CN104152433A - Glucose response microtubule- kinesin transport system and preparation method thereof - Google Patents
Glucose response microtubule- kinesin transport system and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a glucose response microtubule- kinesin transport system and a preparation method thereof. The preparation method for the glucose responding microtubule-kinesin transport system comprises the following steps: the glucose oxidase microspheres or micro capsules are dispersed in a protein liposome solution containing ATP synthetase; after reaction, collection and precipitation, a glucose oxidase microspheres or micro capsules / ATP synthetase assembly system is obtained; the microtubules, the glucose oxidase microsphere or the micro capsules/ ATP synthetase assembly system, ADP, sodium biphosphate, catalase and beta- mercaptoethanol are mixed uniformly, so as to obtain a dispersion liquid; casein water solution, kinesin water solution and the dispersion liquid are sequentially injected into a fluid tank; all the substances inside the fluid tank constitute the microtubule-kinesin transport system. According to the glucose response microtubule- kinesin transport system provided by the invention, the glucose oxidase microspheres or the microcapsules can provide glucose response energy supply to the microtubule kinesin transport system and the glucose oxidase microspheres or the microcapsules can be used as a carrier which transports various substances.
Description
Technical field
The present invention relates to biological technical field, relate in particular to microtubule-kinesin transport system of a kind of glucose responding and preparation method thereof.
Background technology
Zoic motor system all with close the contacting of transporting of energy, this is mainly the result with the high molecular weight protein acting of motor function, these protein are called as molecular motor or motor protein.Up to the present, the mankind have determined hundreds of motor protein, and they are bringing into play various functions in body, by its mode of motion, can be divided into motion of translation motor and the motor that rotatablely moves.Rotation motor ATP synthetic enzyme (F wherein
of
1rotatablely moving-ATPase) can catalyze and synthesize ATP, for cellular activity provides energy, is the core enzyme of Conversion of energy, therefore inspired vast researcher to carry out the bionical research of rotation motor ATP synthetic enzyme.As a kind of transmembrane protein, ATP synthetic enzyme by successful reconstruct in liposome, in this system, the function of ATP synthetic enzyme is similar in organism.In order to improve the stability of system, the Modulatory character of intelligent and size, some investigators successfully cover the reconstruct of ATP synthetic enzyme at phosphatide in recent years polymkeric substance or protein surface of microcapsule, simulate better its function in biomass cells, also expanded the application prospect of ATP synthetic enzyme at aspects such as biological and nano-devices simultaneously.
Kinesin (kinesin) is intracellular a kind of linear motor protein molecular, it can be hydrolyzed Triphosaden (ATP), convert chemical energy to mechanical energy, thereby along the directed transportation of cytoskeletal microtubule (microtubule, MT) nanometer goods (as vesica, karyomit(e), organoid etc.).In view of robustness and the high efficiency of this motor molecular motion, during active biomimetic system on constructing micrometre or nano level yardstick, kinesin becomes a kind of desirable drive element, has attracted more and more scientists' interest.Ten years in the past, transporting cargo (as polystyrene spheres, quantum dot, DNA molecular) on the surface that microtubule is widely used as modifying at kinesin into carrier.Yet, these transport systems are applied in practical application, the system that need to develop some intelligent responses is controlled its transportation better.For example, some investigators use ATP cage compound, and the microtubule that controls kinesin driving by the switch of UV-light moves; The microtubule that also has some seminars to control kinesin driving by the polymkeric substance of the polymkeric substance with electrical response performance or temperature-responsive moves.
Summary of the invention
The preparation method who the object of this invention is to provide a kind of microtubule-kinesin transport system of glucose responding.
Preparation method provided by the invention, comprises the steps:
1) prepare respectively glucose oxidase microballoon or microcapsule, containing the proteolipid liquid solution of ATP synthetic enzyme;
Above-mentioned glucose oxidase microballoon or microcapsule can utilize template, coprecipitation method, self-assembly layer by layer, emulsion method or salting-out process preparation,
Glucose oxidase microballoon is specifically prepared as follows: get Na
2cO
3the aqueous solution, the GOD aqueous solution, CaCl
2the aqueous solution mixes, standing and reacting, and centrifuging and taking throw out, obtains GOD microballoon; In above-mentioned reaction, Na
2cO
3, GOD and CaCl
2quality proportioning is 1:1-10:1, is specially 35:4:37;
Glucose oxidase microcapsule are specifically prepared as follows: first, the manganous carbonate particle that is 2 μ m by particle diameter is distributed in PAH (PAH) solution that contains 0.1M sodium-chlor, after vibration absorption 30min, centrifugation collecting precipitation, fully, after washing, redispersion adsorbs 1h, centrifugation collecting precipitation in the glutaraldehyde that contains 0.025wt% (GA) aqueous solution, wash 3 times, obtain microballoon; Then the microballoon redispersion obtaining is adsorbed to 3h in the 4mg/mL GOD aqueous solution that contains 0.1M sodium-chlor, centrifugation collecting precipitation, washes 3 times; Repeat successively to adsorb the operation of GA, GOD, until the required number of plies; Then to the disodium EDTA solution that adds 0.1M in the microballoon obtaining, concussion reaction 3h, molten except manganous carbonate particle, after centrifugal collecting precipitation, washing 3 times, obtain (GA/GOD)
nmicrocapsule.Every absorption GA, a GOD are one deck.Be 6 layers in an embodiment of the present invention, obtain (GA/GOD)
6microcapsule, particle diameter is 2 μ m.
