CN103408696B - The method of Angiotensin synthesis of receptor and prepared Angiotensin synthesis of receptor is prepared based on engram technology - Google Patents
The method of Angiotensin synthesis of receptor and prepared Angiotensin synthesis of receptor is prepared based on engram technology Download PDFInfo
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Abstract
The present invention discloses a kind of small peptide and derivative thereof as the application of template molecule and the method preparing Angiotensin synthesis of receptor based on engram technology.Described template molecule comprises the one in the nitrogen end of the nitrogen end of angiotensinⅡ or any one section of peptide bond of carbon teminal and derivative or angiotensinⅠ or any one section of peptide bond of carbon teminal and derivative thereof.Described application is for the preparation of Angiotensin synthesis of receptor with above-mentioned template molecule trace.Angiotensin receptor is mixed by a certain percentage by template molecule, function monomer, linking agent and tensio-active agent etc., adds the preparation of initiator post polymerization.This receptor good biocompatibility, can highly selective catch in and angiotensinⅡ and angiotensinⅠ, block angiotensinⅡ and angiotensin receptor (AT
1) and angiotensinⅠ and Zinc metallopeptidase Zace1 (ACE) act on, thus reach the object controlling blood pressure.This receptor also can be used for angiotensinⅠ or II separation and concentration and mensuration.
Description
Technical field
The present invention relates to biomedicine technical field and separation and concentration and technical field of analysis and detection, be specifically related to a kind of small peptide and derivative thereof as the application of template molecule and the method preparing Angiotensin synthesis of receptor based on engram technology.
Background technology
As the primary bioactivity peptide of renin-angiotensin-aldosterone system (RAS), angiotensinⅡ in vivo too expression can cause serious body illness.AngiotensinⅡ can stimulate adrenal cortex glomerular zone cell to synthesize and release aldosterone, and aldosterone acts on uriniferous tubules, plays and protects sodium, water conservation, the effect of row's potassium, cause hypervolemia, vasoconstriction, raising blood pressure; And can I type of vasoactive Angiotensin Ⅱ or II receptor thus raising blood pressure; AngiotensinⅡ also by acting on cell Na-Ca passage, making Ca ionic concn increase, causing vasoconstriction, thus elevation of blood pressure.And feritin energy catalysis blood plasma Angiotensin-Converting changes angiotensinⅠ into, the latter, under the effect of Zinc metallopeptidase Zace1 (ACE), generates angiotensinⅡ.Therefore, blocking-up angiotensinⅠ is converted to angiotensinⅡ or blocks angiotensinⅡ and is attached to angiotensin receptor (AT
1) be the most effectively treat hypertensive method both at home and abroad at present.
Treat hypertensive method for renin-angiotensin-aldosterone system both at home and abroad usually to have: 1.ACE inhibitor, suppress the Zinc metallopeptidase Zace1 (ACE) of RAS system, stop angiotensinⅠ to be converted to angiotensinⅡ, and can Aldosterone Secretion be suppressed, reduce water-sodium retention; 2. the antihypertensive drug of Angiotensin Ⅱ receptor antagonist (AIIA) class, blocks angiotensin receptor (AT
1), avoid angiotensinⅡ to be attached on acceptor, finally reach and prevent vasoconstrictive object.Such as, but these treatments exist serious side effects, birth defects, cough and dizziness, extreme side effect can cause the problems such as the minimizing of renal failure and white cell.
The present invention is intended to design and prepares a kind of trace artificial receptors, makes this artificial receptors Selective recognition catch in body too much angiotensinⅡ (or angiotensinⅠ), just itself and AT capable of blocking
1(with Zinc metallopeptidase Zace1 (ACE)) acts on, thus reaches the object controlling blood pressure further.
Molecular imprinting is a kind of bionics techniques of novel artificial acceptor.Be there is by polyreaction synthesis the polymeric acceptor in specific identification site and hole under template molecule, function monomer and linking agent Coexistence Situation.It is identify organic molecule that current molecular imprinting is the most successfully applied, very limited for identifying the examples of many successful of catching polypeptide and protein, and its reason is structure of biological macromolecule complexity and Thermodynamics etc.; Another major cause is that peptide and protein is expensive, is usually difficult to obtain q.s for the preparation of molecularly imprinted polymer.
