CN103408696A - Application of taking oligopeptide and derivatives thereof as template molecule, and imprinting technology based preparation method of angiotensin synthetic receptor - Google Patents
Application of taking oligopeptide and derivatives thereof as template molecule, and imprinting technology based preparation method of angiotensin synthetic receptor Download PDFInfo
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Abstract
The invention discloses an application of taking oligopeptide and derivatives thereof as a template molecule, and an imprinting technology based preparation method of an angiotensin synthetic receptor. The template molecule comprises any section of a peptide bond at a nitrogen end or a carbon end of an angiotensin II, and derivatives thereof, or any section of the peptide bond at the nitrogen end or the carbon end of an angiotensin I and the derivatives thereof. The application takes the template molecule as an imprint to prepare the angiotensin synthetic receptor; the angiotensin acceptor is prepared in a way that the template molecule, a function monomer, a cross-linking agent, a surface-active agent and the like are mixed according to a certain rate, and then an initiator is added. The acceptor is good in biocompatibility, can high selectively capture and neutralize the angiotensin II and the angiotensin I, and cuts off functions of the angiotensin II and the angiotensin acceptor (AT1) as well as functions of the angiotensin I and angiotensin converzyme (ACE), so as to control the blood pressure. The acceptor can be applied to separation, concentration and measuring of the angiotensin I or II.
Description
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Technical field
The present invention relates to biological medicine technology 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 based on engram technology, prepare the method for Angiotensin synthesis of receptor.
Background technology
As the main biologically active peptides 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 discharge aldosterone, and aldosterone acts on uriniferous tubules, plays and protects sodium, water conservation, the effect of row's potassium, causes hypervolemia, vasoconstriction, rising blood pressure; Thereby but and the I type of vasoactive Angiotensin Ⅱ or II receptor rising blood pressure; AngiotensinⅡ also can increase the Ca ionic concn by acting on cell Na-Ca passage, causes 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, the blocking-up angiotensinⅠ is converted to angiotensinⅡ or blocks angiotensinⅡ and is attached to angiotensin receptor (AT
1) be the hypertensive method of the most effectively treating both at home and abroad at present.
For the hypertensive method of renin-angiotensin-aldosterone system treatment, usually have both at home and abroad: 1. ACE inhibitor, the Zinc metallopeptidase Zace1 (ACE) that suppresses the RAS system, stop angiotensinⅠ to be converted to angiotensinⅡ, and can suppress Aldosterone Secretion, reduce water-sodium retention; 2. the antihypertensive drug of Angiotensin Ⅱ receptor antagonist (AIIA) class, block angiotensin receptor (AT
1), avoid angiotensinⅡ to be attached on acceptor, finally reach and prevent vasoconstrictive purpose.Yet there is serious side effects in these treatments, for example give birth to defect, cough and dizzy, extreme side effect can cause the problems such as minimizing of renal failure and white cell.
The present invention is intended to design and prepares a kind of trace artificial receptors, makes the identification of this artificial receptors selectivity catch in body too much angiotensinⅡ (or angiotensinⅠ), just itself and AT capable of blocking
1(with Zinc metallopeptidase Zace1 (ACE)) acts on, thereby reaches the purpose of further control blood pressure.
Molecular imprinting is a kind of bionics techniques of novel artificial acceptor.Under coexisting situation, template molecule, function monomer and linking agent synthesize the polymeric acceptor with specific identification site and hole by polyreaction.The most successful application of molecular imprinting at present is the identification organic molecule, and the examples of many successful of catching polypeptide and protein for identification is very limited, and its reason is structure of biological macromolecule complexity and Thermodynamics etc.; Another major cause is that peptide and protein is expensive, usually is difficult to obtain q.s for the preparation of molecularly imprinted polymer.
At present, about synthetic, have that (<100nm) similar molecular imprinting nanogel has been reported, and has not yet to see report but take Angiotensin as the trace nanogel acceptor of part to natural enzyme or antibody size.The molecular imprinting nanogel can synthesize in gentle water surrounding, can better retain the three-dimensional conformation of biomolecules, has very high biocompatibility, and specific surface area is large, and the 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, have more the advantage on kinetics of adsorption and thermodynamics than traditional imprinted material in theory, and the cost less of template.