The mass ratio of described manganous carbonate particle, PAH, glutaraldehyde, GOD is 160mg:4mg:1mg:8mg.
Glucose oxidase microcapsule can also have been assembled one deck catalase (CAT) more and obtain (GA/GOD)
6gA/CAT/GA/GOD microcapsule, are specially (GA/GOD)
6microcapsule are distributed in glutaraldehyde (GA) aqueous solution that contains 0.025wt% and adsorb 1h, and centrifugation collecting precipitation, washes 3 times; The microballoon obtaining is distributed in the 4mg/mL CAT aqueous solution that contains 0.1M sodium-chlor and adsorbs 3h again, centrifugation collecting precipitation, washes 3 times; Same operation, then adsorb successively GA, GOD at microsphere surface; Then to the disodium EDTA solution that adds 0.1M in the microballoon obtaining, concussion reaction 3h, molten except manganous carbonate particle, after centrifugal collecting precipitation, washing 3 times, obtain (GA/GOD)
6gA/CAT/GA/GOD microcapsule; Wherein, the proportioning of manganous carbonate particle, PAH, glutaraldehyde, GOD, CAT is that mass ratio is 160mg:4mg:1mg:8mg:8mg.
The above-mentioned proteolipid liquid solution containing ATP synthetic enzyme is prepared according to the method comprising the steps: by ATP synthetic enzyme, washing agent Triton-100, liposome, at damping fluid, (containing final concentration is 40mM NaCl and 5mM MgCl
2the 20mM Tricine damping fluid of pH8.0) in mix, 4 ℃ of stirring reaction 1h; Add Bio-beads stirring at room reaction 1h to remove washing agent, centrifugal collection supernatant liquor, repeats 3 times again; Obtain the proteolipid liquid solution containing ATP synthetic enzyme, in above-mentioned reaction, the quality proportioning of ATP synthetic enzyme, Triton-100, liposome is 0.05mg:16mg:10mg (0.01～0.05mg:8～16mg:1～10mg).
Described liposome obtains liposome for following two kinds of materials are mixed to rear aquation according to following mass ratio: DMPC (DMPC): two myristoyl sodium phosphate (DMPA) quality proportionings are 9:1, specifically be prepared as follows: take DMPC (DMPC) and two myristoyl sodium phosphates (DMPA) (mass ratio 9:1) are dissolved in the mixed solvent of chloroform and methyl alcohol (volume ratio 1:1) simultaneously, ultrasonic it is dissolved completely, 30 ℃ of rotary evaporations obtain even dry adipose membrane, add 60 ℃ of water hydratables, form lipid suspension, ultrasonic until obtain the PA/PC liposome (10mg/mL) of clear to lipid suspension water-bath.
2) described glucose oxidase microballoon or microcapsule are dispersed in the proteolipid liquid solution containing ATP synthetic enzyme, reaction, collecting precipitation, obtains glucose oxidase microballoon or microcapsule/ATP synthetic enzyme assembly system;
3) microtubule, described glucose oxidase microballoon or microcapsule/ATP synthetic enzyme assembly system, ADP, SODIUM PHOSPHATE, MONOBASIC, catalase and beta-mercaptoethanol are mixed, obtain dispersion liquid;
4) caseic aqueous solution, the kinesin aqueous solution and described dispersion liquid are added in reaction vessel and mixed successively, obtain microtubule-kinesin transport system.
In aforesaid method,
Step 1) in, the particle diameter of described glucose oxidase microballoon or microcapsule is 1-4 μ m, and described glucose oxidase microspherulite diameter is specially 2.6 μ m or 4 μ m, and the particle diameter of described microcapsule is concrete 2 μ m or 3 μ m microcapsule;
The described final concentration containing ATP synthetic enzyme in the proteolipid liquid solution of ATP synthetic enzyme is 100-500nM, and the described final concentration containing ATP synthetic enzyme in the proteolipid liquid solution of ATP synthetic enzyme is specially 200nM;
Step 3), in, described microtubule is the microtubule of the biotin modification of rhodamine mark;
The microtubule of the biotin modification of described rhodamine mark is prepared according to the method comprising the steps:: after the microtubule of the microtubule of rhodamine mark, the microtubule of biotin modification and unmodified (volume ratio 1:1:2, the concentration of microtubule is 5mg/mL) mixes, be scattered in and contain 4mM MgCl
2, 1mM GTP (GTP (guanosine triphosphate)) and quality percentage composition 5%DMSO BRB80 damping fluid in, total protein concentration is 2.5mg/ml; The water-bath of then putting into 37 ℃ is hatched after 30min, add 4 μ L containing in the BRB80 damping fluid of 20 μ M taxol (taxol), can obtain the microtubule of the biotin modification of rhodamine mark.
Described glucose oxidase microballoon or microcapsule/ATP synthetic enzyme assembly system are glucose oxidase microballoon or the microcapsule/ATP synthetic enzyme assembly system that Streptavidin is modified.
