CN104152433B - A kind of micro-pipe-kinesin transportation system of glucose responding and preparation method thereof - Google Patents
A kind of micro-pipe-kinesin transportation system of glucose responding and preparation method thereof Download PDFInfo
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Abstract
Micro-pipe-kinesin transportation system that the invention discloses a kind of glucose responding and preparation method thereof. Preparation method provided by the invention, comprise the steps: to be dispersed in the proteolipid liquid solution containing ATP synzyme by described glucoseoxidase microsphere or microcapsule, reaction, collects precipitation, obtains glucoseoxidase microsphere or microcapsule/ATP synzyme assembly system; By micro-pipe, described glucoseoxidase microsphere or microcapsule/ATP synzyme assembly system, ADP, sodium dihydrogen phosphate, catalase and beta-mercaptoethanol mixing, obtain dispersion liquid; Caseic aqueous solution, kinesin aqueous solution and described dispersion liquid are implanted sequentially fluid pool, all substances composition micro-pipe-kinesin transportation system in described fluid pool. Glucoseoxidase microsphere or capsule in the present invention are possible not only to provide the energy supply of glucose responding into micro-pipe-kinesin transportation system, moreover it is possible to as the material that carrier transport is different.
Description
Technical field
The present invention relates to biological technical field, micro-pipe-kinesin transportation system particularly relating to a kind of glucose responding and preparation method thereof.
Background technology
Zoic motor system all with energy transport close contacting, this mainly has the result of high molecular weight protein acting of motor function, and these protein are referred to as molecular motor or motor protein. Up to the present, the mankind have determined that hundreds of motor protein, and they play various function in body, can be divided into linear movement motor and rotary motion motor by its forms of motion. Wherein rotation motor ATP synzyme (FoF1-ATPase) rotary motion can catalyze and synthesize ATP, for cellular activity provide energy, be energy convert core enzyme, therefore inspired vast researcher to carry out the bionics fiber of rotation motor ATP synzyme. As a kind of transmembrane protein, ATP synzyme is successfully reconstructed in liposome, and in this system, the function of ATP synzyme is similar in organism. In order to improve the Modulatory character of the stability of system, intelligent and size, in recent years some researcheres successfully by ATP synzyme reconstruct phospholipid cover polymer or protein microcapsules surface, simulate its function in biological cell better, also extend ATP synzyme application prospect in biology and nano-device etc. simultaneously.
Kinesin (kinesin) is intracellular a kind of linear motor protein molecular, it can be hydrolyzed adenosine triphosphate (ATP), convert chemical energy into mechanical energy, thus transporting nanometer goods (such as vesicle, chromosome, organelle etc.) along cytoskeletal microtubule (microtubule, MT) is directed. In view of robustness and the high efficiency of this motor molecular motion, during active biomimetic system on constructing micrometre or nano-scale dimension, kinesin becomes a kind of desirably driver part, has attracted the interest of more and more scientist. In the ten years in past, micro-pipe is widely used as transporting goods (such as polystyrene spheres, quantum dot, DNA molecular) on the kinesin surface modified for carrier. But, these transportation systems are applied in practical application, it is necessary to develop the system of some intelligent responses to better control over its transport. Such as, some researcheres use ATP cage compound, control the kinesin microtubule driven motion by the switch of ultraviolet light; The polymer also having some seminars polymer or temperature-responsive by using electrical response performance controls the kinesin microtubule driven motion.
Summary of the invention
The preparation method that it is an object of the invention to provide the micro-pipe-kinesin transportation system of a kind of glucose responding.
