacterium anti-adhesive treatment?
Although presently, there are a variety of detection protein glycosylations (such as Protein S er/Thr-O-GlcNAc modification or Asn-N- sugar
Baseization modification) means and tool, but detect and explore special Ser-O-Hep in pathogenetic bacteria and modify the related work of still shortage
Tool is modified using Ser-O-Hep and realizes that anti-adhesive treatment still lacks effective means.
Antibody is sent out in the detection of albumen as a kind of important molecular tool with high sensitivity and specificity
Wave irreplaceable role.Ser-O-Hep modification glycopeptide is synthesized to which the more of selectively targeted Ser-O-Hep modification are capable of in preparation
Clonal antibody can not only accelerate the exploration modified Ser-O-Hep in a variety of pathogenetic bacterias, more develop small molecule of new generation
Antibiotic and antibody class drug provide new opportunity.
In view of the deficienciess of the prior art, we have invented a kind of specifically glycosylated antibody in anti-serine heptan
(Anti-SerHep).The first step has obtained serine heptose using Wittig reaction and Sharpless oxidative synthesis, then benefit
It is obtained with Solid phase peptide synthssis comprising glycosylated polypeptide in serine heptan.Secondly we will pass through the c-terminus and ox blood of the glycopeptide
Pure albumen (BSA) coupling, finally uses it as antigen immune rabbit, and obtained antiserum is carried out affinity purification, thus
To first glycosylated antibody in specifically anti-serine heptan (Anti-SerHep) (Fig. 3).
Summary of the invention
The first purpose of this invention is in view of the deficiencies of the prior art, to provide a kind of glycosylation modified containing serine heptan
Glycopeptide.
Second object of the present invention is in view of the deficiencies of the prior art, to provide one kind and contain serine heptose as described above
The synthetic method of the glycopeptide of baseization modification.
Third object of the present invention is in view of the deficiencies of the prior art, to provide one kind and contain serine heptose as described above
The glycopeptide of baseization modification and the compound of carrier protein crosslinking.
Fourth object of the present invention is in view of the deficiencies of the prior art, to provide a kind of specific recognition and contain as described above
Serine heptan glycosylation modified glycopeptide antiserum or antibody.
5th purpose of the invention is in view of the deficiencies of the prior art, to provide a kind of purifying side of antibody as described above
Method.
6th purpose of the invention is in view of the deficiencies of the prior art, to provide a kind of application of antibody as described above.
To realize above-mentioned first purpose, the technical solution adopted by the present invention is that:
A kind of glycopeptide glycosylation modified containing serine heptan, the structure of the glycopeptide are as follows:
To realize above-mentioned second purpose, the technical solution adopted by the present invention is that:
The synthetic method of glycopeptide as described above, which is characterized in that the synthetic method of the glycopeptide is as follows:
(1) compound 2 obtains compound 3 under the action of acetic anhydride and iodine；
(2) under the catalysis of boron trifluoride ether, end position and toluene-ω-thiol are coupled to obtain compound 4 compound 3；
(3) compound 4 successively reacts to obtain with sodium methoxide and TBDMSSCI compound 5, compound 5 BnBr, NaH,
Compound 6 is obtained under the action of TBAI, DMF；
(4) compound 6 obtains compound 7 under the action of acetic acid and TBAF；
(5) compound 7 obtains compound 8 in the case where wearing the effect of this Martin's oxidant；
(6) compound 8 reacts to obtain thin malt sugar 9 by Wittig；
(7) thin malt sugar 9 obtains compound 10a under the catalysis of AD-mix- α, and thin malt sugar 9 is changed under the catalysis of AD-mix- β
Close object 10b；
(8) compound 10a obtains compound 12a under the action of NB and aceticanhydride pyridine；Compound 10b is in aceticanhydride pyridine
Compound 11b is obtained under effect；
(9) compound 12a obtains compound 14a under the action of acetic acid hydrazine, Tritox and DBU, that is, the trichlorine activated
Imines ester；Compound 11b obtains compound 12b under the action of NIS and silver trifluoromethanesulfonate；
(10) compound 14a obtains compound 15a under the action of triflate；Compound 15a is in trifluoroacetic acid
Under the action of obtain compound 16a；Compound 12b obtains compound 13b under the action of trifluoro formic acid；
(11) using 2- chlorine trityl resin as solid phase carrier, under the action of aceticanhydride pyridine, by Fmoc-Leu-OH,
Fmoc-Gly-OH, 16a and Fmoc-Gly-OH are consecutively connected on carrier, later with soft cutting reagent by glycopeptide together with protection
Base is cut from resin, obtains the glycopeptide 18a of full guard；Using 2- chlorine trityl resin as solid phase carrier, in aceticanhydride pyridine
Under the action of, Fmoc-Leu-OH, Fmoc-Gly-OH, 13b and Fmoc-Gly-OH are consecutively connected on carrier, later with soft
Glycopeptide is cut from resin together with protecting group with cutting reagent, obtains the glycopeptide 18b of full guard；
(12) compound 18a obtains glycopeptide crude product 1 under the action of palladium chloride, hydrogen and sodium ethoxide, and glycopeptide crude product 1 purifies
After obtain glycopeptide 1a；Compound 18b obtains glycopeptide crude product 2 under the action of palladium chloride, hydrogen and sodium ethoxide, and glycopeptide crude product 2 is pure
Glycopeptide 1b is obtained after change；
To realize above-mentioned third purpose, the technical solution adopted by the present invention is that:
Compound as described above containing glycosylation modified glycopeptide and carrier protein crosslinking in serine heptan.