In an embodiment of the present invention, glucose oxidase microcapsule/ATP synthetic enzyme the assembly system that adopts Streptavidin to modify, specifically be prepared as follows: 500 μ L (GOD content is 0.2mg/mL) GOD microcapsule/ATP synthetic enzyme assembly system is distributed in the aqueous solution of 10 μ L 50 μ M Streptavidins, absorption 30min, centrifugal collecting precipitation, wash three times, obtain GOD microcapsule/ATP synthetic enzyme assembly system dispersion liquid that Streptavidin is modified, be glucose oxidase microcapsule/ATP synthetic enzyme assembly system that Streptavidin is modified.
In aforesaid method,
Step 2) in, described glucose oxidase microballoon and the described proteolipid weight proportioning containing in the proteolipid liquid solution of ATP synthetic enzyme are 10-16:5, and described glucose oxidase microballoon and the described proteolipid weight proportioning containing in the proteolipid liquid solution of ATP synthetic enzyme are specially 16:5;
Or in described glucose oxidase microcapsule glucose oxidase content and described containing the proteolipid weight proportioning in the proteolipid liquid solution of ATP synthetic enzyme, be 1:10-25, in described glucose oxidase microcapsule, glucose oxidase content is specially 1:25 with the described proteolipid weight proportioning containing in the proteolipid liquid solution of ATP synthetic enzyme;
Step 4) in,
The proportioning of described casein, described kinesin, described microtubule, described glucose oxidase microballoon/ATP synthetic enzyme assembly system, described ADP, described SODIUM PHOSPHATE, MONOBASIC, described catalase, described beta-mercaptoethanol is 0.025mg:1-1.5pmol:1-5 μ g:50-100 μ g:0.1-0.5 μ mol:0.1-0.5 μ mol:0.01-0.05 μ g:1-5 μ g;
The proportioning of described casein, described kinesin, described microtubule, described glucose oxidase microballoon/ATP synthetic enzyme assembly system, described ADP, described SODIUM PHOSPHATE, MONOBASIC, described catalase, described beta-mercaptoethanol is specially 0.025mg:1.5pmol:5 μ g:80 μ g:0.5 μ mol:0.25 μ mol:0.04 μ g:2.5 μ g;
Or the proportioning of described casein, described kinesin, described microtubule, described glucose oxidase microcapsule/ATP synthetic enzyme assembly system, described ADP, described SODIUM PHOSPHATE, MONOBASIC, described catalase, described beta-mercaptoethanol is 0.03mg:1-1.5pmol:1-5 μ g:1-5 μ g:0.1-0.5 μ mol:0.1-0.5 μ mol:0.01-0.05 μ g:1-5 μ g;
The proportioning of described casein, described kinesin, described microtubule, described glucose oxidase microcapsule/ATP synthetic enzyme assembly system, described ADP, described SODIUM PHOSPHATE, MONOBASIC, described catalase, described beta-mercaptoethanol is specially 0.03mg:1.35pmol:5 μ g:1 μ g:0.5 μ mol:0.25 μ mol:0.04 μ g:2.5 μ g;
Described glucose oxidase microcapsule/ATP synthetic enzyme assembly system is glucose oxidase microcapsule/ATP synthetic enzyme assembly system that Streptavidin is modified.
In aforesaid method,
Step 2), in, the described reaction times is 30min;
Step 4), in, the described kinesin aqueous solution joining day is 5min after caseic aqueous solution adds;
The described dispersion liquid joining day adds rear 5min for stating the kinesin aqueous solution.
In aforesaid method,
Step 1), in, described glucose oxidase microballoon or microcapsule are for loading microballoon or the microcapsule of polyelectrolyte, protein, polysaccharide or medicine.
Step 4) in, described kinesin is kinesin-1, and its aminoacid sequence is sequence 1 in sequence table.
Microtubule-kinesin transport system of glucose responding prepared by aforesaid method is also the scope of protection of the invention.
The application of microtubule-kinesin transport system of above-mentioned glucose responding in preparing nano-device is also the scope of protection of the invention.
Microtubule-kinesin transport system of above-mentioned glucose responding is also the scope of protection of the invention in the application as in transport agent.
The present invention of experiment showed, of the present invention prepares a kind of microtubule-kinesin transport system of glucose responding, and this transport system tool has the following advantages:
(1) microtubule-kinesin transport system of the present invention has glucose responding, and in having the solution of glucose, ATP synthetic enzyme can change into ATP by the ADP in system, and kinesin can drive microtubule transportation; In the solution without glucose, without ATP, produce, kinesin can not drive microtubule transportation.
(2) microtubule-kinesin transport system of the present invention can be supplied with energy certainly, without additionally adding ATP.
(3) microtubule-kinesin transport system of the present invention can realize the regeneration of ATP, the concentration of ATP in buffer system, thereby the sustainable supply of assurance ATP.
(4) the glucose oxidase microballoon in the present invention or capsule not only can, for microtubule-kinesin transport system continues to provide energy, can also transport different materials as carrier.