At present, about synthesis, there is the molecular imprinting nanogel similar to natural enzyme or antibody size (<100nm) and have been reported, but be that the trace nanogel acceptor of part has not yet to see report with Angiotensin.Molecular imprinting nanogel can synthesize in the water surrounding of gentleness, better can retain the three-dimensional conformation of biomolecules, have very high biocompatibility, and specific surface area is large, and action site embedding is little, and carrying capacity is high, and stability is high.Refine Angiotensin Target Recognition determining area as imprinted templates, the molecular imprinting nanogel of preparation, has more the advantage on kinetics of adsorption and thermodynamics than traditional imprinted material in theory, and the cost less of template.
Summary of the invention
The object of the present invention is to provide a kind of small peptide and derivative thereof as the application of template molecule and the method preparing Angiotensin synthesis of receptor based on engram technology, propose to utilize the angiotensinⅠ or II in this receptor neutralization (identify and combine) body simultaneously, realize the new way controlling blood pressure.This receptor good biocompatibility, catching of highly selective can neutralize and separation and concentration angiotensinⅠ or II, has the application of potential control blood pressure.
A kind of small peptide and derivative thereof are as the application of template molecule, namely for preparing Angiotensin synthesis of receptor with engram technology, described template molecule comprises the one in the nitrogen end of the nitrogen end of angiotensinⅡ or any one section of peptide bond of carbon teminal and derivative or angiotensinⅠ or any one section of peptide bond of carbon teminal and derivative thereof.
As preferably, prepare the method for Angiotensin synthesis of receptor based on engram technology, comprise following concrete steps
(1) utilize and comprise one in the nitrogen end of the nitrogen end of angiotensinⅡ or any one section of peptide bond of carbon teminal and derivative or angiotensinⅠ or any one section of peptide bond of carbon teminal and derivative thereof as template molecule;
(2) by template molecule, function monomer A, gel skeleton monomer is dissolved in distilled water and obtains solution 1; Function monomer B is dissolved in dehydrated alcohol and obtains solution 2; Be mixed into by solution 2 in above-mentioned solution 1, vibration preact, obtains solution 3;
(3) crosslinkers monomers and Surfactant SDS are added in the solution 3 of step (2), logical nitrogen 15 ~ 60min, adds initiator, sealing, and nitrogen atmosphere reacts, and obtains molecular imprinting nanogel;
(4) purifying of imprinted polymer: proceed in dialysis tubing by the molecular imprinting nanogel obtained in step (3), dialyse in distilled water, dialysis postlyophilization, obtains nanogel acceptor and Angiotensin synthesis of receptor.
In above-mentioned preparation method, the C that template molecule described in step (1) comprises following (1) angiotensinⅡ holds tripeptide derivative template (Ac-His-Pro-Phe), (2) C of angiotensinⅠ holds tripeptide derivative template (Ac-Phe-His-Leu), and the N of (3) angiotensinⅡ holds tripeptide derivative template (Asp-Arg-Val-NHC
2h
5), the N of (4) angiotensinⅡ holds a peptide derivant template (Asp-NHC
2h
5), the C of (5) angiotensinⅡ holds dipeptidase derivant template (Ac-Pro-Phe), and the N of (6) angiotensinⅡ holds pentapeptide derivative template (Asp-Arg-Val-Tyr-Ile-NHC
2h
5) in more than one.
In above-mentioned preparation method, described in step (2), function monomer A is vinylformic acid, and described gel skeleton monomer is NIPA, and described function monomer B is N tert butyl acrylamide.
In above-mentioned preparation method, in step (2), the molar ratio of template molecule, function monomer A, gel skeleton monomer and distilled water is 1:(7 ~ 9): (11 ~ 18): 2780.
In above-mentioned preparation method, template molecule described in step (2), the mol ratio of function monomer A, function monomer B tri-kinds of materials is: Mo plate Fen ︰ function monomer A ︰ function monomer B=1 ︰ (7 ~ 9) ︰ (1.6 ~ 9); In described solution 3, three kinds of total monomer are 5.9 ~ 9.8mM, and wherein each monomer concentration is than being function monomer A ︰ function monomer B ︰ backbone monomer=1:(0.17 ~ 1) ︰ (1.3 ~ 2.1).
In above-mentioned preparation method, dehydrated alcohol volume described described in step (2) is 2% ~ 5% of volume in solution 1; Adopt the purity of all ingredients be analytical pure and more than, distilled water is secondary redistilled water; The time of vibration preact is 4-6 hour.