As a preferred embodiment of the invention, the carrier protein is BSA, and the crosslinking is the carboxyl of glycopeptide
End is coupled with carrier protein BSA.
To realize above-mentioned 4th purpose, the technical solution adopted by the present invention is that:
Antiserum or antibody of the specific recognition as described above containing glycosylation modified glycopeptide in serine heptan.
To realize above-mentioned 5th purpose, the technical solution adopted by the present invention is that:
The purification process of antibody as described above, the purification process include the following steps:
A) ProteinA is purified: purification system: AKTApurifier10, filler: ProteinA Sepharose 4Fast
Flow；Equilibration buffer: 20mM PBS, 0.15M NaCl, pH7.2, elution buffer: 0.1M glycine-HCl, pH2.8,
Loading flow velocity: 1ml/min, process: balance pillar → loading → rebalancing → elution, sample 1M Tris-HCl tune after elution
PH is to neutrality, PBS dialysed overnight；
B) specificity purifying: purification system: AKTA purifier 10, filler: BSA Sepharose 4Fast Flow&
CL5 (SH)-BSA Sepharose 4Fast Flow, equilibration buffer: 20mM PBS, 0.15M NaCl, pH 7.2, elution are slow
Fliud flushing: 0.1M glycine-HCl, loading flow velocity: 1ml/min, process: balance pillar → loading → collection flows through → loading again
→ collection flows through, sample 1M Tris-HCl tune pH to neutrality after elution.
To realize above-mentioned 6th purpose, the technical solution adopted by the present invention is that:
The antibody as described above albumen and 6 D type serine heptose bases glycosylation modified in detection 6 L-type serine heptan
Change the application in the albumen of modification.
Antibody as described above is preparing the application in antibody class antibiotic.
Application of the antibody in small molecule antibiotic-screening as described above.
The invention has the advantages that:
1, the present invention provides first specifically glycosylated antibody in anti-serine heptan, preparation-obtained antibody to have
Production cost is low, specific good, high sensitivity, the high advantage of potency, in addition, the antibody can be used for finding to include more silk ammonia
Sour heptan glycosylation modified native protein, and the sieve comprising new antibiotic of both antibody drug and small-molecule drug
Choosing.
2, the preparation method of antibody provided by the invention is high-efficient, and required antibody can largely be prepared, and has good
Application prospect.
Detailed description of the invention
The Ser-O-Hep modification and its field planting in mouse that attached drawing 1 is the AIDA-I/TibA that BAHT is mediated.
Attached drawing 2 is the glycosyl transferase configuration reversal reaction that BAHT is mediated.
Attached drawing 3 is the synthesis of serine glycosylated polypeptide in heptan and the preparation of anti-glycosylated antibody in serine heptan.
Attached drawing 4 is the synthetic route chart of key intermediate 10a and 10b in embodiment 1.
Attached drawing 5 is the synthetic route chart of key intermediate 16a in embodiment 2.
Attached drawing 6 is the synthetic route chart of key intermediate 13b in embodiment 2.
Attached drawing 7 is the synthetic route chart of glycopeptide 1a and 1b in embodiment 3.
Attached drawing 8 is the result figure for the potency that indirect elisa method measures thick serum and purified antibodies in embodiment 6.
Attached drawing 9 is Anti-Ser in embodiment 7HepThe internal external serine for detecting various configuration is glycosylation modified heptan
Result figure.