(5) microtubule-kinesin transport system in the present invention can realize Partial controll (, only in the region that has glucose, microtubule can move; The region that there is no glucose, microtubule can not move) microtubule-kinesin transport system.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscopy figure (Figure 1A) of ATP synthetase albumen liposome of the embodiment of the present invention 1 preparation and the transmission electron microscopy figure (Figure 1B) that ATP synthetase albumen liposome adsorbs at glucose oxidase microsphere surface.
Fig. 2 is the F that recombinated in embodiment 1
of
1the ATP temporal evolution graphic representation that the glucose oxidase microballoon of-ATPase proteoliposome produces.
Fig. 3 is the velocity-time relation curve moving on the surface that in embodiment 1, microtubule is modified at kinesin.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, fluid pool used is prepared as follows:
Common fluid pond forms (Scotch 3M, 0.1mm is thick) by slide glass, cover glass and double faced adhesive tape.Slide glass and cover glass be ultrasonic 10min in saturated KOH solution first, then uses ethanol and washed with de-ionized water, then dries up with liquid nitrogen.If no special instructions, experiment use is all common fluid pond.
In following embodiment, the preparation method of the microtubule of the biotin modification of rhodamine mark used is: after the microtubule of the microtubule of rhodamine mark, the microtubule of biotin modification and unmodified (volume ratio 1:1:2, the concentration of microtubule is 5mg/mL) mixes, be scattered in and contain 4mM MgCl
2, 1mM GTP (GTP (guanosine triphosphate)) and quality percentage composition 5%DMSO BRB80 damping fluid in, total protein concentration is 2.5mg/ml; The water-bath of then putting into 37 ℃ is hatched after 30min, add 4 μ L containing in the BRB80 damping fluid of 20 μ M taxol (taxol), can obtain the microtubule of the biotin modification of rhodamine mark.
Wherein, the microtubule of the microtubule of rhodamine mark, the microtubule of biotin modification and unmodified is all purchased from Cytoskeleton company limited, and cat. no is respectively TL590M, T333P and TL238.
In following embodiment, kinesin used is that Kinesin-1 (its aminoacid sequence is sequence 1 in sequence table :) is prepared as follows: the total length drosophila melanogaster C end kinesin heavy chain of histidine mark and the kinesin plasmid of light chain are expressed in intestinal bacteria Escherichia coli (purchased from the calm and peaceful Bioisystech Co., Ltd of Sino-U.S.), then the albumen of using nickel-nitrilotriacetic acid agarose resin (nickel-nitrilotriacetic acid agarose resin, Ni-NTA) post and phosphocellurose column to purify and express.
Embodiment 1, prepare microtubule-kinesin transport system of glucose responding
1, microtubule-kinesin transport system of glucose responding preparation
(1) prepare glucose oxidase (GOD) microballoon
Getting concentration is 0.33M Na
2cO
3water liquid and concentration be the 4mg/ml GOD aqueous solution in round-bottomed flask, it is disposable subsequently that to add fast isopyknic concentration be 0.33M CaCl
2the aqueous solution in flask, stirring and evenly mixing 20s immediately, the about 2min of standing and reacting, centrifuging and taking throw out, 3 washings, obtain the GOD microballoon of 4 μ m.
In above-mentioned reaction, Na
2cO
3, GOD and CaCl
2quality proportioning is 1:1-10:1, is specially 35:4:37;
(2) preparation is containing the proteolipid liquid solution of ATP synthetic enzyme
ATP synthetic enzyme is prepared as follows: by fresh spinach leaves, and arteries and veins, cleaning in removal; In stirrer, stir leaf to being grain of rice shape size completely, with Nylon Bag, filter, remove filter residue; Get filtrate centrifugal, collecting precipitation thing, puts into hypotonic buffer solution (10mM Tris-HCl pH 8.0 and 0.5mM MgCl
2) in, stir, centrifugal, taking precipitate is put into high ionic strength washing lotion damping fluid (0.4M sucrose, 10mM Tris-HCl pH 8.0,150mM NaCl and 0.5mM MgCl
2) in, stir, centrifugal, taking precipitate adds buffer suspension liquid (0.4M sucrose, 50mM Tricine-NaOH pH 8.0,0.2mM MgCl
2) in.Add wherein solid DTT (final concentration 50mM), stir; Add subsequently isopyknic Extraction buffer, stir 30min at 4 ℃, centrifugal, get supernatant liquor and add (NH4) 2SO4 powder, centrifugal, collecting precipitation thing; The crude protein solution obtaining is mixed with isopyknic density gradient buffered soln, be followed successively by: 60%, centrifugal in 52%, 44%, 36%, 28%, 20% sucrose density gradient, collect 44% sucrose layer, be ATP synthetic enzyme, final buffered soln is: 1.25mM sucrose, 30mM Na
2hPO
4-NaOH pH 7.2,2mM MgCl
2, 0.5mM Na
2eDTA, 4mM dodecyl maltoside, stores in liquid nitrogen.