Preparation method provided by the invention, comprises the steps:
1) glucoseoxidase microsphere or microcapsule, proteolipid liquid solution containing ATP synzyme are prepared respectively;
Prepared by above-mentioned glucoseoxidase microsphere or the available template of microcapsule, coprecipitation, LBL self-assembly, emulsion method or salting out method,
Glucoseoxidase microsphere is specifically prepared as follows: take Na2CO3Aqueous solution, GOD aqueous solution, CaCl2Aqueous solution mixes, standing and reacting, and namely centrifuging and taking precipitate obtains GOD microsphere; In above-mentioned reaction, Na2CO3, GOD and CaCl2Quality proportioning is 1:1-10:1, is specially 35:4:37;
Glucoseoxidase microcapsule is specifically prepared as follows: first, the manganese carbonate particle that particle diameter is 2 ��m is distributed in PAH (PAH) solution containing 0.1M sodium chloride, after vibration absorption 30min, precipitation is collected in centrifugation, fully after washing, redispersion adsorbs 1h in glutaraldehyde (GA) aqueous solution containing 0.025wt%, and precipitation is collected in centrifugation, wash 3 times, obtain microsphere; Then adsorbing 3h in the microsphere redispersion obtained to the 4mg/mLGOD aqueous solution containing 0.1M sodium chloride, precipitation is collected in centrifugation, washes 3 times; It is repeated in the operation of absorption GA, GOD, until the required number of plies; Then in the microsphere obtained, add the disodium EDTA solution of 0.1M, concussion reaction 3h, molten except manganese carbonate particle, after centrifugal collecting precipitation, washing 3 times, obtain (GA/GOD)nMicrocapsule. Often GA, GOD of absorption is one layer. It is 6 layers in an embodiment of the present invention, obtains (GA/GOD)6Microcapsule, particle diameter is 2 ��m.
Described manganese carbonate particle, PAH, glutaraldehyde, GOD mass ratio be 160mg:4mg:1mg:8mg.
Glucoseoxidase microcapsule can also assemble one layer of catalase (CAT) more and obtain (GA/GOD)6GA/CAT/GA/GOD microcapsule, is specially (GA/GOD)6Microcapsule is distributed in glutaraldehyde (GA) aqueous solution containing 0.025wt% and adsorbs 1h, and precipitation is collected in centrifugation, washes 3 times; Being distributed to by the microsphere obtained in the 4mg/mLCAT aqueous solution containing 0.1M sodium chloride again and adsorb 3h, precipitation is collected in centrifugation, washes 3 times;Same operation, then adsorb GA, GOD successively at microsphere surface; Then in the microsphere obtained, add the disodium EDTA solution of 0.1M, concussion reaction 3h, molten except manganese carbonate particle, after centrifugal collecting precipitation, washing 3 times, obtain (GA/GOD)6GA/CAT/GA/GOD microcapsule; Wherein, manganese carbonate particle, PAH, glutaraldehyde, GOD, CAT proportioning be mass ratio be 160mg:4mg:1mg:8mg:8mg.
The above-mentioned proteolipid liquid solution containing ATP synzyme is prepared according to the method comprised the steps: by ATP synzyme, detergent Triton-100, liposome at buffer (containing final concentration of 40mMNaCl and 5mMMgCl2The 20mMTricine buffer of pH8.0) in mixing, 4 DEG C of stirring reaction 1h; Add Bio-beads and reaction 1h removal detergent, centrifugal collection supernatant are stirred at room temperature, repeat 3 times; Namely obtain the proteolipid liquid solution containing ATP synzyme, ATP synzyme in above-mentioned reaction, Triton-100, liposome quality proportioning be 0.05mg:16mg:10mg (0.01��0.05mg:8��16mg:1��10mg).
Described liposome obtains liposome for aquation after being mixed according to following mass ratio by the following two kinds material: DMPC (DMPC): two myristoyl sodium phosphate (DMPA) quality proportionings are 9:1, specifically it is prepared as follows: weigh DMPC (DMPC) and two myristoyl sodium phosphates (DMPA) (mass ratio 9:1) are dissolved in the mixed solvent of chloroform and methanol (volume ratio 1:1) simultaneously, ultrasonic it is made to be completely dissolved, 30 DEG C of rotary evaporations obtain uniform dry adipose membrane, add 60 DEG C of water hydratables, form lipid suspension, to lipid suspension water bath sonicator until obtaining the PA/PC liposome (10mg/mL) of clear.
