CN106645743A - Technology for quickly constructing carbohydrate chip through neoglycoprotein - Google Patents
Technology for quickly constructing carbohydrate chip through neoglycoprotein Download PDFInfo
- Publication number
- CN106645743A CN106645743A CN201610905225.0A CN201610905225A CN106645743A CN 106645743 A CN106645743 A CN 106645743A CN 201610905225 A CN201610905225 A CN 201610905225A CN 106645743 A CN106645743 A CN 106645743A
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- China
- Prior art keywords
- neoglycoproteins
- technology
- carbohydrate
- carbohydrate chip
- sugar
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
- G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
Abstract
The invention discloses a technology for quickly constructing a carbohydrate chip through neoglycoprotein. The technology includes the steps of: 1) under a proper pH value, connecting an aminated carbohydrate derivative and bovine serum albumin through diethyl squarate to prepare a corresponding neoglycoprotein; and 2) dissolving the neoglycoprotein in a phosphate buffer solution, so that the neoglycoprotein is adsorbed on a surface of a material directly to form the carbohydrate chip within 1 h. Through control on addition ratio of the carbohydrate to the protein, neoglycoproteins, of which the outer sides are connected to carbohydrate chains in different numbers, can be produced. The technology has very simple process and employs easy-to-obtain raw materials, is low in cost and is suitable for large-scale production.
Description
Technical field
The present invention relates to a kind of technology of utilization Neoglycoproteins rapid build carbohydrate chip, belongs to technical field of biological.
Background technology
Saccharide compound is that all living things body sustains life the main source of energy needed for activity.It is not only nutrients
Matter, but also with special physiologically active.For example：Heparin in liver has blood coagulation resisting function；Glycophorin is lived with immunity
Property is relevant；Contain saccharide compound in the constituent of nucleic acid --- ribose and deoxyribose；Cell recognition also with cell membrane table
The glycoprotein in face is relevant.Therefore, saccharide compound is studied for medical science and life science, with extremely important meaning
Justice.
Carbohydrate chip originates from the research of DNA or protein-chip, and technology maturation is the sugared and various biological substance of present analysis
The effective tool of the interphase interaction of (such as agglutinin, bacterium, virus, antibody).Carbohydrate chip is typically first solid carbohydrate probe
Host material (metal, glass, nitrocellulose or polystyrene etc.) surface is scheduled on, the biological substance of fluorogen then will be marked with
Injection surface, if there is corresponding bonding behavior, fluorogen is also carried with reference to after by part, can be sent under ultraviolet lighting
Fluorescence.So, traditional carbohydrate chip is required for being marked sample in detection, and labeling process is possible to that combination can be changed
It is selective；In addition, the removal of fluorogen is also a problem for needing to consider.
Based on the biology sensor of surface plasma body resonant vibration (SPR) phenomenon, due to its have without the need for mark, to sample without
The many merits such as damage, dynamic realtime detection, the focus of the research in 30 years that becomes history is widely used in various biological points
The real-time detection in situ of son and the research of biological substance interphase interaction.Applied in the research of saccharide compound, had
The advantage that traditional carbohydrate chip cannot replace.SPR carbohydrate chips are also required to for carbohydrate probe to be fixed on sensor surface, allow testing sample
Flows through sensor surface, if there is material to have an effect with the probe on surface in sample, will cause the phase of detector signal
Should change.
So whether traditional carbohydrate chip or SPR carbohydrate chips, sugared probe is in efficient, the quick and stable fixation of material surface
It is emphasis.Although the larger polysaccharide probe of molecular weight can easily be adsorbed by hydrophobic interaction or Van der Waals force passing
Sensor surfaces, but for monose or this kind of small molecule carbohydrate of disaccharides, it is impossible to sensor surface is directly anchored to, is needed in sugar
- SH ,-NH in end group modification2Deng functional group, sensor surface is fixed in the form of covalently or non-covalently；Can also synthesize
Various saccharide complexes (such as dendrimer, DNA, lipid).And in order to preferably simulate biological internal sugared structure, sometimes
We also want to control the quantity of sugared probe and distribution on surface.By ATRP (ATRP),
Sensor surface forms polymer brush and can realize this purpose.But due to preparation process it is relatively complicated, and synthetic polymer brush
Process need suitable monomer material and specific catalyst, relatively costly, application has certain limitation, it is difficult to adapt to large quantities of
Amount production.
Here, a kind of it is proposed that new method of quick fixed monose probe.Using bovine serum albumin (BSA) in various materials
The material good adsorption capacity in (gold, glass, polystyrene etc.) surface, is directly adsorbed in material list by the BSA for being connected with sugared probe
Face, forms carbohydrate chip.Because protein generally has three-dimensional structure, and monose probe is and exposed ammonia on the outside of BSA albumen
Base connects, and institute in this way can also be in a certain degree control sugar quantity and distribution of the probe on BSA.Due to the fixation side
Method process is simple, and raw material is easy to get and with low cost, and usability is strong, is adapted to produce in enormous quantities, and application prospect is boundless.