In above-mentioned preparation method, in step (2) and (3), function monomer A, function monomer B, backbone monomer and cross-linking monomer totally four kinds of monomers total concn in solution 3 are 6 ~ 10mM, wherein crosslinkers monomers concentration is total concn 2% ~ 4%, function monomer B concentration is total concn 5% ~ 30%, and function monomer A concentration is total concn 30%; The temperature of described nitrogen atmosphere reaction is 23 ~ 25 DEG C, and the reaction times is 24 ~ 36 hours.
In above-mentioned preparation method, described in step (3), crosslinkers monomers is N, N-methylene diacrylamide, and its concentration is 2% ~ 4% of three kinds of total monomer in step (2); Described initiator is that ammonium persulphate and N, N, N, N-tetramethyl-diethylamine combine, both add after concentration in solution 3 be respectively 0.6 ~ 1.0mg/mL, 0.24 ~ 0.48mg/mL; Concentration after described Surfactant SDS adds in solution 3 is 0.2 ~ 0.4mg/mL.
In above-mentioned preparation method, described in step (4), dialysis tubing specification is MWCO10000, and dialysis tubing molecular weight cut-off is 10000; Described dialysis time at least four days, every day at least changes twice water; Described sublimation drying is 2-3 days.
The present invention is with angiotensinⅡ and derivative, angiotensinⅠ and derivative thereof, the nitrogen end of angiotensinⅡ or the nitrogen end of any one section of peptide bond of carbon teminal and derivative or angiotensinⅠ or any one section of peptide bond of carbon teminal and derivative thereof, below a kind of Angiotensin nanogel acceptor prepared by template molecule wherein has high imprinting effect, all has high adsorptive capacity and selectivity to angiotensinⅠ and II.
The invention has the advantages that: process is simple, the three-dimensional conformation of polypeptide better can be retained in preparation process, template cost is low, prepared nanogel acceptor has avidity to target polypeptides and selectivity is high, specific surface area is large, action site embedding is few, charge capacity and stability high, and there is the advantages such as very high biocompatibility.
Accompanying drawing explanation
Fig. 1 is that the low multiple TEM of Angiotensin synthesis of receptor that embodiment 1 is synthesized schemes.
Fig. 2 is that the Angiotensin synthesis of receptor height multiple TEM that embodiment 1 is synthesized schemes.
Fig. 3 is the blood pressure figure of the 1st group of anesthetized rat intravenous injection angiotensinⅡ in example 2.
Fig. 4 is the blood pressure figure of the 2nd group of anesthetized rat intravenous injection angiotensinⅡ mixing acceptor in example 2.
Embodiment
Following embodiment further illustrates of the present invention, instead of limit the scope of the invention.
embodiment 1
The N of 0.0045g template molecule and angiotensinⅡ is held tripeptide derivative template (Asp-Arg-Val-NHC
2h
5), 0.098mmol vinylformic acid, 0.156mmolN-N-isopropylacrylamide is dissolved in 50mL distilled water and obtains solution 1.0.065mmolN-N-tert-butyl acrylamide is dissolved in 1mL dehydrated alcohol, obtains solution 2, is mixed into by solution 2 in above-mentioned solution 1, and vibration preact, after 4 hours, obtains solution 3.1mgN, N-methylene diacrylamide and 10mg sodium lauryl sulphate are added in above-mentioned solution 3, logical nitrogen 30min, adds initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, N, N, N-tetramethyl-diethylamine, and sealing, ambient temperature under nitrogen atmosphere reacts 24 hours.Proceeded to by reaction solution in dialysis tubing (MWCO10000), in a large amount of distilled water, carry out dialysis four days, every day at least changes twice water, to remove unreacted monomer and template molecule.Solution after dialysis is carried out lyophilize 3 days, obtains nanogel acceptor and Angiotensin synthesis of receptor.
Fig. 1 and Fig. 2 is the TEM figure of the Angiotensin synthesis of receptor that embodiment 1 is synthesized respectively.As can be seen from the figure, this Angiotensin synthesis of receptor particle diameter narrow distribution, median size is about 25nm.