2) described glucoseoxidase microsphere or microcapsule are dispersed in the proteolipid liquid solution containing ATP synzyme, reaction, collect precipitation, obtain glucoseoxidase microsphere or microcapsule/ATP synzyme assembly system;
3) by micro-pipe, described glucoseoxidase microsphere or microcapsule/ATP synzyme assembly system, ADP, sodium dihydrogen phosphate, catalase and beta-mercaptoethanol mixing, dispersion liquid is obtained;
4) caseic aqueous solution, kinesin aqueous solution and described dispersion liquid are sequentially added in reaction vessel and mix, obtain micro-pipe-kinesin transportation system.
In said method,
Step 1) in, the particle diameter of described glucoseoxidase microsphere or microcapsule is 1-4 ��m, and described glucoseoxidase microspherulite diameter is specially 2.6 ��m or 4 ��m, and the particle diameter of described microcapsule is concrete 2 ��m or 3 ��m of microcapsules;
The final concentration of 100-500nM of ATP synzyme in the described proteolipid liquid solution containing ATP synzyme, in the described proteolipid liquid solution containing ATP synzyme, the final concentration of ATP synzyme is specially 200nM;
Step 3) in, described micro-pipe is micro-pipe of the biotin modification of rhodamine labelling;
Micro-pipe of the biotin modification of described rhodamine labelling is prepared according to the method comprised the steps:: micro-pipe of rhodamine labelling, micro-pipe of biotin modification and micro-pipe (volume ratio 1:1:2, the concentration of micro-pipe is 5mg/mL) of unmodified are scattered in containing 4mMMgCl after mixing2, 1mMGTP (GTP (guanosine triphosphate)) and weight/mass percentage composition 5%DMSO BRB80 buffer in, total protein concentration is 2.5mg/ml; After being then placed in 30min is hatched in the water-bath of 37 DEG C, add in the BRB80 buffer containing 20 ��Ms of taxol (paclitaxel) of 4 �� L, micro-pipe of the biotin modification of rhodamine labelling can be obtained.
Described glucoseoxidase microsphere or microcapsule/ATP synzyme assembly system are glucoseoxidase microsphere or the microcapsule/ATP synzyme assembly system of Streptavidin modification.
In an embodiment of the present invention, adopt glucoseoxidase microcapsule/ATP synzyme assembly system that Streptavidin is modified, specifically it is prepared as follows: 500 �� L (GOD content is 0.2mg/mL) GOD microcapsule/ATP synzyme assembly system is distributed in the aqueous solution of 10 L50 ��M of Streptavidins of ��, absorption 30min, centrifugal collecting precipitation, wash three times, obtain GOD microcapsule/ATP synzyme assembly system dispersion liquid that Streptavidin is modified, be glucoseoxidase microcapsule/ATP synzyme assembly system that Streptavidin is modified.
In said method,
Step 2) in, described glucoseoxidase microsphere is 10-16:5 with the proteolipid weight proportioning in the described proteolipid liquid solution containing ATP synzyme, and described glucoseoxidase microsphere is specially 16:5 with the proteolipid weight proportioning in the described proteolipid liquid solution containing ATP synzyme;
Or glucoseoxidase content is 1:10-25 with the proteolipid weight proportioning in the described proteolipid liquid solution containing ATP synzyme in described glucoseoxidase microcapsule, in described glucoseoxidase microcapsule, glucoseoxidase content is specially 1:25 with the proteolipid weight proportioning in the described proteolipid liquid solution containing ATP synzyme;
Step 4) in,
Described casein, described kinesin, described micro-pipe, described glucoseoxidase microsphere/ATP synzyme assembly system, described ADP, described sodium dihydrogen phosphate, described catalase, described beta-mercaptoethanol proportioning be g:0.1-0.5 ��m ol:0.1-0.5 ��m ol:0.01-0.05 �� g:1-5 �� g of 0.025mg:1-1.5pmol:1-5 �� g:50-100 ��;
Described casein, described kinesin, described micro-pipe, described glucoseoxidase microsphere/ATP synzyme assembly system, described ADP, described sodium dihydrogen phosphate, described catalase, described beta-mercaptoethanol proportioning be specially g:0.5 ��m ol:0.25 ��m ol:0.04 �� g:2.5 �� g of 0.025mg:1.5pmol:5 �� g:80 ��;
Or the proportioning of described casein, described kinesin, described micro-pipe, described glucoseoxidase microcapsule/ATP synzyme assembly system, described ADP, described sodium dihydrogen phosphate, described catalase, described beta-mercaptoethanol is g:0.1-0.5 ��m ol:0.1-0.5 ��m ol:0.01-0.05 �� g:1-5 �� g of 0.03mg:1-1.5pmol:1-5 �� g:1-5 ��;
Described casein, described kinesin, described micro-pipe, described glucoseoxidase microcapsule/ATP synzyme assembly system, described ADP, described sodium dihydrogen phosphate, described catalase, described beta-mercaptoethanol proportioning be specially g:0.5 ��m ol:0.25 ��m ol:0.04 �� g:2.5 �� g of 0.03mg:1.35pmol:5 �� g:1 ��;
Described glucoseoxidase microcapsule/ATP synzyme assembly system is glucoseoxidase microcapsule/ATP synzyme assembly system that Streptavidin is modified.