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Summary of the invention
The object of the present invention is to provide a kind of small peptide and derivative thereof to reach as the application of template molecule the method for preparing the Angiotensin synthesis of receptor based on engram technology, propose simultaneously to utilize angiotensinⅠ or the II in this receptor neutralization (identification and combination) body, realize controlling the new way of blood pressure.This receptor good biocompatibility, but highly selective catch neutralization and separation and concentration angiotensinⅠ or II, have the application of potential control blood pressure.
A kind of small peptide and derivative thereof are as the application of template molecule, namely for engram technology, preparing the Angiotensin synthesis of receptor, described template molecule comprises a kind of in any one section peptide bond of the nitrogen end of any one section peptide bond of the nitrogen end of angiotensinⅡ or carbon teminal and derivative or angiotensinⅠ or carbon teminal and derivative thereof.
As preferably, based on engram technology, prepare the method for Angiotensin synthesis of receptor, comprise following concrete steps
(1) utilize a kind of as template molecule in any one section peptide bond of the nitrogen end of any one section peptide bond of the nitrogen end comprise angiotensinⅡ or carbon teminal and derivative or angiotensinⅠ or carbon teminal and derivative thereof;
(2) by template molecule, function monomer A, gel skeleton monomer are dissolved in distilled water and obtain solution 1; Function monomer B is dissolved in dehydrated alcohol and obtains solution 2; Solution 2 is mixed in above-mentioned solution 1, and vibration preact, obtain solution 3;
(3) linking agent monomer and Surfactant SDS are added in the solution 3 of step (2), logical nitrogen 15～60min, add initiator, sealing, and the nitrogen atmosphere reaction, obtain the molecular imprinting nanogel;
(4) purifying of imprinted polymer: the molecular imprinting nanogel obtained in step (3) is proceeded in dialysis tubing, dialyse in distilled water, the dialysis postlyophilization, obtaining the nanogel acceptor is the Angiotensin synthesis of receptor.
In above-mentioned preparation method, template molecule described in step (1) comprises the C end tripeptide derivative template (Ac-His-Pro-Phe) of following (1) angiotensinⅡ, (2) C of angiotensinⅠ end tripeptide derivative template (Ac-Phe-His-Leu), the N end tripeptide derivative template (Asp-Arg-Val-NHC of (3) angiotensinⅡ
2H
5), the N end one peptide derivant template (Asp-NHC of (4) angiotensinⅡ
2H
5), the C end dipeptidase derivant template (Ac-Pro-Phe) of (5) angiotensinⅡ, the N end pentapeptide derivative template (Asp-Arg-Val-Tyr-Ile-NHC of (6) angiotensinⅡ
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), function monomer A, the mol ratio of tri-kinds of materials of function monomer B 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 ︰ skeleton monomer=1:(0.17～1) ︰ (1.3～2.1).
In above-mentioned preparation method, described dehydrated alcohol volume described in step (2) be in solution 1 volume 2%～5%; The purity of all ingredients that adopts be analytical pure and more than, distilled water is the 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, skeleton monomer and cross-linking monomer totally four kinds of monomers total concn in solution 3 are 6～10mM, wherein the linking agent monomer 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 ℃, and the reaction times is 24～36 hours.
In above-mentioned preparation method, described in step (3), the linking agent monomer is N, the N-methylene diacrylamide, its concentration be in step (2) three kinds of total monomer 2%～4%; Described initiator is ammonium persulphate and N, N, N, N-tetramethyl-diethylamine combination, 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), the dialysis tubing specification is MWCO 10000, and the dialysis tubing molecular weight cut-off is 10000; Described dialysis time at least four days, change twice water every day at least; 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 any one section peptide bond and the nitrogen end of derivative or angiotensinⅠ or any one section peptide bond and the derivative thereof of carbon teminal of carbon teminal, below wherein a kind of has high trace effect for the prepared Angiotensin nanogel acceptor of template molecule, and angiotensinⅠ and II are all had to high adsorptive capacity and selectivity.