The Angiotensin synthesis of receptor obtained with aforesaid method carries out selective adsorption experiment, and experimentation is: the concentration of 2mL accurate formulation is 1.2 × 10
-4the angiotensinⅠ of M, angiotensinⅡ and [Sar1, Val5, Ala8] the Angiotensin synthesis of receptor that obtains of the aqueous solution (SDS concentration is 1mg/ml) of-angiotensinⅡ and 5mg aforesaid method puts into the centrifuge tube of 5mL, at 25 DEG C shaking table vibration about 12 hours after the centrifugal 30min of 16000rpm, get the supernatant liquor membrane filtration of 0.45 μm, and detect strength of solution with HPLC.Experimental result shows that this Angiotensin synthesis of receptor optionally can adsorb angiotensinⅠ and II in three peptide species mixing solutionss simultaneously, it is to angiotensinⅠ, angiotensinⅡ and [Sar1, Val5, Ala8] adsorptive capacity of-angiotensinⅡ is respectively 15.4 μm of ol/g, 14.2 μm of ol/g, 1.2 μm of ol/g.
embodiment 2
The N of 0.0045g template molecule and angiotensinⅡ is held tripeptide derivative template (Asp-Arg-Val-NHC
2h
5), 0.098mmol vinylformic acid, 0.156mmolN-N-isopropylacrylamide is dissolved in 50mL distilled water and obtains solution 1.0.065mmolN-N-tert-butyl acrylamide is dissolved in 1mL dehydrated alcohol, obtains solution 2, is mixed into by solution 2 in above-mentioned solution 1, and vibration preact, after 4 hours, obtains solution 3.1mgN, N-methylene diacrylamide and 10mg sodium lauryl sulphate are added in above-mentioned solution 3, logical nitrogen 30min, adds initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, N, N, N-tetramethyl-diethylamine, and sealing, ambient temperature under nitrogen atmosphere reacts 24 hours.Proceeded to by reaction solution in dialysis tubing (MWCO10000), in a large amount of distilled water, carry out dialysis four days, every day at least changes twice water, to remove unreacted monomer and template molecule.Solution after dialysis is carried out lyophilize 3 days, obtains nanogel acceptor and Angiotensin synthesis of receptor.
Rat is divided into 2 groups at random, often organizes 8, that is: the 1st group, angiotensinⅡ group (0.1mg/kg); 2nd group, angiotensinⅡ (0.1mg/kg)+acceptor (2.4mg/kg) group.Rat, after 3% vetanarcol (45mg/kg) intraperitoneal injection of anesthesia, is fixed on operation plate.Row operation on neck is separated right common carotid artery and right external jugular vein, and threading is for subsequent use.Right common carotid artery intubate is connected with pressure transducer and measures arteriotony.Side external jugular vein intubate is used as intravenous administration.Stablize after 15min until blood markers, intravenous administration, with the physiological saline washing pipe of 0.4ml after administration, observe medicine to the impact of mean arterial pressure, irregular pulse occurrence degree.
Fig. 3 is the blood pressure (the 1st group) to anesthetized rat intravenous injection angiotensinⅡ.Fig. 4 is the blood pressure (the 2nd group) to anesthetized rat intravenous injection angiotensinⅡ mixing acceptor.
Rat mean arterial blood pressure rising 48 ± 9mmHg (P<0.001) of experimental result display injection angiotensinⅡ, occurs that the probability of irregular pulse phenomenon is 60.0%; And inject rat mean arterial blood pressure rising about 29 ± 8mmHg (P<0.001) of angiotensinⅡ mixing acceptor, occur that ARR probability is only 36.0%.
Result shows that its mean arterial blood pressure value risen of rat of the 2nd group is 60% of the rat of the 1st group, more than 60%. experimental results that irregular pulse probability is only the appearance irregular pulse probability of the 1st group of rat illustrate that Angiotensin synthesis of receptor has high avidity to angiotensinⅡ, after receptors bind angiotensinⅡ, combined part cannot with the AT in rat body
1receptors bind and promote elevation of blood pressure.
embodiment 3
The N of 0.0045g template molecule and angiotensinⅡ is held tripeptide derivative template (Asp-Arg-Val-NHC
2h
5), 0.098mmol vinylformic acid, 0.156mmolN-N-isopropylacrylamide is dissolved in 50mL distilled water and obtains solution 1.0.065mmolN-N-tert-butyl acrylamide is dissolved in 1mL dehydrated alcohol, obtains solution 2, is mixed into by solution 2 in above-mentioned solution 1, and vibration preact, after 4 hours, obtains solution 3.1mgN, N-methylene diacrylamide and 10mg sodium lauryl sulphate are added in above-mentioned solution 3, logical nitrogen 30min, adds initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, N, N, N-tetramethyl-diethylamine, and sealing, ambient temperature under nitrogen atmosphere reacts 24 hours.Proceeded to by reaction solution in dialysis tubing (MWCO10000), in a large amount of distilled water, carry out dialysis four days, every day at least changes twice water, to remove unreacted monomer and template molecule.Solution after dialysis is carried out lyophilize 3 days, obtains nanogel acceptor and Angiotensin synthesis of receptor.