Liposome obtains liposome for following two kinds of materials are mixed to rear aquation according to following mass ratio: DMPC (DMPC): two myristoyl sodium phosphate (DMPA) quality proportionings are 9:1, specifically be prepared as follows: take DMPC (DMPC) and two myristoyl sodium phosphates (DMPA) (mass ratio 9:1) are dissolved in the mixed solvent of chloroform and methyl alcohol (volume ratio 1:1) simultaneously, ultrasonic it is dissolved completely, 30 ℃ of rotary evaporations obtain even dry adipose membrane, add 60 ℃ of water hydratables, form lipid suspension, ultrasonic until obtain the PA/PC liposome (10mg/mL) of clear to lipid suspension water-bath.
By ATP synthetic enzyme, washing agent Triton-100, liposome, at damping fluid, (containing final concentration is 40mM NaCl and 5mM MgCl
2the 20mM Tricine damping fluid of pH8.0) in mix, 4 ℃ of stirring reaction 1h; Add Bio-beads stirring at room reaction 1h to remove washing agent, centrifugal collection supernatant liquor, repeats 3 times again; Obtain the proteolipid liquid solution containing ATP synthetic enzyme, concentration is 5mg/ml, in liquid nitrogen, preserves, and ATP synthetic enzyme is 200nM at the final concentration containing in the proteolipid liquid solution of ATP synthetic enzyme.
In above-mentioned reaction, the quality proportioning of ATP synthetic enzyme, Triton-100, liposome is 0.05mg:16mg:10mg (0.01～0.05mg:8～16mg:1～10mg).
As shown in Figure 1A, the median size of proteoliposome is 180nm to the transmission electron microscopy figure of the proteoliposome of above-mentioned preparation.
(3) prepare GOD microballoon/ATP synthetic enzyme assembly system
The GOD microballoon of 500 μ L 16mg/mL prepared by step (1) is distributed to 500 μ L 5mg/mL containing in the proteolipid liquid solution of ATP synthetic enzyme, concussion evenly, absorption 30min, 4 ℃ of centrifuge washings, collecting precipitation is GOD microballoon/ATP synthetic enzyme assembly system (transmission electron microscopy figure as shown in Figure 1B).
The content that detects GOD microballoon/ATP synthetic enzyme assembly system ATP is as follows:
By the GOD microballoon/ATP synthetic enzyme assembly system obtaining be distributed to the buffered soln that 2mL contains 18wt% glucose (pH 8.0, containing 10mM tricine, 30mM NaCl, 2.5mM MgCl
2, 5mM NaH
2pO
4with 0.2mM ADP) in, from system, take out the sample of 20 μ L, join in fluorescein-luciferase ATP measurement system, with light-emitting appearance, measure luminous signal, thereby calculate the content of ATP in system.
Result as shown in Figure 2, adds glucose, and along with the increase in reaction times, ATP concentration continues to rise, and has realized the controlledly synthesis of the ATP of glucose responding.
(4) acquisition of microtubule-kinesin transport system of glucose responding
Prepare dispersion liquid: the microtubule of the biotin modification of 2 μ L 2.5mg/mL rhodamine marks, 5 μ L 16mg/mLGOD microballoon/ATP synthetic enzyme assembly systems, 25 μ L 20mM ADP, 25 μ L 10mM SODIUM PHOSPHATE, MONOBASIC (NaH
2pO
4), 5 μ L 0.008mg/mL catalases and the 0.5 μ L 0.5wt% beta-mercaptoethanol aqueous solution mixes, and obtains dispersion liquid.
The preparation of microtubule-kinesin transport system:
First, casein (1mg/mL) solution is injected to fluid pool, absorption 5min; Then the kinesin-1 aqueous solution (20nM) is injected to fluid pool, absorption 5min; Then, above-mentioned dispersion liquid is slowly injected to fluid pool, obtain microtubule-kinesin transport system.
In microtubule-kinesin transport system, each material is as follows: the microtubule of the biotin modification of 50 μ L 0.5mg/mL caseins, 50 μ L 30nM kinesin-1,2 μ L 2.5mg/mL rhodamine marks, 5 μ L 16mg/mL GOD microballoon/ATP synthetic enzyme assembly systems, 25 μ L 20mM ADP, 25 μ L 10mM SODIUM PHOSPHATE, MONOBASIC (NaH
2pO
4), 5 μ L 0.008mg/mL catalases, the 0.5 μ L 0.5wt% beta-mercaptoethanol aqueous solution;
In microtubule-kinesin transport system, the proportioning of each material is as follows: the microtubule of the biotin modification of casein, kinesin-1, rhodamine mark, GOD microballoon/ATP synthetic enzyme assembly system, ADP, SODIUM PHOSPHATE, MONOBASIC (NaH
2pO
4), the proportioning of catalase, beta-mercaptoethanol is 0.025mg:1.5pmol:5 μ g:80 μ g:0.5 μ mol:0.25 μ mol:0.04 μ g:2.5 μ g.
2, detect microtubule-kinesin transport system to glucose responding ability
By adding final concentration in above-mentioned microtubule-kinesin transport system, be the glucose of 26wt% (quality percentage composition), mix, fluid pool is got up with silica gel sealing, move under Laser Scanning Confocal Microscope and observe.Take and do not add glucose as contrast.
Record MT moving image in time, result as shown in Figure 3, does not add in the system of glucose, and microtubule does not move; And add the system of glucose, and As time goes on, on the surface that microtubule MT can successfully modify at kinesin-1, slide, sliding velocity average out to 19nm/s.