The invention has the advantages that: process is simple, in preparation process, can better retain the three-dimensional conformation of polypeptide, the template cost is low, target polypeptides is had to avidity to prepared nanogel acceptor and selectivity is high, specific surface area is large, the action site embedding is few, and charge capacity and stability are high, and has the very high advantages such as biocompatibility.
The accompanying drawing explanation
Fig. 1 is the low multiple TEM figure of the synthetic Angiotensin synthesis of receptor of embodiment 1.
Fig. 2 is the high multiple TEM figure of the synthetic Angiotensin synthesis of receptor of embodiment 1.
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, rather than limits the scope of the invention.
Embodiment 1
By the 0.0045g template molecule, be the N end tripeptide derivative template (Asp-Arg-Val-NHC of angiotensinⅡ
2H
5), 0.098 mmol vinylformic acid, 0.156 mmol NIPA is dissolved in 50 mL distilled water and obtains solution 1.0.065 the mmol N tert butyl acrylamide is dissolved in the 1mL dehydrated alcohol, obtains solution 2, and solution 2 is mixed in above-mentioned solution 1, vibration preact, after 4 hours, obtains solution 3.By 1 mg N, N-methylene diacrylamide and 10 mg sodium lauryl sulphate are added in above-mentioned solution 3, and logical nitrogen 30min, add initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, and N, N, N-tetramethyl-diethylamine, sealing, under room temperature, the nitrogen atmosphere reaction is 24 hours.Reaction solution is proceeded in dialysis tubing (MWCO 10000), in a large amount of distilled water, dialysed four days, change at least twice water every day, to remove unreacted monomer and template molecule.Solution after dialysis was carried out to lyophilize 3 days, and obtaining the nanogel acceptor is the Angiotensin synthesis of receptor.
Fig. 1 and Fig. 2 are respectively the TEM figure of the synthetic Angiotensin synthesis of receptor of embodiment 1.As can be seen from the figure, this Angiotensin synthesis of receptor particle diameter narrow distribution, median size is 25 nm approximately.
With the Angiotensin synthesis of receptor that aforesaid method makes, carry out the selective adsorption experiment, experimentation is: the concentration that 2mL is accurately prepared is 1.2 * 10
-4The angiotensinⅠ of M, angiotensinⅡ and [Sar1, Val5, Ala8]-aqueous solution (SDS concentration is 1mg/ml) of angiotensinⅡ and the Angiotensin synthesis of receptor that the 5mg aforesaid method makes put into the centrifuge tube of 5mL, at the about centrifugal 30min of 16000rpm after 12 hours of 25 ℃ of lower shaking tables vibrations, get the membrane filtration of supernatant liquor with 0.45 μ m, and detect strength of solution with HPLC.Experimental result shows that this Angiotensin synthesis of receptor can optionally adsorb angiotensinⅠ and II simultaneously in three peptide species mixing solutionss, it is to angiotensinⅠ, angiotensinⅡ and [Sar1, Val5, Ala8]-adsorptive capacity of angiotensinⅡ is respectively 15.4 μ mol/g, 14.2 μ mol/g, 1.2 μ mol/g.
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Embodiment 2
By the 0.0045g template molecule, be the N end tripeptide derivative template (Asp-Arg-Val-NHC of angiotensinⅡ
2H
5), 0.098 mmol vinylformic acid, 0.156 mmol NIPA is dissolved in 50 mL distilled water and obtains solution 1.0.065 the mmol N tert butyl acrylamide is dissolved in the 1mL dehydrated alcohol, obtains solution 2, and solution 2 is mixed in above-mentioned solution 1, vibration preact, after 4 hours, obtains solution 3.By 1 mg N, N-methylene diacrylamide and 10 mg sodium lauryl sulphate are added in above-mentioned solution 3, and logical nitrogen 30min, add initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, and N, N, N-tetramethyl-diethylamine, sealing, under room temperature, the nitrogen atmosphere reaction is 24 hours.Reaction solution is proceeded in dialysis tubing (MWCO 10000), in a large amount of distilled water, dialysed four days, change at least twice water every day, to remove unreacted monomer and template molecule.Solution after dialysis was carried out to lyophilize 3 days, and obtaining the nanogel acceptor is the Angiotensin synthesis of receptor.