Specific embodiment
The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair
It is bright rather than limit the scope of the invention.In addition, it should also be understood that, after having read the content of the invention recorded, art technology
Personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Fixed range.
The synthesis of 1 key intermediate 6 of embodiment
It is raw material with mannose 2, the mannose 3 of full acetyl group protection, mannose 3 is obtained under the action of acetic anhydride and iodine
Under the catalysis of boron trifluoride ether, end position and toluene-ω-thiol are coupled to obtain compound 4, and subsequent compound 4 is in sodium methoxide
After sloughing hydroxyacetyl protection under effect, 6 primary hydroxyl groups are selectively protected to obtain compound 5, compound 5 with TBDMS
With obtaining compound 6 after benzyl protection.In order to selectively expose 6 primary hydroxyl groups, selected under conditions of acetic acid and TBAF
Property slough primary hydroxyl group, TBDMS protects to obtain compound 7, and compound 7 is in the case where wearing the effect of this Martin's oxidant through endless total oxygen
To change, hydroxyl is oxidized to aldehyde and obtains compound 8, it is thin malt sugar 9 that subsequent compound 8, which is reacted by Wittig by convert aldehyde groups, one
The introducing of new carbon atom realizes conversion of the hexose to seven carbon sugar.Catalysis of the double bond contained in thin malt sugar 9 in AD-mix- α
6 D type compound 10a are obtained by Sharpless asymmetric oxidation with high specificity down；The double bond contained in thin malt sugar 9 is in AD-
Pass through Sharpless asymmetric oxidation under the catalysis of mix- β and obtain 6 L-type compound 10b with high specificity, specific route is shown in
Fig. 4.
The synthesis of embodiment 2 heptose amino acid 1 6a and 13b
Glycosylation based on three chlorimide esters synthesizes the heptose amino acid of 6 D types, the glycosylation based on sulphur glycosides
Synthesize the heptose amino acid of 6 L-types.
Specifically, the synthesis of heptose amino acid intermediate 16a is as follows: it is right that compound 10a sloughs end position under the action of NBS
After thiocresols, 1,6,7 three hydroxyls are subjected to acetyl group position under conditions of aceticanhydride pyridine and protect to obtain compound 12a,
Compound 12a selectively sloughs the acetyl group protection of end position hydroxyl under the catalysis of acetic acid hydrazine, compound 13a is obtained, in trichlorine
13a is converted into three chlorimide esters of activation under the action of acetonitrile and DBU, using three chlorimide esters of activation as saccharide donor, three
Under the action of fluorine methanesulfonates, (amino Fmoc is protected exposed hydroxyl, carboxyl tBu in the three chlorimide esters and serine of activation
Protection) it carries out glycosylation and obtains compound 15a, crucial sugar is obtained after the tBu protection that 15a passes through trifluoroacetic acid decarboxylize
Amino acid intermediate 16a, the Peptide systhesis (Fig. 5) for next step.
Heptose amino acid 1 3b synthesizes following (Fig. 6): first being protected 6,7 two hydroxyls of 10b by aceticanhydride pyridine
Shield obtains compound 11b, and 11b directly carries out the glycosylation of next step as sulphur glycosides, in urging for NIS and silver trifluoromethanesulfonate
Under change, it is anti-that glycosylation occurs for the hydroxyl (amino Fmoc is protected, carboxyl tBu protection) exposed in the sulphur glycosides and serine in 11b
Compound 12b should be obtained, tBu protecting group of 12b under the action of trifluoro formic acid on decarboxylize obtains in crucial glycoprotein amino acid
Mesosome 13b.
The synthesis of 3 serine glycosylated polypeptide 1a and 1b in heptan of embodiment
The synthetic route of haptens 1a, 1b are shown in Fig. 7.