In said method,
Step 2) in, the described response time 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 is for stating 5min after kinesin aqueous solution adds.
In said method,
Step 1) in, described glucoseoxidase microsphere or microcapsule are the microsphere or the microcapsule that load polyelectrolyte, protein, polysaccharide or medicine.
Step 4) in, described kinesin is kinesin-1, and its aminoacid sequence is sequence 1 in sequence table.
Micro-pipe-kinesin the transportation system of glucose responding prepared by said method is also the scope of protection of the invention.
Micro-pipe of above-mentioned glucose responding-kinesin transportation system application in preparing nano-device is also the scope of protection of the invention.
Micro-pipe-kinesin the transportation system of above-mentioned glucose responding is also being the scope of protection of the invention as the application in transport agent.
The experiment proves that, the present invention prepares the micro-pipe-kinesin transportation system of a kind of glucose responding, and this transportation system has the advantage that
(1) the micro-pipe-kinesin transportation system of the present invention has glucose responding, and in the solution having glucose, the ADP in system can be changed into ATP, kinesin and micro-pipe can be driven to transport by ATP synzyme; Without, in the solution of glucose, producing without ATP, kinesin can not drive micro-pipe to transport.
(2) the micro-pipe-kinesin transportation system of the present invention can automatically supply energy, it is not necessary to additionally adds ATP.
(3) the micro-pipe-kinesin transportation system of the present invention is capable of the regeneration of ATP, and the concentration of ATP in buffer system, thus ensureing the sustainable supply of ATP.
(4) the glucoseoxidase microsphere in the present invention or capsule are possible not only to continue to provide energy for micro-pipe-kinesin transportation system, moreover it is possible to as the material that carrier transport is different.
(5) the micro-pipe-kinesin transportation system in the present invention is capable of Partial controll (namely only in the region having glucose, micro-pipe can move; Not having the region of glucose, micro-pipe can not move) micro-pipe-kinesin transportation system.
Accompanying drawing explanation
The transmission electron microscopy figure (Figure 1B) that 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 ATP synthetase albumen liposome adsorbs at glucoseoxidase microsphere surface.
Fig. 2 is the F that recombinated in embodiment 1oF1The ATP that the glucoseoxidase microsphere of-ATPase proteoliposome produces changes over curve chart.
Fig. 3 is the Velocity-time relation curve that in embodiment 1, micro-pipe moves on the surface that kinesin is modified.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
In following embodiment, fluid pool used is prepared as follows:
Common fluid pond is formed (Scotch3M, 0.1mm are thick) by microscope slide, coverslip and double faced adhesive tape. Microscope slide and coverslip be ultrasonic 10min in saturated KOH solution first, then cleans with ethanol and deionized water, then dries up with liquid nitrogen. If no special instructions, experiment is all common fluid pond.