In order to prove the imprinting effect of the Angiotensin synthesis of receptor obtained, spy does following experiment: the centrifuge tube Angiotensin synthesis of receptor that the template molecule aqueous solution of the 0.1mmol/mL of 2mL accurate formulation and 5mg aforesaid method obtain being put into 5mL, at 25 DEG C shaking table vibration about 12 hours after the centrifugal 30min of 16000rpm, get the supernatant liquor membrane filtration of 0.45 μm, and detect strength of solution with HPLC.Experiment shows, this Angiotensin synthesis of receptor static adsorbance reaches 130mg/g; Its imprinting factor (IF=Q
mIPs/ Q
nIPs; Q
mIPsthe adsorptive capacity of blot gel to target molecule, Q
nIPsthe adsorptive capacity of non-blot gel to target molecule) be 4.9.
embodiment 4
The N of 0.0045g template molecule and angiotensinⅡ is held tripeptide derivative template (Asp-Arg-Val-NHC
2h
5), 0.098mmol vinylformic acid, 0.156mmolN-N-isopropylacrylamide is dissolved in 50mL distilled water and obtains solution 1.0.065mmolN-N-tert-butyl acrylamide is dissolved in 1mL dehydrated alcohol, obtains solution 2, is mixed into by solution 2 in above-mentioned solution 1, and vibration preact, after 4 hours, obtains solution 3.1mgN, N-methylene diacrylamide and 10mg sodium lauryl sulphate are added in above-mentioned solution 3, logical nitrogen 30min, adds initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, N, N, N-tetramethyl-diethylamine, and sealing, ambient temperature under nitrogen atmosphere reacts 24 hours.Proceeded to by reaction solution in dialysis tubing (MWCO10000), in a large amount of distilled water, carry out dialysis four days, every day at least changes twice water, to remove unreacted monomer and template molecule.Solution after dialysis is carried out lyophilize 3 days, obtains nanogel acceptor and Angiotensin synthesis of receptor.
In order to prove the biocompatibility of the Angiotensin synthesis of receptor obtained, employment mesentery cavity of resorption arterial endothelium cells (Ealy926) carries out external cytotoxicity experiment, test as follows: after cell recovery, by the DMEM culture medium culturing containing 10%FBS to logarithmic phase cell, use trysinization sucking-off.After centrifugal, with cultivation keynote cell concn to 4 × 10
4cell/mL.Add 96 well culture plates with 100 μ L/ holes, cultivate 24h.Sucking-off substratum, every hole adds the substratum of each drug level and each positive drug concentration prepared, 100 μ L/ holes, establish the substratum control wells (not adding cell) of blank control wells (not adding cell and liquid), negative control hole (not adding liquid) and each drug level simultaneously.Each concentration does 3 multiple holes.After hatching 48h, add CCK-8,100uL/ hole, after hatching 4h, read plate in 450nm microplate reader.
Final concentration after Angiotensin synthesis of receptor adds is respectively 0.01,0.1,1,10,100,1000 μ g/mL.Final concentration after cisplatin for inj adds is 0.003,0.03,0.3,3,30,300 μ g/mL.
Experimental result is as shown in table 1: the concentration of cell toxicant positive drug cisplatin for inj, within the scope of 30-0.03 μ g/mL, has obvious cytotoxicity, and has obvious dose-response relationship.And Ealy926 Growth of Cells is good, after giving the Angiotensin synthesis of receptor of different concns, cell has no obvious exception, in 100 ~ 0.01 μ g/mL concentration ranges, the OD value of each drug level has no notable difference, when 1000 μ g/mL concentration, OD value increases on the contrary, and the OD value not containing the contrast of this concentration liquid of cell also increases, and shows itself make CCK8 produce reaction at the Angiotensin synthesis of receptor of this concentration and absorbancy is increased.Therefore angiotensin receptor has no in 1000 ~ 0.01 μ g/mL concentration ranges has cytotoxic effect to people's mesentery cavity of resorption arterial endothelium cells (Ealy926).