Rat is divided into to 2 groups at random, 8 every group, that is: the 1st group, angiotensinⅡ group (0.1mg/kg); The 2nd group, angiotensinⅡ (0.1mg/kg)+acceptor (2.4mg/kg) group.Rat, after 3% vetanarcol (45mg/kg) intraperitoneal injection of anesthesia, is fixed on the operation plate.The row operation on neck separates right common carotid artery and right external jugular vein, and threading is standby.The right common carotid artery intubate is connected with pressure transducer and measures arteriotony.One side external jugular vein intubate is used as intravenous administration.After the blood pressure index was stablized 15min, intravenous administration, with the physiological saline washing pipe of 0.4ml, observed the impact of medicine on mean arterial pressure, irregular pulse occurrence degree after administration.
Fig. 3 is to the blood pressure of anesthetized rat intravenous injection angiotensinⅡ (the 1st group).Fig. 4 is to the blood pressure of anesthetized rat intravenous injection angiotensinⅡ mixing acceptor (the 2nd group).
Experimental result shows rat mean arterial blood pressure rising 48 ± 9 mmHg (P<0.001) of injection angiotensinⅡ, and the probability that the irregular pulse phenomenon occurs is 60.0%; And the about 29 ± 8mmHg (P<0.001) of the rat mean arterial blood pressure rising of injection angiotensinⅡ mixings acceptor occurs that ARR probability is only 36.0%.
Result show the mean arterial blood pressure value of its rising of rat of the 2nd group be the 1st group rat 60%, the irregular pulse probability is only that 60%. above experimental result explanation Angiotensin synthesis of receptor of the appearance irregular pulse probability of the 1st group of rat have high avidity to angiotensinⅡ, receptors bind after angiotensinⅡ, combined part can't with the rat body in AT
1Receptors bind and promote elevation of blood pressure.
Embodiment 3
By the 0.0045g template molecule, be the N end tripeptide derivative template (Asp-Arg-Val-NHC of angiotensinⅡ
2H
5), 0.098 mmol vinylformic acid, 0.156 mmol NIPA is dissolved in 50 mL distilled water and obtains solution 1.0.065 the mmol N tert butyl acrylamide is dissolved in the 1mL dehydrated alcohol, obtains solution 2, and solution 2 is mixed in above-mentioned solution 1, vibration preact, after 4 hours, obtains solution 3.By 1 mg N, N-methylene diacrylamide and 10 mg sodium lauryl sulphate are added in above-mentioned solution 3, and logical nitrogen 30min, add initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, and N, N, N-tetramethyl-diethylamine, sealing, under room temperature, the nitrogen atmosphere reaction is 24 hours.Reaction solution is proceeded in dialysis tubing (MWCO 10000), in a large amount of distilled water, dialysed four days, change at least twice water every day, to remove unreacted monomer and template molecule.Solution after dialysis was carried out to lyophilize 3 days, and obtaining the nanogel acceptor is the Angiotensin synthesis of receptor.
In order to prove the trace effect of the Angiotensin synthesis of receptor made, the spy does following experiment: by 2mL accurately the template molecule aqueous solution of the 0.1mmol/mL of preparation and the centrifuge tube that Angiotensin synthesis of receptor that the 5mg aforesaid method makes is put into 5mL, at the about centrifugal 30min of 16000rpm after 12 hours of 25 ℃ of lower shaking tables vibrations, get the membrane filtration of supernatant liquor with 0.45 μ m, and detect strength of solution with HPLC.Experiment shows, this Angiotensin synthesis of receptor static adsorbance reaches 130mg/g; Its trace factor (IF=Q
MIPs/ Q
NIPsQ
MIPsThe adsorptive capacity of blot gel to target molecule, Q
NIPsThe adsorptive capacity of non-blot gel to target molecule) be 4.9.