In following embodiment, the preparation method of micro-pipe of the biotin modification of rhodamine labelling used is: micro-pipe of rhodamine labelling, micro-pipe of biotin modification and micro-pipe (volume ratio 1:1:2, the concentration of micro-pipe is 5mg/mL) of unmodified are scattered in containing 4mMMgCl after mixing2, 1mMGTP (GTP (guanosine triphosphate)) and weight/mass percentage composition 5%DMSO BRB80 buffer in, total protein concentration is 2.5mg/ml;After being then placed in 30min is hatched in the water-bath of 37 DEG C, add in the BRB80 buffer containing 20 ��Ms of taxol (paclitaxel) of 4 �� L, micro-pipe of the biotin modification of rhodamine labelling can be obtained.
Wherein, micro-Guan Jun of micro-pipe of rhodamine labelling, micro-pipe of biotin modification and unmodified is purchased from Cytoskeleton company limited, and catalogue No is TL590M, T333P and TL238 respectively.
In following embodiment, kinesin used is that Kinesin-1 (its aminoacid sequence is sequence 1 in sequence table :) is prepared as follows: the kinesin heavy chain of total length Drosophila melanogaster C end histidine mark and the kinesin plasmid of light chain are expressed in escherichia coli Escherichiacoli (purchased from calm and peaceful Bioisystech Co., Ltd of Sino-U.S.), then purify, with nickel-aminotriacetic acid agarose resin (nickel-nitrilotriaceticacidagaroseresin, Ni-NTA) post and phosphocellurose column, the albumen expressed.
Embodiment 1, prepare the micro-pipe-kinesin transportation system of glucose responding
1, prepared by the micro-pipe-kinesin transportation system of glucose responding
(1) glucoseoxidase (GOD) microsphere is prepared
Taking concentration is 0.33MNa2CO3Water liquid and concentration be 4mg/mlGOD aqueous solution in round-bottomed flask, it is disposable subsequently that to rapidly join isopyknic concentration be 0.33MCaCl2Aqueous solution is in flask, and stirring and evenly mixing 20s immediately, standing and reacting is about 2min, centrifuging and taking precipitate, 3 washings, obtains the GOD microsphere of 4 ��m.
In above-mentioned reaction, Na2CO3, GOD and CaCl2Quality proportioning is 1:1-10:1, is specially 35:4:37;
(2) preparation proteolipid liquid solution containing ATP synzyme
ATP synzyme is prepared as follows: by fresh spinach leaves, arteries and veins, cleaning in removal; Blender stirs leaf to completely in grain of rice shape size, filter with Nylon Bag, remove filtering residue; Take filtrate to be centrifuged, collect precipitate, put into hypotonic buffer solution (10mMTris-HClpH8.0 and 0.5mMMgCl2) in, stirring, centrifugal, taking precipitate puts into high ionic strength wash buffer (0.4M sucrose, 10mMTris-HClpH8.0,150mMNaCl and 0.5mMMgCl2) in, stirring, centrifugal, taking precipitate adds buffer suspension liquid (0.4M sucrose, 50mMTricine-NaOHpH8.0,0.2mMMgCl2) in. It is added thereto to solid DTT (final concentration 50mM), stirring; It is subsequently added isopyknic Extraction buffer, at 4 DEG C, stirs 30min, centrifugal, take supernatant and add (NH4) 2SO4 powder, centrifugal, collect precipitate; The crude protein solution obtained is mixed with isopyknic density gradient buffer solution, it is being followed successively by: 60%, centrifugal in the sucrose density gradient of 52%, 44%, 36%, 28%, 20%, collect 44% sucrose layer, it is ATP synzyme, final buffer solution is: 1.25mMsucrose, 30mMNa2HPO4-NaOHpH7.2,2mMMgCl2,0.5mMNa2EDTA, 4mMdodecylmaltoside, store in liquid nitrogen.
Liposome obtains liposome for aquation after being mixed according to following mass ratio by the following two kinds material: DMPC (DMPC): two myristoyl sodium phosphate (DMPA) quality proportionings are 9:1, specifically it is prepared as follows: weigh DMPC (DMPC) and two myristoyl sodium phosphates (DMPA) (mass ratio 9:1) are dissolved in the mixed solvent of chloroform and methanol (volume ratio 1:1) simultaneously, ultrasonic it is made to be completely dissolved, 30 DEG C of rotary evaporations obtain uniform dry adipose membrane, add 60 DEG C of water hydratables, form lipid suspension, to lipid suspension water bath sonicator until obtaining the PA/PC liposome (10mg/mL) of clear.