First using 2- chlorine trityl resin as solid phase carrier, by Fmoc-Leu-OH, Fmoc-Gly-OH, 16aFmoc-
Gly-OH is consecutively connected on carrier, and terminal amino group uses acetyl group end socket under the action of aceticanhydride pyridine, using soft when cutting
Cutting reagent (acetic acid/TFE/DCM=1:1:8) cuts glycopeptide together with protecting group from resin, obtains the glycopeptide of full guard
18a；Using 2- chlorine trityl resin as solid phase carrier, by Fmoc-Leu-OH, Fmoc-Gly-OH, 13b, Fmoc-Gly-OH
It is consecutively connected on carrier, terminal amino group uses acetyl group end socket under the action of aceticanhydride pyridine, and soft cutting examination is utilized when cutting
Agent (acetic acid/TFE/DCM=1:1:8) cuts glycopeptide together with protecting group from resin, obtains the glycopeptide 18b of full guard.Chemical combination
Object 18a directly sloughs the benzyl protecting group on hydroxyl under the catalysis of palladium chloride and hydrogen without further purification, and reaction solution is filtered to remove
After PdCl2, the acetyl group protection of residual hydroxyl obtains glycopeptide crude product 1 after sloughing under conditions of sodium ethoxide, and glycopeptide crude product 1 is through anti-
Mutually preparation liquid phase obtains the heptose serine glycopeptide 1a of respective configuration after purification；
Compound 18b directly sloughs the benzyl protecting group on hydroxyl under the catalysis of palladium chloride and hydrogen without further purification, instead
Liquid is answered to be filtered to remove PdCl2Afterwards, the acetyl group protection of residual hydroxyl obtains glycopeptide crude product 2 after sloughing under conditions of sodium ethoxide,
The inverted preparation liquid phase of glycopeptide crude product 2 obtains the heptose serine glycopeptide 1b of respective configuration after purification, with heptose serine glycopeptide
1a and heptose serine glycopeptide 1b carries out the Antibody preparation of next step.
4 glycopeptide of embodiment is immune to be prepared with antiserum
The immune method of glycopeptide is specific as follows:
(1) it is mixed with antigen with Freund ' s adjuvant, after emulsification, subcutaneous and intradermal multi-point injection is immunized, antigen
Dosage is about 1mg/ times；
(2) interval time is 3 weeks；
(3) it is immunized again and cannots be used up full Freund ' s adjuvant, after emulsification, subcutaneous and intradermal multi-point injection is immunized, and is resisted
Former dosage is about 1g/ times；
(4) interval time is 3 weeks；
(5) it is immunized three times and cannots be used up full Freund ' s adjuvant, after emulsification, subcutaneous and intradermal multi-point injection is immunized, and is resisted
Former dosage is about 1mg/ times.
Antiserum the preparation method is as follows:
(1) 1 week after third time is immune, antiserum is collected in blood drawing；
(2) next day separates serum, -20 DEG C of preservations.
The purifying of 5 antibody of embodiment
Using the method antibody purification of affinity chromatography, be utilized respectively ProteinA filler and BSA Sepharose 4 and
The affine column purification three times of CL5 (SH)-BSA Sepharose 4.
The method of antibody purification is specific as follows:
(1) ProteinA is purified: purification system: AKTApurifier10 (Amersham Biosciences), filler:
ProteinA Sepharose 4FastFlow, equilibration buffer: 20mM PBS, 0.15M NaCl, pH7.2, elution buffer:
0.1M glycine-HCl, pH2.8, loading flow velocity: 1ml/min, process: balance pillar → loading → rebalancing → elution is washed
Sample adjusts pH to neutrality, PBS dialysed overnight with 1M Tris-HCl (pH=9) after de-；
(2) specificity purifying: purification system: AKTApurifier10 (Amersham Biosciences), filler: BSA
Sepharose4Fast Flow&CL5 (SH)-BSASepharose 4Fast Flow, equilibration buffer: 20mM PBS, 0.15M
NaCl, pH7.2, elution buffer: 0.1M glycine-HCl (pH=2.8), loading flow velocity: 1ml/min, process: balance columns
Son → loading → collection flows through → again loading → collection flow through, sample adjusts pH to neutrality with 1MTris-HCl (pH=9) after elution.
6 indirect elisa method of embodiment measures the potency of antiserum and purified antibodies
1 experimental method
(1) BSA or BSA-1 (5 μ g/mL) envelope antigen: are dissolved in coating buffer (0.032M Na2CO3, 0.068M
NaHCO3, pH9.6), 100 μ L samples are added in 96 orifice plates, and after 37 DEG C incubate 1 hour, 4 DEG C of refrigerators are placed 16~18 hours；
(2) it washs: using up liquid in plate hole, fill it up with cleaning solution, it is quiet to put three minutes, repeatedly for three times, finally reaction plate is fallen
It sets on blotting paper, flows to end cleaning solution in hole；
(3) add 200 μ L of confining liquid, 37 DEG C are placed 1 hour；
(4) washing is the same as 2；
(5) it plus is detected serum: tested serum being diluted with dilution, every 200 μ L of hole, while making dilution control, 37 DEG C
It places 2 hours；
(6) washing is the same as 2；
(7) plus horseradish peroxidase goat anti-rabbit igg, every 200 μ L of hole place 37 DEG C 1 hour；
(8) washing is the same as 2；
(9) add substrate: o-phenylenediamine solution adds 200ml, room temperature dark place 10-15 minutes；
(10) add terminate liquid: every 50 μ L of hole；
(11) result is observed: with enzyme-linked immunosorbent assay instrument record 490nm reading.