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Embodiment 4
By the 0.0045g template molecule, be the N end tripeptide derivative template (Asp-Arg-Val-NHC of angiotensinⅡ
2H
5), 0.098 mmol vinylformic acid, 0.156 mmol NIPA is dissolved in 50 mL distilled water and obtains solution 1.0.065 the mmol N tert butyl acrylamide is dissolved in the 1mL dehydrated alcohol, obtains solution 2, and solution 2 is mixed in above-mentioned solution 1, vibration preact, after 4 hours, obtains solution 3.By 1 mg N, N-methylene diacrylamide and 10 mg sodium lauryl sulphate are added in above-mentioned solution 3, and logical nitrogen 30min, add initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, and N, N, N-tetramethyl-diethylamine, sealing, under room temperature, the nitrogen atmosphere reaction is 24 hours.Reaction solution is proceeded in dialysis tubing (MWCO 10000), in a large amount of distilled water, dialysed four days, change at least twice water every day, to remove unreacted monomer and template molecule.Solution after dialysis was carried out to lyophilize 3 days, and obtaining the nanogel acceptor is the Angiotensin synthesis of receptor.
In order to prove the biocompatibility of the Angiotensin synthesis of receptor made, employment mesentery cavity of resorption arteries endotheliocyte (Ealy926) carries out external cytotoxicity experiment, test as follows: after cell recovery, by the DMEM culture medium culturing that contains 10%FBS, to the logarithmic phase cell, use the trysinization sucking-off.After centrifugal, with cultivating keynote cell concn to 4 * 10
4Cell/mL.With 100 μ L/ holes, add 96 well culture plates, cultivate 24h.The sucking-off substratum, every hole adds the substratum of each drug level of having prepared and each positive drug concentration, 100 μ L/ holes, the substratum control wells (not adding cell) of establishing simultaneously blank hole (not adding cell and liquid), negative control hole (not adding liquid) and each drug level.Each concentration is done 3 multiple holes.After hatching 48h, add CCK-8, the 100uL/ hole, after hatching 4h, read plate in the 450nm microplate reader.
Final concentration after the 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, in 30-0.03 μ g/mL scope, has obvious cytotoxicity, and obvious dose-response relationship is arranged.And the Ealy926 Growth of Cells is good, cell has no significantly abnormal after giving the Angiotensin synthesis of receptor of different concns, 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, the OD value increases on the contrary, and the OD value that does not contain this concentration liquid contrast of cell also increases, and shows that the Angiotensin synthesis of receptor in this concentration itself makes CCK8 produce reaction and absorbancy is increased.Therefore angiotensin receptor has no people's mesentery cavity of resorption arteries endotheliocyte (Ealy926) is had to cytotoxic effect in 1000～0.01 μ g/mL concentration ranges.
Table 1. hypertensin 2 artificial receptors cellulotoxic experiment respectively detects the OD value (mean value) in hole
Embodiment 5
By the 0.0045g template molecule, be the C end tripeptide derivative template (Ac-His-Pro-Phe) of angiotensinⅡ, 0.098 mmol vinylformic acid, 0.124 mmol NIPA is dissolved in 50 mL distilled water and obtains solution 1.0.098 the mmol N tert butyl acrylamide is dissolved in the 1mL dehydrated alcohol, obtains solution 2, and solution 2 is mixed in above-mentioned solution 1, vibration preact, after 4 hours, obtains solution 3.By 1 mg N, N-methylene diacrylamide and 10 mg sodium lauryl sulphate are added in above-mentioned solution 3, and logical nitrogen 30min, add initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, and N, N, N-tetramethyl-diethylamine, sealing, under room temperature, the nitrogen atmosphere reaction is 24 hours.Reaction solution is proceeded in dialysis tubing (MWCO 10000), in a large amount of distilled water, dialysed four days, change at least twice water every day, to remove unreacted monomer and template molecule.Solution after dialysis was carried out to lyophilize 3 days, and obtaining the nanogel acceptor is the Angiotensin synthesis of receptor.