Embodiment 2, prepare microtubule-kinesin transport system of glucose responding
1, microtubule-kinesin transport system of glucose responding preparation
In step prepared by microtubule-kinesin transport system of the present embodiment glucose responding and parameter and embodiment 1,1 is basic identical, and difference is step (1):
(1) preparation loads glucose oxidase (GOD) microballoon of medicine
Getting concentration is the Na that 0.33M contains 2mg/mL fluorescein isothiocyanate-dextran (FITC-Dextran)
2cO
3water liquid and concentration be the 4mg/mL GOD aqueous solution in round-bottomed flask, it is disposable subsequently that to add fast isopyknic concentration be 0.33M CaCl
2the aqueous solution, in flask, stirs 20s immediately, the about 2min of standing and reacting, and centrifuging and taking throw out, 3 washings, obtain GOD-medicine complex microsphere (particle diameter is 2.6 μ m).
In above-mentioned reaction, Na
2cO
3, FITC-Dextran, GOD and CaCl
2quality proportioning be 35mg:2mg:4mg:37mg;
(2) preparation is containing the proteoliposome of ATP synthetic enzyme
Identical with embodiment 11 (2);
(3) prepare GOD microballoon/ATP synthetic enzyme assembly system
Identical with embodiment 11 (3), obtain GOD microballoon/ATP synthetic enzyme assembly system;
(4) acquisition of microtubule-kinesin transport system of glucose responding
Identical with embodiment 11 (4), obtain microtubule-kinesin transport system;
2, detect microtubule-kinesin transport system to glucose responding ability
2 identical with embodiment 1, does not add in the system of glucose, and microtubule does not move; And add the system of glucose, and As time goes on, on the surface that microtubule MT can successfully modify at kinesin, slide, sliding velocity average out to 30nm/s.
3, the application of microtubule-kinesin transport system in transport agent
Microtubule-kinesin transport system of above-mentioned 2 preparations is moved under Laser Scanning Confocal Microscope and observed, and copolymerization Jiao tests and shows, microtubule-kinesin transport system can be transported fluorescein isothiocyanate-dextran.
Therefore, can think that microtubule-kinesin transport system can directed transportation medicine, microtubule-kinesin the transport system that is loaded with medicine under glucose stimulates is moving to not mobile containing stopping in glucose system, thereby unloading medicine is realized directed transportation medicine.
Embodiment 3, prepare microtubule-kinesin transport system of glucose responding
1, microtubule-kinesin transport system of glucose responding preparation
(1) prepare GOD microcapsule (this embodiment of microcapsule prepares microcapsule, also can prepare Nano capsule, and just template is not manganous carbonate, changes nano silicon or nano-calcium carbonate into)
First, the manganous carbonate particle that is 2 μ m by particle diameter is distributed in the PAH solution (sigma company buys) that contains 0.1M sodium-chlor, after vibration absorption 30min, centrifugation collecting precipitation, fully, after washing, redispersion adsorbs 1h, centrifugation collecting precipitation in the glutaraldehyde that contains 0.025wt% (GA) aqueous solution, wash 3 times, obtain microballoon;
Then the microballoon redispersion obtaining is adsorbed to 3h in the 4mg/mL GOD aqueous solution that contains 0.1M sodium-chlor, centrifugation collecting precipitation, washes 3 times; Repeat successively to adsorb the operation of GA, GOD, until the required number of plies; Then to the disodium EDTA solution that adds 0.1M in the microballoon obtaining, concussion reaction 3h, molten except manganous carbonate particle, after centrifugal collecting precipitation, washing 3 times, obtain (GA/GOD)
nmicrocapsule.
Every absorption GA, a GOD are one deck, and the present embodiment is 6 layers, obtains (GA/GOD)
6microcapsule, particle diameter is 2 μ m.
The mass ratio of described manganous carbonate particle, PAH, glutaraldehyde, GOD is 160mg:4mg:1mg:8mg.
(2) preparation is containing the proteolipid liquid solution of ATP synthetic enzyme
Identical with embodiment 11 (2);
(3) prepare GOD microcapsule/ATP synthetic enzyme assembly system
500 μ L prepared by step (1) (GOD content is 0.2mg/mL) GOD microcapsule are distributed to 500 μ L5mg/mL containing in the proteolipid liquid solution of ATP synthetic enzyme, concussion evenly, absorption 30min, 4 ℃ of centrifuge washings, collecting precipitation is GOD microcapsule/ATP synthetic enzyme assembly system.
(4) acquisition of microtubule-kinesin transport system of glucose responding
(3) 500 μ L (GOD content is 0.2mg/mL) GOD microcapsule/ATP synthetic enzyme assembly system is distributed in the aqueous solution of 10 μ L 50 μ M Streptavidins, absorption 30min, centrifugal collecting precipitation, wash three times, obtain GOD microcapsule/ATP synthetic enzyme assembly system dispersion liquid that Streptavidin is modified; GOD microcapsule/ATP synthetic enzyme assembly system dispersion liquid of again 5 μ L (GOD content is 0.2mg/mL) Streptavidin being modified dropwise joins in the microtubule solution of biotin modification of 2 μ L 2.5mg/mL rhodamine marks, the lower jog 30min of room temperature (25 ℃), then add 25 μ L 20mM ADP, 25 μ L 10mM SODIUM PHOSPHATE, MONOBASIC (NaH
2pO
4), 5 μ L 0.008mg/mL catalases and 0.5 μ L 0.5wt% beta-mercaptoethanol mix, and obtains dispersion liquid.