The content of the invention
It is an object of the invention to provide a kind of new simple, quick, the monose chip technology of preparing that applicability is wide.
The know-why of the present invention
Under certain pH conditions, by the monosaccharide derivatives and bovine serum albumin of the side's of being terminated with diethyl phthalate by different proportion
Reaction, can obtain the Neoglycoproteins that outside is connected to different sugar chain numbers.This Neoglycoproteins can directly be adsorbed in various materials
Surface, it is not necessary to any material surface pretreatment.
To realize above-mentioned purpose, the technical solution used in the present invention is：
(1) preparation of Neoglycoproteins
Amidized sugar derivatives is dissolved in the phosphate buffer of pH=7, is then added in solution a certain amount of
Square diethyl phthalate, is slowly stirred at a room temperature 14h.After reaction terminates, it is evaporated in vacuo solvent, using the method for rapid column chromatography
Purifying obtains the sugar derivatives of the side's of being terminated with diethyl phthalate.A certain amount of bovine serum albumin (BSA) is taken afterwards is dissolved in pH=9's
Borate buffer solution, sugar derivatives obtained in the previous step is added in solution, is slowly stirred at a room temperature 18h.After reaction terminates,
With sephadex (Sephadex G-75) purified product, Neoglycoproteins is obtained after freeze-drying, and (i.e. BSA- sugar is combined
Body).By the addition mass ratio for controlling bovine serum albumin and sugar derivatives, the plan sugar that outside is connected to different sugar chain numbers can be obtained
Albumen.
(2) in the absorption of material surface
The Neoglycoproteins for preparing is dissolved in the phosphate buffer of pH=7, then material surface is immersed directly in
In the solution, you can obtain being adsorbed with the material surface of Neoglycoproteins.In the environment of circulating, the time of general 1h intends sugar
The absorption of albumen can reach saturation.
The invention has the beneficial effects as follows：
(1) present invention can obtain outside and be connected to varying number monose probe by control sugar and the additional proportion of albumen
Neoglycoproteins.
(2) sugared probe prepared by the present invention is in distributed in three dimensions on bovine serum albumin, and can directly adsorb in various materials
Material surface.
(3) preparation process of the invention is very simple, and needed raw material is easy to get, and preparation cost is low, is adapted to produce in enormous quantities.
Description of the drawings
Fig. 1 is the principle signal that utilization side of the invention diethyl phthalate connection monosaccharide derivatives and protein prepare Neoglycoproteins
Figure；
Fig. 2 is the schematic flow sheet that the present invention is rebuild using Neoglycoproteins rapid build carbohydrate chip and carbohydrate chip；
Fig. 3 is the SPR kinetic curves that the present invention is rebuild using Neoglycoproteins rapid build carbohydrate chip and carbohydrate chip.
Specific embodiment
For a better understanding of the present invention, present disclosure is further elucidated with reference to embodiment, but the present invention
Content is not limited solely to the following examples.
Embodiment 1：
The amidized mannose derivative for taking 220mg is dissolved in the phosphate buffer of 10ml pH=7, then to molten
A number of side's diethyl phthalate is added in liquid, 14h is slowly stirred at a room temperature.After reaction terminates, it is evaporated in vacuo solvent, makes
Purify the mannose derivative for obtaining the side's of being terminated with diethyl phthalate with the method for rapid column chromatography.The ox blood of 54.89mg is taken again
Albumin is dissolved in the borate buffer solution of 10ml pH=9, and the side's of being terminated with diethyl phthalate of 10mg is then added in solution
Mannose derivative, be slowly stirred at a room temperature 18h.After reaction terminates, with sephadex (Sephadex G-75)
Purified product, obtains prepared mannose-bovine serum albumin after freeze-drying.According to substance assistant laser desorpted ionized flight
The test result of the molecular weight of albumen that time mass spectrum is obtained is inferred, sweet in mannose obtained above-bovine serum albumin structure
The sugared number of probes of dew is 11.
Take mannose obtained above-bovine serum albumin 1.4mg to be dissolved in the phosphate buffer of 1.4ml pH=7, lead to
The conveying of peristaltic pump is crossed, with the flow velocity of 700 μ L/min in golden film surface circulation flowing 1h, Neoglycoproteins can be directly adsorbed to gold
Surface, formation can be used for the carbohydrate chip of SPR detections.