In order to prove the trace effect of the Angiotensin synthesis of receptor made, the spy does following experiment: by 2mL accurately the template molecule aqueous solution of the 0.1mmol/mL of preparation and the centrifuge tube that Angiotensin synthesis of receptor that the 5mg aforesaid method makes is put into 5mL, at the about centrifugal 30min of 16000rpm after 12 hours of 25 ℃ of lower shaking tables vibrations, get the membrane filtration of supernatant liquor with 0.45 μ m, and detect strength of solution with HPLC.Experiment shows, this Angiotensin synthesis of receptor static adsorbance reaches 110 mg/g; Its trace factor (IF=Q
MIPs/ Q
NIPsQ
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
By the 0.0056g template molecule, be the C end tripeptide derivative template (Ac-Phe-His-Leu) of angiotensinⅠ, 0.098 mmol vinylformic acid, 0.124 mmol NIPA is dissolved in 50 mL distilled water and obtains solution 1.0.098 the mmol N tert butyl acrylamide is dissolved in the 1mL dehydrated alcohol, obtains solution 2, and solution 2 is mixed in above-mentioned solution 1, vibration preact, after 4 hours, obtains solution 3.By 1 mg N, N-methylene diacrylamide and 10 mg sodium lauryl sulphate are added in above-mentioned solution 3, and logical nitrogen 30min, add initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, and N, N, N-tetramethyl-diethylamine, sealing, under room temperature, the nitrogen atmosphere reaction is 24 hours.Reaction solution is proceeded in dialysis tubing (MWCO 10000), in a large amount of distilled water, dialysed four days, change at least twice water every day, to remove unreacted monomer and template molecule.Solution after dialysis was carried out to lyophilize 3 days, and obtaining the nanogel acceptor is the Angiotensin synthesis of receptor.
In order to prove the trace effect of the Angiotensin synthesis of receptor made, the spy does following experiment: by 2mL accurately the template molecule aqueous solution of the 0.1mmol/mL of preparation and the centrifuge tube that Angiotensin synthesis of receptor that the 5mg aforesaid method makes is put into 5mL, at the about centrifugal 30min of 16000rpm after 12 hours of 25 ℃ of lower shaking tables vibrations, get the membrane filtration of supernatant liquor with 0.45 μ m, and detect strength of solution with HPLC.Experiment shows, this Angiotensin synthesis of receptor static adsorbance reaches 94 mg/g; Its trace factor (IF=Q
MIPs/ Q
NIPsQ
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
By the 0.0035g template molecule, be the C end dipeptidase derivant template (Ac-Pro-Phe) of angiotensinⅡ, 0.065 mmol vinylformic acid, 0.156 mmol NIPA is dissolved in 50 mL distilled water and obtains solution 1.0.098 the mmol N tert butyl acrylamide is dissolved in the 1mL dehydrated alcohol, obtains solution 2, and solution 2 is mixed in above-mentioned solution 1, vibration preact, after 4 hours, obtains solution 3.By 1 mg N, N-methylene diacrylamide and 10 mg sodium lauryl sulphate are added in above-mentioned solution 3, and logical nitrogen 60min, add initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, and N, N, N-tetramethyl-diethylamine, sealing, under room temperature, the nitrogen atmosphere reaction is 24 hours.Reaction solution is proceeded in dialysis tubing (MWCO 10000), in a large amount of distilled water, dialysed four days, change at least twice water every day, to remove unreacted monomer and template molecule.Solution after dialysis was carried out to lyophilize 3 days, and obtaining the nanogel acceptor is the Angiotensin synthesis of receptor.