By ATP synzyme, detergent Triton-100, liposome at buffer (containing final concentration of 40mMNaCl and 5mMMgCl2The 20mMTricine buffer of pH8.0) in mixing, 4 DEG C of stirring reaction 1h; Add Bio-beads and reaction 1h removal detergent, centrifugal collection supernatant are stirred at room temperature, repeat 3 times; Namely obtaining the proteolipid liquid solution containing ATP synzyme, concentration is 5mg/ml, preserves, the ATP synzyme final concentration of 200nM in the proteolipid liquid solution containing ATP synzyme in liquid nitrogen.
ATP synzyme in above-mentioned reaction, Triton-100, liposome quality proportioning be 0.05mg:16mg:10mg (0.01��0.05mg:8��16mg:1��10mg).
As shown in Figure 1A, the mean diameter of proteoliposome is 180nm to the transmission electron microscopy figure of the proteoliposome of above-mentioned preparation.
(3) GOD microsphere/ATP synzyme assembly system is prepared
The GOD microsphere of 500 �� L16mg/mL prepared by step (1) is distributed in the 500 �� L5mg/mL proteolipid liquid solution containing ATP synzyme, concussion is uniformly, absorption 30min, 4 DEG C of centrifuge washings, collect precipitation and are GOD microsphere/ATP synzyme assembly system (transmission electron microscopy figure is as shown in Figure 1B).
The content of detection GOD microsphere/ATP synzyme assembly system ATP is as follows:
The GOD obtained microsphere/ATP synzyme assembly system is distributed to the 2mL buffer solution containing 18wt% glucose, and (pH8.0, containing 10mMtricine, 30mMNaCl, 2.5mMMgCl2, 5mMNaH2PO4And 0.2mMADP) in, from system, take out the sample of 20 �� L, join in fluorescein-luciferase ATP measurement system, measure luminous signal with light-emitting appearance, thus calculating the content of ATP in system.
Result is as in figure 2 it is shown, add glucose, and along with the increase in response time, ATP concentration continues to rise, it is achieved that the controlledly synthesis of the ATP of glucose responding.
(4) acquisition of the micro-pipe-kinesin transportation system of glucose responding
Prepare dispersion liquid: micro-pipe of biotin modification of 2 �� L2.5mg/mL rhodamine labellings, 5 �� L16mg/mLGOD microspheres/ATP synzyme assembly system, 25 �� L20mMADP, 25 �� L10mM sodium dihydrogen phosphate (NaH2PO4), 5 �� L0.008mg/mL catalases and 0.5 �� L0.5wt% beta-mercaptoethanol aqueous solution mixing, obtain dispersion liquid.
Prepared by micro-pipe-kinesin transportation system:
First, casein (1mg/mL) solution is injected in fluid pool, adsorb 5min; Then kinesin-1 aqueous solution (20nM) is injected in fluid pool, adsorb 5min; Then, above-mentioned dispersion liquid is slowly injected in fluid pool, obtains micro-pipe-kinesin transportation system.
In micro-pipe-kinesin transportation system, each material is as follows: 50 �� L0.5mg/mL caseins, 50 �� L30nMkinesin-1, micro-pipe of biotin modification of 2 �� L2.5mg/mL rhodamine labellings, 5 �� L16mg/mLGOD microspheres/ATP synzyme assembly system, 25 �� L20mMADP, 25 �� L10mM sodium dihydrogen phosphate (NaH2PO4), 5 �� L0.008mg/mL catalases, 0.5 �� L0.5wt% beta-mercaptoethanol aqueous solution;
In micro-pipe-kinesin transportation system, the proportioning of each material is as follows: casein, kinesin-1, micro-pipe of biotin modification of rhodamine labelling, GOD microsphere/ATP synzyme assembly system, ADP, sodium dihydrogen phosphate (NaH2PO4), catalase, beta-mercaptoethanol proportioning be g:0.5 ��m ol:0.25 ��m ol:0.04 �� g:2.5 �� g of 0.025mg:1.5pmol:5 �� g:80 ��.