the OD value (mean value) of each detect aperture of table 1. hypertensin 2 artificial receptors cellulotoxic experiment
embodiment 5
The C of 0.0045g template molecule and angiotensinⅡ is held tripeptide derivative template (Ac-His-Pro-Phe), and 0.098mmol vinylformic acid, 0.124mmolN-N-isopropylacrylamide is dissolved in 50mL distilled water and obtains solution 1.0.098mmolN-N-tert-butyl acrylamide is dissolved in 1mL dehydrated alcohol, obtains solution 2, is mixed into by solution 2 in above-mentioned solution 1, and vibration preact, after 4 hours, obtains solution 3.1mgN, N-methylene diacrylamide and 10mg sodium lauryl sulphate are added in above-mentioned solution 3, logical nitrogen 30min, adds initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, N, N, N-tetramethyl-diethylamine, and sealing, ambient temperature under nitrogen atmosphere reacts 24 hours.Proceeded to by reaction solution in dialysis tubing (MWCO10000), in a large amount of distilled water, carry out dialysis four days, every day at least changes twice water, to remove unreacted monomer and template molecule.Solution after dialysis is carried out lyophilize 3 days, obtains nanogel acceptor and Angiotensin synthesis of receptor.
In order to prove the imprinting effect of the Angiotensin synthesis of receptor obtained, spy does following experiment: the centrifuge tube Angiotensin synthesis of receptor that the template molecule aqueous solution of the 0.1mmol/mL of 2mL accurate formulation and 5mg aforesaid method obtain being put into 5mL, at 25 DEG C shaking table vibration about 12 hours after the centrifugal 30min of 16000rpm, get the supernatant liquor membrane filtration of 0.45 μm, and detect strength of solution with HPLC.Experiment shows, this Angiotensin synthesis of receptor static adsorbance reaches 110mg/g; Its imprinting factor (IF=Q
mIPs/ Q
nIPs; Q
mIPsthe adsorptive capacity of blot gel to target molecule, Q
nIPsthe adsorptive capacity of non-blot gel to target molecule) be 4.6.
embodiment 6
The C of 0.0056g template molecule and angiotensinⅠ is held tripeptide derivative template (Ac-Phe-His-Leu), and 0.098mmol vinylformic acid, 0.124mmolN-N-isopropylacrylamide is dissolved in 50mL distilled water and obtains solution 1.0.098mmolN-N-tert-butyl acrylamide is dissolved in 1mL dehydrated alcohol, obtains solution 2, is mixed into by solution 2 in above-mentioned solution 1, and vibration preact, after 4 hours, obtains solution 3.1mgN, N-methylene diacrylamide and 10mg sodium lauryl sulphate are added in above-mentioned solution 3, logical nitrogen 30min, adds initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, N, N, N-tetramethyl-diethylamine, and sealing, ambient temperature under nitrogen atmosphere reacts 24 hours.Proceeded to by reaction solution in dialysis tubing (MWCO10000), in a large amount of distilled water, carry out dialysis four days, every day at least changes twice water, to remove unreacted monomer and template molecule.Solution after dialysis is carried out lyophilize 3 days, obtains nanogel acceptor and Angiotensin synthesis of receptor.
In order to prove the imprinting effect of the Angiotensin synthesis of receptor obtained, spy does following experiment: the centrifuge tube Angiotensin synthesis of receptor that the template molecule aqueous solution of the 0.1mmol/mL of 2mL accurate formulation and 5mg aforesaid method obtain being put into 5mL, at 25 DEG C shaking table vibration about 12 hours after the centrifugal 30min of 16000rpm, get the supernatant liquor membrane filtration of 0.45 μm, and detect strength of solution with HPLC.Experiment shows, this Angiotensin synthesis of receptor static adsorbance reaches 94mg/g; Its imprinting factor (IF=Q
mIPs/ Q
nIPs; Q
mIPsthe adsorptive capacity of blot gel to target molecule, Q
nIPsthe adsorptive capacity of non-blot gel to target molecule) be 3.8.
embodiment 7
The C of 0.0035g template molecule and angiotensinⅡ is held dipeptidase derivant template (Ac-Pro-Phe), and 0.065mmol vinylformic acid, 0.156mmolN-N-isopropylacrylamide is dissolved in 50mL distilled water and obtains solution 1.0.098mmolN-N-tert-butyl acrylamide is dissolved in 1mL dehydrated alcohol, obtains solution 2, is mixed into by solution 2 in above-mentioned solution 1, and vibration preact, after 4 hours, obtains solution 3.1mgN, N-methylene diacrylamide and 10mg sodium lauryl sulphate are added in above-mentioned solution 3, logical nitrogen 60min, adds initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, N, N, N-tetramethyl-diethylamine, and sealing, ambient temperature under nitrogen atmosphere reacts 24 hours.Proceeded to by reaction solution in dialysis tubing (MWCO10000), in a large amount of distilled water, carry out dialysis four days, every day at least changes twice water, to remove unreacted monomer and template molecule.Solution after dialysis is carried out lyophilize 3 days, obtains nanogel acceptor and Angiotensin synthesis of receptor.