In order to prove the trace effect of the Angiotensin synthesis of receptor made, the spy does following experiment: by 2mL accurately the template molecule aqueous solution of the 0.1mmol/mL of preparation and the centrifuge tube that Angiotensin synthesis of receptor that the 5mg aforesaid method makes is put into 5mL, at the about centrifugal 30min of 16000rpm after 12 hours of 25 ℃ of lower shaking tables vibrations, get the membrane filtration of supernatant liquor with 0.45 μ m, and detect strength of solution with HPLC.Experiment shows, this Angiotensin synthesis of receptor static adsorbance reaches 78 mg/g; Its trace factor (IF=Q
MIPs/ Q
NIPsQ
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
By the 0.0045g template molecule, be the N end one peptide derivant template (Asp-NHC of angiotensinⅡ
2H
5), 0.098 mmol vinylformic acid, 0.201 mmol NIPA is dissolved in 50 mL distilled water and obtains solution 1.0.016 the mmol N tert butyl acrylamide is dissolved in the 1mL dehydrated alcohol, obtains solution 2, and solution 2 is mixed in above-mentioned solution 1, vibration preact, after 4 hours, obtains solution 3.By 1 mg N, N-methylene diacrylamide and 10 mg sodium lauryl sulphate are added in above-mentioned solution 3, and logical nitrogen 15min, add initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, and N, N, N-tetramethyl-diethylamine, sealing, under room temperature, the nitrogen atmosphere reaction is 24 hours.Reaction solution is proceeded in dialysis tubing (MWCO 10000), in a large amount of distilled water, dialysed four days, change at least twice water every day, to remove unreacted monomer and template molecule.Solution after dialysis was carried out to lyophilize 3 days, and obtaining the nanogel acceptor is the Angiotensin synthesis of receptor.
In order to prove the trace effect of the Angiotensin synthesis of receptor made, the spy does following experiment: by 2mL accurately the template molecule aqueous solution of the 0.1mmol/mL of preparation and the centrifuge tube that Angiotensin synthesis of receptor that the 5mg aforesaid method makes is put into 5mL, at the about centrifugal 30min of 16000rpm after 12 hours of 25 ℃ of lower shaking tables vibrations, get the membrane filtration of supernatant liquor with 0.45 μ m, and detect strength of solution with HPLC.Experiment shows, this Angiotensin synthesis of receptor static adsorbance reaches 60 mg/g; Its trace factor (IF=Q
MIPs/ Q
NIPsQ
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
By the 0.0078g template molecule, be the N end pentapeptide derivative template (Asp-Arg-Val-Tyr-Ile-NHC of angiotensinⅡ
2H
5), 0.098 mmol vinylformic acid, 0.124 mmol NIPA is dissolved in 50 mL distilled water and obtains solution 1.0.098 the mmol N tert butyl acrylamide is dissolved in the 1mL dehydrated alcohol, obtains solution 2, and solution 2 is mixed in above-mentioned solution 1, vibration preact, after 4 hours, obtains solution 3.By 1 mg N, N-methylene diacrylamide and 10 mg sodium lauryl sulphate are added in above-mentioned solution 3, and logical nitrogen 30min, add initiator peroxosulphuric ammonium 30mg, the N of 15 μ L, and N, N, N-tetramethyl-diethylamine, sealing, under room temperature, the nitrogen atmosphere reaction is 36 hours.Reaction solution is proceeded in dialysis tubing (MWCO 10000), in a large amount of distilled water, dialysed four days, change at least twice water every day, to remove unreacted monomer and template molecule.Solution after dialysis was carried out to lyophilize 3 days, and obtaining the nanogel acceptor is the Angiotensin synthesis of receptor.
In order to prove the trace effect of the Angiotensin synthesis of receptor made, the spy does following experiment: by 2mL accurately the template molecule aqueous solution of the 0.1mmol/mL of preparation and the centrifuge tube that Angiotensin synthesis of receptor that the 5mg aforesaid method makes is put into 5mL, at the about centrifugal 30min of 16000rpm after 12 hours of 25 ℃ of lower shaking tables vibrations, get the membrane filtration of supernatant liquor with 0.45 μ m, and detect strength of solution with HPLC.Experiment shows, this Angiotensin synthesis of receptor static adsorbance reaches 150 mg/g; Its trace factor (IF=Q
MIPs/ Q
NIPsQ
MIPsThe adsorptive capacity of blot gel to target molecule, Q
NIPsThe adsorptive capacity of non-blot gel to target molecule) be 4.0.