2, micro-pipe-kinesin transportation system is detected to glucose responding ability
Above-mentioned micro-pipe-kinesin transportation system will add the glucose of final concentration of 26wt% (weight/mass percentage composition), mixing, fluid pool silica gel sealing is got up, moves to and observe under Laser Scanning Confocal Microscope. Not add glucose for comparison.
Recording MT moving image in time, result is not as it is shown on figure 3, add in the system of glucose, and micro-pipe does not move; And add the system of glucose, As time goes on, micro-pipe MT can successfully slide on the kinesin-1 surface modified, sliding speed average out to 19nm/s.
Embodiment 2, prepare the micro-pipe-kinesin transportation system of glucose responding
1, prepared by the micro-pipe-kinesin transportation system of glucose responding
In step prepared by the micro-pipe-kinesin transportation system of the present embodiment glucose responding and parameter and embodiment 1,1 is essentially identical, and difference is in that step (1):
(1) preparation loads glucoseoxidase (GOD) microsphere of medicine
Taking concentration is the 0.33M Na containing 2mg/mL FITC-dextran (FITC-Dextran)2CO3Water liquid and concentration be 4mg/mLGOD aqueous solution in round-bottomed flask, it is disposable subsequently that to rapidly join isopyknic concentration be 0.33MCaCl2Aqueous solution, in flask, stirs 20s immediately, and standing and reacting is about 2min, centrifuging and taking precipitate, 3 washings, obtains GOD-medicine complex microsphere (particle diameter is 2.6 ��m).
In above-mentioned reaction, Na2CO3, FITC-Dextran, GOD and CaCl2Quality proportioning be 35mg:2mg:4mg:37mg;
(2) preparation proteoliposome containing ATP synzyme
Identical with (2) of the 1 of embodiment 1;
(3) GOD microsphere/ATP synzyme assembly system is prepared
Identical with (3) of the 1 of embodiment 1, obtain GOD microsphere/ATP synzyme assembly system;
(4) acquisition of the micro-pipe-kinesin transportation system of glucose responding
Identical with (4) of the 1 of embodiment 1, obtain micro-pipe-kinesin transportation system;
2, micro-pipe-kinesin transportation system is detected to glucose responding ability
Identical with the 2 of embodiment 1, it is not added with in the system of glucose, micro-pipe does not move; And add the system of glucose, As time goes on, micro-pipe MT can successfully slide on the kinesin surface modified, sliding speed average out to 30nm/s.
3, micro-pipe-kinesin transportation system application in transport agent
Micro-pipe-kinesin the transportation system of above-mentioned 2 preparations being moved to and observe under Laser Scanning Confocal Microscope, confocal experiments shows, micro-pipe-kinesin transportation system can transport FITC-dextran.
Therefore, it is believed that micro-pipe-kinesin transportation system can directed transporting drugs, micro-pipe-kinesin the transportation system being loaded with medicine under glucose stimulates is moving to stopping movement in without glucose system, thus unloading medicine, it is achieved directed transporting drugs.
Embodiment 3, prepare the micro-pipe-kinesin transportation system of glucose responding
1, prepared by the micro-pipe-kinesin transportation system of glucose responding
(1) GOD microcapsule (this embodiment of microcapsule is to prepare microcapsule, it is also possible to prepare Nano capsule, and simply template is not manganese carbonate, changes nano silicon or nano-calcium carbonate into) is prepared
First, the manganese carbonate particle that particle diameter is 2 ��m is distributed in the PAH solution (sigma company buys) containing 0.1M sodium chloride, after vibration absorption 30min, precipitation is collected in centrifugation, fully after washing, redispersion adsorbs 1h in glutaraldehyde (GA) aqueous solution containing 0.025wt%, and precipitation is collected in centrifugation, wash 3 times, obtain microsphere;
Then adsorbing 3h in the microsphere redispersion obtained to the 4mg/mLGOD aqueous solution containing 0.1M sodium chloride, precipitation is collected in centrifugation, washes 3 times; It is repeated in the operation of absorption GA, GOD, until the required number of plies; Then in the microsphere obtained, add the disodium EDTA solution of 0.1M, concussion reaction 3h, molten except manganese carbonate particle, after centrifugal collecting precipitation, washing 3 times, obtain (GA/GOD)nMicrocapsule.