2 experimental results
Testing result is as shown in Figure 8.
The specificity of 7 immunoblot experiment of embodiment confirmation antibody
1 experimental method
(1) PAGE gel is configured according to standard method, after adjusting sample protein concentration, and isometric 2* loading buffer
It boils 10 minutes, is centrifuged 1 minute (3000rpm/min) after albuminous degeneration, 200V constant pressure electrophoresis 20 divides in 95 DEG C of water after liquid mixing
Clock after stopping electrophoresis, using 300mA120 minutes transferring films of electric transferring film method constant current to pvdf membrane, is closed 1 hour with 3%BSA room temperature,
In washing film three times with TBST on shaking table, it is added in incubation bags after the antibody that embodiment 5 obtains is diluted to required concentration with TBST,
4 DEG C of overnight incubations are used after the anti-rabbit secondary antibody of horseradish peroxidase (HRP) label is incubated at room temperature 2 hours after TBST washes film three times
GelDocTM(BioRad) it carries out immune detection and quantifies；
(2) DAEC, ETEC, C.Rodentium, bulkholderia cepasea, Shigella shigae, Salmonella are cultivated respectively
Cell pyrolysis liquid is carried out immunoblotting assay by bacterium and Hong Kong Public Library.
2 experimental results
(1) as shown in figure 9, Anti-SerHepIt is able to detect the Ser-O-Hep modification recognized in bacterium, Anti-
SerHep1aAnd Anti-SerHep1bTo Ser-O-D, D-Hep and the Ser-O-L of 6 various configurations of detection, D-Hep modification has structure
Type specificity.AAH includes two kinds of heptose of 6 L-types and D type to the Ser-O-Hep modified ligand of AIDA-1 in DAEC, and ETEC
In TibC can only be with D when shifting heptose, D-Hep coincide as ligand with this, modifies glycopeptide with Ser-O-D, D-Hep and makees
For Anti-Ser obtained by haptensHep1aIt can identify the glycosylation modified albumen in DAEC and ETEC bacterium, and with Ser-O-
L, D-Hep modify glycopeptide as the obtained Anti-Ser of haptensHep1bIt can only identify the glycosylation modified egg in DAEC bacterium
It is white, and cannot identify the albumen in ETEC.
(2) testing result that Ser-O-Hep is modified in cell lysate is as shown in Figure 9.
The present invention provides first specifically glycosylated antibody in anti-serine heptan, preparation-obtained antibody has life
Produce at low cost, specific good, high sensitivity, the high advantage of potency, in addition, the antibody can be used for finding to include more serine
Heptan glycosylation modified native protein, and the sieve comprising new antibiotic of both antibody drug and small-molecule drug
Choosing.The preparation method of antibody provided by the invention is high-efficient, and required antibody can largely be prepared, before having application well
Scape.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, without departing from the principle of the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (9)
1. a kind of glycopeptide glycosylation modified containing serine heptan, which is characterized in that the structure of the glycopeptide is as follows:
2. the synthetic method of glycopeptide as described in claim 1, which is characterized in that the synthetic method of the glycopeptide is as follows:
(1) compound 2 obtains compound 3 under the action of acetic anhydride and iodine；
(2) under the catalysis of boron trifluoride ether, end position and toluene-ω-thiol are coupled to obtain compound 4 compound 3；
(3) compound 4 successively reacts to obtain with sodium methoxide and TBDMSSCI compound 5, compound 5 BnBr, NaH, TBAI,
Compound 6 is obtained under the action of DMF；
(4) compound 6 obtains compound 7 under the action of acetic acid and TBAF；
(5) compound 7 obtains compound 8 in the case where wearing the effect of this Martin's oxidant；
(6) compound 8 reacts to obtain thin malt sugar 9 by Wittig；
(7) thin malt sugar 9 obtains compound 10a under the catalysis of AD-mix- α, and thin malt sugar 9 obtains compound under the catalysis of AD-mix- β
10b；
(8) compound 10a obtains compound 12a under the action of NB and aceticanhydride pyridine；Effect of the compound 10b in aceticanhydride pyridine
Under obtain compound 11b；
(9) compound 12a obtains compound 14a under the action of acetic acid hydrazine, Tritox and DBU, that is, three chlorimides activated