The preparation of microtubule-kinesin transport system:
First, casein (1mg/mL) solution is injected to fluid pool, absorption 5min; Then the kinesin aqueous solution (20nM) is injected to fluid pool, absorption 5min; Then, above-mentioned dispersion liquid is slowly injected to fluid pool, obtain microtubule-kinesin transport system.
In microtubule-kinesin transport system, each material is as follows: GOD microcapsule/ATP synthetic enzyme assembly system, 25 μ L 20mM ADP, 25 μ L 10mM SODIUM PHOSPHATE, MONOBASIC (NaH that the microtubule of the biotin modification of 60 μ L 0.5mg/mL caseins, 45 μ L 30nM kinesin-1,2 μ L 2.5mg/mL rhodamine marks, 5 μ L (GOD content is 0.2mg/mL) Streptomycin sulphate are modified
2pO
4), 5 μ L 0.008mg/mL catalases, the 0.5 μ L 0.5wt% beta-mercaptoethanol aqueous solution;
In microtubule-kinesin transport system, the proportioning of each material is the microtubule of the biotin modification of casein, kinesin-1, rhodamine mark, GOD microcapsule/ATP synthetic enzyme assembly system, ADP, the SODIUM PHOSPHATE, MONOBASIC (NaH that Streptomycin sulphate is modified
2pO
4), the proportioning of catalase, beta-mercaptoethanol is as follows: 0.03mg:1.35pmol:5 μ g:1 μ g:0.5 μ mol:0.25 μ mol:0.04 μ g:2.5 μ g.
2, detect microtubule-kinesin transport system to glucose responding ability
By adding final concentration in above-mentioned microtubule-kinesin transport system, be the glucose of 18wt%, mix, fluid pool is got up with silica gel sealing, move under Laser Scanning Confocal Microscope and observe.Take and do not add glucose sugar as contrast.
Fluid pool is got up with silica gel sealing, move under Laser Scanning Confocal Microscope and observe, 2 identical with embodiment 1, does not add in the system of glucose, and microtubule does not move; And add the system of glucose, and As time goes on, on the surface that microtubule MT can successfully modify at kinesin, slide, sliding velocity average out to 50nm/s.
Embodiment 4, prepare microtubule-kinesin transport system of glucose responding
In step prepared by microtubule-kinesin transport system of the present embodiment glucose responding and parameter and embodiment 3,1 is basic identical, and difference is step (1):
(1) (GA/GOD)
ngA/CAT/GA/GOD microcapsule
Basic identical in the step of the present embodiment and parameter and embodiment 3, difference is: when preparation GOD microcapsule, assembled one deck catalase (CAT) more, obtained (GA/GOD)
ngA/CAT/GA/GOD microcapsule, specific as follows:
By (GA/GOD) that obtain in (1) step of 1 of embodiment 3
6microballoon is distributed in glutaraldehyde (GA) aqueous solution that contains 0.025wt% and adsorbs 1h, and centrifugation collecting precipitation, washes 3 times; The microballoon obtaining is distributed in the 4mg/mL CAT aqueous solution that contains 0.1M sodium-chlor and adsorbs 3h again, centrifugation collecting precipitation, washes 3 times; Same operation, then adsorb successively GA, GOD at microsphere surface; Then to the disodium EDTA solution that adds 0.1M in the microballoon obtaining, concussion reaction 3h, molten except manganous carbonate particle, after centrifugal collecting precipitation, washing 3 times, obtain (GA/GOD)
6gA/CAT/GA/GOD microcapsule (particle diameter is 3 μ m).
The proportioning of described manganous carbonate particle, PAH, glutaraldehyde, GOD, CAT is that mass ratio is 160mg:4mg:1mg:8mg:8mg.
(GA/GOD)
ngA/CAT/GA/GOD microcapsule can effectively be removed the hydrogen peroxide that catalytic oxidation of glucose produces, and the active oxygen that can reduce the concentration of hydrogen peroxide in system as far as possible and further generate, reduces the damage of active oxygen to microtubule or kinesin.
(2) preparation is containing the proteoliposome of ATP synthetic enzyme
Identical with embodiment 31 (2);
(3) prepare GOD microcapsule/ATP synthetic enzyme assembly system
Identical with embodiment 31 (3);
(4) prepare microtubule-kinesin transport system
Identical with embodiment 31 (4); Obtain microtubule-kinesin transport system.
2, detect microtubule-kinesin transport system to glucose responding ability
By adding final concentration in above-mentioned microtubule-kinesin transport system, be the glucose of 20wt%, mix, fluid pool is got up with silica gel sealing, move under Laser Scanning Confocal Microscope and observe.Take and do not add glucose sugar as contrast.Result shows, does not add in the system of glucose, and microtubule does not move; And add the system of glucose, and As time goes on, on the surface that microtubule MT can successfully modify at kinesin, slide, sliding velocity average out to 26nm/s.