Claims (10)
1. a small peptide and derivative thereof are as the application of template molecule, it is characterized in that for engram technology, preparing the Angiotensin synthesis of receptor, described template molecule comprises a kind of in any one section peptide bond of the nitrogen end of any one section peptide bond of the nitrogen end of angiotensinⅡ or carbon teminal and derivative or angiotensinⅠ or carbon teminal and derivative thereof.
2. based on engram technology, prepare the method for Angiotensin synthesis of receptor, its feature comprises the steps:
(1) utilize a kind of as template molecule in any one section peptide bond of the nitrogen end of any one section peptide bond of the nitrogen end comprise angiotensinⅡ or carbon teminal and derivative or angiotensinⅠ or carbon teminal and derivative thereof;
(2) by template molecule, function monomer A, gel skeleton monomer are dissolved in distilled water and obtain solution 1; Function monomer B is dissolved in dehydrated alcohol and obtains solution 2; Solution 2 is mixed in above-mentioned solution 1, and vibration preact, obtain solution 3;
(3) linking agent monomer and Surfactant SDS are added in the solution 3 of step (2), logical nitrogen 15～60min, add initiator, sealing, and the nitrogen atmosphere reaction, obtain the molecular imprinting nanogel;
(4) purifying of imprinted polymer: the molecular imprinting nanogel obtained in step (3) is proceeded in dialysis tubing, dialyse in distilled water, the dialysis postlyophilization, obtaining the nanogel acceptor is the Angiotensin synthesis of receptor.
3. method as claimed in claim 2, it is characterized in that: template molecule described in step (1) comprises the C end tripeptide derivative template (Ac-His-Pro-Phe) of following (1) angiotensinⅡ, (2) C of angiotensinⅠ end tripeptide derivative template (Ac-Phe-His-Leu), the N end tripeptide derivative template (Asp-Arg-Val-NHC of (3) angiotensinⅡ
2H
5), the N end one peptide derivant template (Asp-NHC of (4) angiotensinⅡ
2H
5), the C end dipeptidase derivant template (Ac-Pro-Phe) of (5) angiotensinⅡ, the N end pentapeptide derivative template (Asp-Arg-Val-Tyr-Ile-NHC of (6) angiotensinⅡ
2H
5) in more than one.
4. method as claimed in claim 2, it is characterized in that: 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.
5. method as claimed in claim 2, it is characterized in that: 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; The mol ratio of described template molecule, function monomer A, tri-kinds of materials of function monomer B 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 ︰ skeleton monomer=1:(0.17～1) ︰ (1.3～2.1).
6. method as claimed in claim 2 is characterized in that: described dehydrated alcohol volume described in step (2) be in solution 1 volume 2%～5%; The purity of all ingredients that adopts be analytical pure and more than, distilled water is the secondary redistilled water; The time of vibration preact is 4-6 hour.
7. method as claimed in claim 2, it is characterized in that: in step (2) and (3), function monomer A, function monomer B, skeleton monomer and cross-linking monomer totally four kinds of monomers total concn in solution 3 are 6～10mM, wherein the linking agent monomer 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 ℃, and the reaction times is 24～36 hours.
8. method as claimed in claim 2, it is characterized in that: described in step (3), the linking agent monomer is N, the N-methylene diacrylamide, its concentration be in step (2) three kinds of total monomer 2%～4%; Described initiator is ammonium persulphate and N, N, N, N-tetramethyl-diethylamine combination, 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.
9. method as claimed in claim 2, it is characterized in that: described in step (4), the dialysis tubing specification is MWCO 10000, the dialysis tubing molecular weight cut-off is 10000; Described dialysis time at least four days, change twice water every day at least; Described sublimation drying is 2-3 days.
10. by the described method of claim 1 ~ 9 or apply resulting Angiotensin synthesis of receptor.
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| CN110563883A (en) * | 2019-07-21 | 2019-12-13 | 北京化工大学 | Preparation method and application of nano artificial antibody of targeting brain natriuretic peptide |
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