Ester；Compound 11b obtains compound 12b under the action of NIS and silver trifluoromethanesulfonate；
(10) compound 14a obtains compound 15a under the action of triflate；Work of the compound 15a in trifluoroacetic acid
Compound 16a is obtained under；Compound 12b obtains compound 13b under the action of trifluoro formic acid；
(11) using 2- chlorine trityl resin as solid phase carrier, under the action of aceticanhydride pyridine, by Fmoc-Leu-OH, Fmoc-
Gly-OH, 16a and Fmoc-Gly-OH are consecutively connected on carrier, later with soft cutting reagent by glycopeptide together with protecting group from
It is cut on resin, obtains the glycopeptide 18a of full guard；Using 2- chlorine trityl resin as solid phase carrier, in the work of aceticanhydride pyridine
Under, Fmoc-Leu-OH, Fmoc-Gly-OH, 13b and Fmoc-Gly-OH are consecutively connected on carrier, cut later with soft
It cuts reagent and cuts glycopeptide from resin together with protecting group, obtain the glycopeptide 18b of full guard；
(12) compound 18a obtains glycopeptide crude product 1 under the action of palladium chloride, hydrogen and sodium ethoxide, and glycopeptide crude product 1 obtains after purification
To glycopeptide 1a；Compound 18b obtains glycopeptide crude product 2 under the action of palladium chloride, hydrogen and sodium ethoxide, and glycopeptide crude product 2 is after purification
Obtain glycopeptide 1b；
3. the compound containing glycosylation modified glycopeptide and carrier protein crosslinking in serine heptan as described in claim 1.
4. compound according to claim 3, which is characterized in that the carrier protein is BSA, and the crosslinking is glycopeptide
C-terminus and carrier protein BSA are coupled.
5. antiserum or antibody containing glycosylation modified glycopeptide in serine heptan described in specific recognition claim 1.
6. the purification process of antibody as claimed in claim 5, which is characterized in that the purification process includes the following steps:
A) ProteinA is purified: purification system: AKTA purifier 10, filler: ProteinA Sepharose 4Fast
Flow；Equilibration buffer: 20mM PBS, 0.15M NaCl, pH7.2, elution buffer: 0.1M glycine-HCl, pH2.8,
Loading flow velocity: 1ml/min, process: balance pillar → loading → rebalancing → elution, sample 1M Tris-HCl tune after elution
PH is to neutrality, PBS dialysed overnight；
B) specificity purifying: purification system: AKTA purifier 10, filler: BSA Sepharose 4Fast Flow&CL5
(SH)-BSA Sepharose 4Fast Flow, equilibration buffer: 20mM PBS, 0.15M NaCl, pH 7.2, elution buffer
Liquid: 0.1M glycine-HCl, loading flow velocity: 1ml/min, process: balance pillar → loading → collection flow through → again loading →
Collection flows through, sample 1M Tris-HCl tune pH to neutrality after elution.
7. antibody described in claim 5 albumen and 6 D type serine heptose glycosylation modified in detection 6 L-type serine heptan
Application in the albumen of baseization modification.
8. antibody described in claim 5 is preparing the application in antibody class antibiotic.
9. application of the antibody described in claim 5 in small molecule antibiotic-screening.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110684188A (en) * | 2019-10-31 | 2020-01-14 | 北京纳百生物科技有限公司 | Nonylphenol polyoxyethylene ether hapten and holoantigen as well as preparation method and application thereof |
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| CN105524145A (en) * | 2015-12-07 | 2016-04-27 | 中国人民解放军第二军医大学 | Polyclonal antibody for efficient recognition of arginine rhamanopyranosylation of proteins and preparation method thereof |
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| CN105524145A (en) * | 2015-12-07 | 2016-04-27 | 中国人民解放军第二军医大学 | Polyclonal antibody for efficient recognition of arginine rhamanopyranosylation of proteins and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684188A (en) * | 2019-10-31 | 2020-01-14 | 北京纳百生物科技有限公司 | Nonylphenol polyoxyethylene ether hapten and holoantigen as well as preparation method and application thereof |
| CN110684188B (en) * | 2019-10-31 | 2022-02-22 | 北京纳百生物科技有限公司 | Nonylphenol polyoxyethylene ether hapten and holoantigen as well as preparation method and application thereof |
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