In order to prove the imprinting effect of the Angiotensin synthesis of receptor obtained, spy does following experiment: the centrifuge tube Angiotensin synthesis of receptor that the template molecule aqueous solution of the 0.1mmol/mL of 2mL accurate formulation and 5mg aforesaid method obtain being put into 5mL, at 25 DEG C shaking table vibration about 12 hours after the centrifugal 30min of 16000rpm, get the supernatant liquor membrane filtration of 0.45 μm, and detect strength of solution with HPLC.Experiment shows, this Angiotensin synthesis of receptor static adsorbance reaches 78mg/g; Its imprinting factor (IF=Q
mIPs/ Q
nIPs; Q
mIPsthe adsorptive capacity of blot gel to target molecule, Q
nIPsthe adsorptive capacity of non-blot gel to target molecule) be 4.2.
embodiment 8
The N of 0.0045g template molecule and angiotensinⅡ is held a peptide derivant template (Asp-NHC
2h
5), 0.098mmol vinylformic acid, 0.201mmolN-N-isopropylacrylamide is dissolved in 50mL distilled water and obtains solution 1.0.016mmolN-N-tert-butyl acrylamide is dissolved in 1mL dehydrated alcohol, obtains solution 2, is mixed into by solution 2 in above-mentioned solution 1, and vibration preact, after 4 hours, obtains solution 3.1mgN, N-methylene diacrylamide and 10mg sodium lauryl sulphate are added in above-mentioned solution 3, logical nitrogen 15min, adds initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, N, N, N-tetramethyl-diethylamine, and sealing, ambient temperature under nitrogen atmosphere reacts 24 hours.Proceeded to by reaction solution in dialysis tubing (MWCO10000), in a large amount of distilled water, carry out dialysis four days, every day at least changes twice water, to remove unreacted monomer and template molecule.Solution after dialysis is carried out lyophilize 3 days, obtains nanogel acceptor and Angiotensin synthesis of receptor.
In order to prove the imprinting effect of the Angiotensin synthesis of receptor obtained, spy does following experiment: the centrifuge tube Angiotensin synthesis of receptor that the template molecule aqueous solution of the 0.1mmol/mL of 2mL accurate formulation and 5mg aforesaid method obtain being put into 5mL, at 25 DEG C shaking table vibration about 12 hours after the centrifugal 30min of 16000rpm, get the supernatant liquor membrane filtration of 0.45 μm, and detect strength of solution with HPLC.Experiment shows, this Angiotensin synthesis of receptor static adsorbance reaches 60mg/g; Its imprinting factor (IF=Q
mIPs/ Q
nIPs; Q
mIPsthe adsorptive capacity of blot gel to target molecule, Q
nIPsthe adsorptive capacity of non-blot gel to target molecule) be 3.5.
embodiment 9
The N of 0.0078g template molecule and angiotensinⅡ is held pentapeptide derivative template (Asp-Arg-Val-Tyr-Ile-NHC
2h
5), 0.098mmol vinylformic acid, 0.124mmolN-N-isopropylacrylamide is dissolved in 50mL distilled water and obtains solution 1.0.098mmolN-N-tert-butyl acrylamide is dissolved in 1mL dehydrated alcohol, obtains solution 2, is mixed into by solution 2 in above-mentioned solution 1, and vibration preact, after 4 hours, obtains solution 3.1mgN, N-methylene diacrylamide and 10mg sodium lauryl sulphate are added in above-mentioned solution 3, logical nitrogen 30min, adds initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, N, N, N-tetramethyl-diethylamine, and sealing, ambient temperature under nitrogen atmosphere reacts 36 hours.Proceeded to by reaction solution in dialysis tubing (MWCO10000), in a large amount of distilled water, carry out dialysis four days, every day at least changes twice water, to remove unreacted monomer and template molecule.Solution after dialysis is carried out lyophilize 3 days, obtains nanogel acceptor and Angiotensin synthesis of receptor.
In order to prove the imprinting effect of the Angiotensin synthesis of receptor obtained, spy does following experiment: the centrifuge tube Angiotensin synthesis of receptor that the template molecule aqueous solution of the 0.1mmol/mL of 2mL accurate formulation and 5mg aforesaid method obtain being put into 5mL, at 25 DEG C shaking table vibration about 12 hours after the centrifugal 30min of 16000rpm, get the supernatant liquor membrane filtration of 0.45 μm, and detect strength of solution with HPLC.Experiment shows, this Angiotensin synthesis of receptor static adsorbance reaches 150mg/g; Its imprinting factor (IF=Q
mIPs/ Q
nIPs; Q
mIPsthe adsorptive capacity of blot gel to target molecule, Q
nIPsthe adsorptive capacity of non-blot gel to target molecule) be 4.0.