Then evaluation is made to the performance of SPR carbohydrate chips using ConA (ConA).Fig. 2, Fig. 3 are respectively carbohydrate chip
Structure and reconstruction schematic flow sheet and SPR kinetic curves.The SPR kinetic curves result of Fig. 3 is it can be shown that the method
The SPR carbohydrate chips of preparation can be used for the research that sugar-agglutinin interacts, and can intuitively reflect whole association and dissociation
Process.And only chip surface just can need to be effectively rebuild using the sodium hydroxide solution of low concentration, chip can repeat to make
With.
Claims (5)
1. a kind of technology of utilization Neoglycoproteins rapid build carbohydrate chip, it is characterised in that preparation method is carried out according to the following steps：
(1) preparation of Neoglycoproteins
Amidized sugar derivatives is dissolved in the phosphate buffer of pH=7, a certain amount of side's acid is then added in solution
Diethylester, is slowly stirred at a room temperature 14h.After reaction terminates, it is evaporated in vacuo solvent, is purified using the method for rapid column chromatography
Obtain the sugar derivatives of the side's of being terminated with diethyl phthalate.The boric acid that a certain amount of bovine serum albumin (BSA) is dissolved in pH=9 is taken afterwards
Salt buffer, sugar derivatives obtained in the previous step is added in solution, is slowly stirred at a room temperature 18h.After reaction terminates, with friendship
Connection sephadex (Sephadex G-75) purified product, obtains Neoglycoproteins (i.e. BSA- sugar combinations) after freeze-drying.It is logical
The addition mass ratio of control bovine serum albumin and sugar derivatives is crossed, the Neoglycoproteins that outside is connected to different sugar chain numbers can be obtained.
(2) in the absorption of material surface
The Neoglycoproteins for preparing is dissolved in the phosphate buffer of pH=7, then material surface this is immersed directly in molten
In liquid, you can obtain being adsorbed with the material surface of Neoglycoproteins.In the environment of circulating, the time Neoglycoproteins of general 1h
Absorption can reach saturation, form the carbohydrate chip containing unskilled sugared probe.
2. according to claim 1 using the technology of Neoglycoproteins rapid build carbohydrate chip, it is characterised in that：Albumen used is
Bovine serum albumin.
3. according to claim 1 using the technology of Neoglycoproteins rapid build carbohydrate chip, it is characterised in that：Connection sugar used
The material of derivative and albumen is square diethyl phthalate.
4. according to claim 1 using the technology of Neoglycoproteins rapid build carbohydrate chip, it is characterised in that：By reacting
Sugar derivatives is different with the additional proportion of bovine serum albumin in journey, obtains the Neoglycoproteins of different sugar chain numbers.
5. according to claim 1 using the technology of Neoglycoproteins rapid build carbohydrate chip, it is characterised in that：By the plan for obtaining
Glycoprotein is dissolved in phosphate buffer, you can directly adsorb in material surface, quickly prepares carbohydrate chip.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610905225.0A CN106645743A (en) | 2016-10-17 | 2016-10-17 | Technology for quickly constructing carbohydrate chip through neoglycoprotein |
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| CN201610905225.0A CN106645743A (en) | 2016-10-17 | 2016-10-17 | Technology for quickly constructing carbohydrate chip through neoglycoprotein |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109107620A (en) * | 2017-06-23 | 2019-01-01 | 天津科技大学 | A method of quickly preparing carbohydrate chip |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050221337A1 (en) * | 2003-10-02 | 2005-10-06 | Massachusetts Institute Of Technology | Microarrays and microspheres comprising oligosaccharides, complex carbohydrates or glycoproteins |
| CN104977417A (en) * | 2015-05-15 | 2015-10-14 | 西北大学 | Glycopeptide chip and preparation method thereof |
-
2016
- 2016-10-17 CN CN201610905225.0A patent/CN106645743A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050221337A1 (en) * | 2003-10-02 | 2005-10-06 | Massachusetts Institute Of Technology | Microarrays and microspheres comprising oligosaccharides, complex carbohydrates or glycoproteins |
| CN104977417A (en) * | 2015-05-15 | 2015-10-14 | 西北大学 | Glycopeptide chip and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| OYINDASOLA OYELARAN等: "（Microarrays with varying Carbohydrate Density Reveal Distinct Subpopulations of Serum Antibodies", 《JOURNAL OF PROTEOME》 * |
| TIETZE等: "Conjugation of p-Aminophenyl Glycosides with Squaric Acid Diester to a Carrier Protein and the Use of Neoglycoprotein in the Histochemical Detection of Lectins1", 《BIOCONJUGATE CHEM.》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109107620A (en) * | 2017-06-23 | 2019-01-01 | 天津科技大学 | A method of quickly preparing carbohydrate chip |
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Application publication date: 20170510 |