Claims (8)
1. a preparation method for microtubule-kinesin transport system of glucose responding, comprises the steps:
1) prepare respectively glucose oxidase microballoon or microcapsule, containing the proteolipid liquid solution of ATP synthetic enzyme;
2) described glucose oxidase microballoon or microcapsule are dispersed in the proteolipid liquid solution containing ATP synthetic enzyme, reaction, collecting precipitation, obtains glucose oxidase microballoon or microcapsule/ATP synthetic enzyme assembly system;
3) microtubule, described glucose oxidase microballoon or microcapsule/ATP synthetic enzyme assembly system, ADP, SODIUM PHOSPHATE, MONOBASIC, catalase and beta-mercaptoethanol are mixed, obtain dispersion liquid;
4) caseic aqueous solution, the kinesin aqueous solution and described dispersion liquid are added in reaction vessel and mixed successively, obtain microtubule-kinesin transport system.
2. method according to claim 1, is characterized in that:
Step 1), in, the particle diameter of described glucose oxidase microballoon or microcapsule is 1-4 μ m;
The described final concentration containing ATP synthetic enzyme in the proteolipid liquid solution of ATP synthetic enzyme is 100-500nM, and the described final concentration containing ATP synthetic enzyme in the proteolipid liquid solution of ATP synthetic enzyme is specially 200nM;
Step 3), in, described microtubule is the microtubule of the biotin modification of rhodamine mark;
Described glucose oxidase microballoon or microcapsule/ATP synthetic enzyme assembly system are glucose oxidase microballoon or the microcapsule/ATP synthetic enzyme assembly system that Streptavidin is modified.
3. method according to claim 1 and 2, is characterized in that:
Step 2) in, described glucose oxidase microballoon and the described proteolipid weight proportioning containing in the proteolipid liquid solution of ATP synthetic enzyme are 10-16:5, and described glucose oxidase microballoon and the described proteolipid weight proportioning containing in the proteolipid liquid solution of ATP synthetic enzyme are specially 16:5;
Or in described glucose oxidase microcapsule glucose oxidase content and described containing the proteolipid weight proportioning in the proteolipid liquid solution of ATP synthetic enzyme, be 1:10-25, in described glucose oxidase microcapsule, glucose oxidase content is specially 1:25 with the described proteolipid weight proportioning containing in the proteolipid liquid solution of ATP synthetic enzyme;
Step 4) in,
The proportioning of described casein, described kinesin, described microtubule, described glucose oxidase microballoon/ATP synthetic enzyme assembly system, described ADP, described SODIUM PHOSPHATE, MONOBASIC, described catalase, described beta-mercaptoethanol is 0.025mg:1-1.5pmol:1-5 μ g:50-100 μ g:0.1-0.5 μ mol:0.1-0.5 μ mol:0.01-0.05 μ g:1-5 μ g;
The proportioning of described casein, described kinesin, described microtubule, described glucose oxidase microballoon/ATP synthetic enzyme assembly system, described ADP, described SODIUM PHOSPHATE, MONOBASIC, described catalase, described beta-mercaptoethanol is specially 0.025mg:1.5pmol:5 μ g:80 μ g:0.5 μ mol:0.25 μ mol:0.04 μ g:2.5 μ g;
Or the proportioning of described casein, described kinesin, described microtubule, described glucose oxidase microcapsule/ATP synthetic enzyme assembly system, described ADP, described SODIUM PHOSPHATE, MONOBASIC, described catalase, described beta-mercaptoethanol is 0.03mg:1-1.5pmol:1-5 μ g:1-5 μ g:0.1-0.5 μ mol:0.1-0.5 μ mol:0.01-0.05 μ g:1-5 μ g;
The proportioning of described casein, described kinesin, described microtubule, described glucose oxidase microcapsule/ATP synthetic enzyme assembly system, described ADP, described SODIUM PHOSPHATE, MONOBASIC, described catalase, described beta-mercaptoethanol is specially 0.03mg:1.35pmol:5 μ g:1 μ g:0.5 μ mol:0.25 μ mol:0.04 μ g:2.5 μ g;
Described glucose oxidase microcapsule/ATP synthetic enzyme assembly system is glucose oxidase microcapsule/ATP synthetic enzyme assembly system that Streptavidin is modified.
4. according to arbitrary described method in claim 1-3, it is characterized in that:
Step 2), in, the described reaction times is 30min;
Step 4), in, the described kinesin aqueous solution joining day is 5min after caseic aqueous solution adds;
The described dispersion liquid joining day adds rear 5min for stating the kinesin aqueous solution.
5. according to arbitrary described method in claim 1-4, it is characterized in that:
Step 1), in, described glucose oxidase microballoon or microcapsule are for loading microballoon or the microcapsule of polyelectrolyte, protein, polysaccharide or medicine.
6. microtubule-kinesin transport system of the glucose responding that in claim 1-5, described in any one prepared by method.
7. the application of microtubule-kinesin transport system of glucose responding in preparing nano-device described in claim 6.
Described in claim 6 microtubule-kinesin transport system of glucose responding in the application as in transport agent.
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