Claims (5)
1. prepare the method for Angiotensin synthesis of receptor based on engram technology, its feature comprises the steps:
(1) more than one in following six kinds of utilization are as template molecule: the C of (1) angiotensinⅡ holds tripeptide derivative template Ac-His-Pro-Phe, (2) C of angiotensinⅠ holds tripeptide derivative template Ac-Phe-His-Leu, and the N of (3) angiotensinⅡ holds tripeptide derivative template Asp-Arg-Val-NHC
2h
5, the N of (4) angiotensinⅡ holds a peptide derivant template Asp-NHC
2h
5, the C of (5) angiotensinⅡ holds dipeptidase derivant template Ac-Pro-Phe, and the N of (6) angiotensinⅡ holds pentapeptide derivative template Asp-Arg-Val-Tyr-Ile-NHC
2h
5;
(2) by template molecule, function monomer A, gel skeleton monomer is dissolved in distilled water and obtains solution 1; Function monomer B is dissolved in dehydrated alcohol and obtains solution 2; Be mixed into by solution 2 in above-mentioned solution 1, vibration preact, obtains solution 3; Described function monomer A is vinylformic acid, and described gel skeleton monomer is NIPA, and described function monomer B is N tert butyl acrylamide; The molar ratio of template molecule, function monomer A, gel skeleton monomer and distilled water is 1:(7 ~ 9): (11 ~ 18): 2780; The mol ratio of described template molecule, function monomer A, function monomer B tri-kinds of materials is: template molecule: function monomer A: function monomer B=1:(7 ~ 9): (1.6 ~ 9); In described solution 3, three kinds of total monomer are 5.9 ~ 9.8mM, and wherein each monomer concentration ratio is function monomer A: function monomer B: backbone monomer=1:(0.17 ~ 1): (1.3 ~ 2.1);
(3) crosslinkers monomers and Surfactant SDS are added in the solution 3 of step (2), logical nitrogen 15 ~ 60min, adds initiator, sealing, and nitrogen atmosphere reacts, and obtains molecular imprinting nanogel; Function monomer A, function monomer B, backbone monomer and cross-linking monomer totally four kinds of monomers total concn in solution 3 are 6 ~ 10mM, wherein crosslinkers monomers concentration is total concn 2% ~ 4%, function monomer B concentration is total concn 5% ~ 30%, and function monomer A concentration is total concn 30%; The temperature of described nitrogen atmosphere reaction is 23 ~ 25 DEG C, and the reaction times is 24 ~ 36 hours;
(4) purifying of imprinted polymer: proceed in dialysis tubing by the molecular imprinting nanogel obtained in step (3), dialyse in distilled water, dialysis postlyophilization, obtains nanogel acceptor and Angiotensin synthesis of receptor.
2. the method preparing Angiotensin synthesis of receptor based on engram technology as claimed in claim 1, is characterized in that: the dehydrated alcohol volume described in step (2) is 2% ~ 5% of solution 1 volume; Adopt the purity of all ingredients be analytical pure and more than, distilled water is secondary redistilled water; The time of vibration preact is 4-6 hour.
3. the method preparing Angiotensin synthesis of receptor based on engram technology as claimed in claim 1, it is characterized in that: described in step (3), crosslinkers monomers is N, N-methylene diacrylamide, its concentration is 2% ~ 4% of three kinds of total monomer in step (2); Described initiator is that ammonium persulphate and N, N, N, N-tetramethyl-diethylamine combine, both add after concentration in solution 3 be respectively 0.6 ~ 1.0mg/mL, 0.24 ~ 0.48mg/mL; Concentration after described Surfactant SDS adds in solution 3 is 0.2 ~ 0.4mg/mL.
4. the method preparing Angiotensin synthesis of receptor based on engram technology as claimed in claim 1, it is characterized in that: described in step (4), dialysis tubing specification is MWCO10000, dialysis tubing molecular weight cut-off is 10000; Described dialysis time at least four days, every day at least changes twice water; Described sublimation drying is 2-3 days.
5. the Angiotensin synthesis of receptor obtained by the method preparing Angiotensin synthesis of receptor based on engram technology described in any one of claim 1 ~ 4.
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