Often GA, GOD of absorption is one layer, and the present embodiment is 6 layers, obtains (GA/GOD)6Microcapsule, particle diameter is 2 ��m.
Described manganese carbonate particle, PAH, glutaraldehyde, GOD mass ratio be 160mg:4mg:1mg:8mg.
(2) preparation proteolipid liquid solution containing ATP synzyme
Identical with (2) of the 1 of embodiment 1;
(3) GOD microcapsule/ATP synzyme assembly system is prepared
500 �� L (GOD content is 0.2mg/mL) GOD microcapsule prepared by step (1) is distributed in the 500 �� L5mg/mL proteolipid liquid solution containing ATP synzyme, concussion is uniformly, absorption 30min, 4 DEG C of centrifuge washings, collect precipitation and are GOD microcapsule/ATP synzyme assembly system.
(4) acquisition of the micro-pipe-kinesin transportation system of glucose responding
(3) 500 �� L (GOD content is 0.2mg/mL) GOD microcapsule/ATP synzyme assembly system is distributed in the aqueous solution of 10 L50 ��M of Streptavidins of ��, absorption 30min, centrifugal collecting precipitation, wash three times, obtain GOD microcapsule/ATP synzyme assembly system dispersion liquid that Streptavidin is modified; Again GOD microcapsule/ATP synzyme assembly system dispersion liquid that 5 �� L (GOD content is 0.2mg/mL) Streptavidin is modified is added dropwise in micro-pipe solution of biotin modification of 2 �� L2.5mg/mL rhodamine labellings, jog 30min under room temperature (25 DEG C), adds 25 �� L20mMADP, 25 �� L10mM sodium dihydrogen phosphate (NaH2PO4), 5 �� L0.008mg/mL catalases and 0.5 �� L0.5wt% beta-mercaptoethanol mixing, obtain dispersion liquid.
Prepared by micro-pipe-kinesin transportation system:
First, casein (1mg/mL) solution is injected in fluid pool, adsorb 5min; Then kinesin aqueous solution (20nM) is injected in fluid pool, adsorb 5min; Then, above-mentioned dispersion liquid is slowly injected in fluid pool, obtains micro-pipe-kinesin transportation system.
In micro-pipe-kinesin transportation system, each material is as follows: the GOD microcapsule/ATP synzyme assembly system of 60 �� L0.5mg/mL caseins, 45 �� L30nMkinesin-1, micro-pipe of biotin modification of 2 �� L2.5mg/mL rhodamine labellings, 5 �� L (GOD content is 0.2mg/mL) streptomycin modification, 25 �� L20mMADP, 25 �� L10mM sodium dihydrogen phosphate (NaH2PO4), 5 �� L0.008mg/mL catalases, 0.5 �� L0.5wt% beta-mercaptoethanol aqueous solution;
In micro-pipe-kinesin transportation system, the proportioning of each material is micro-pipe of the biotin modification of casein, kinesin-1, rhodamine labelling, the GOD microcapsule/ATP synzyme assembly system of streptomycin modification, ADP, sodium dihydrogen phosphate (NaH2PO4), catalase, beta-mercaptoethanol proportioning as follows: g:0.5 ��m ol:0.25 ��m ol:0.04 �� g:2.5 �� g of 0.03mg:1.35pmol:5 �� g:1 ��.
2, micro-pipe-kinesin transportation system is detected to glucose responding ability
Above-mentioned micro-pipe-kinesin transportation system will add the glucose of final concentration of 18wt%, mixing, fluid pool silica gel sealing is got up, moves to and observe under Laser Scanning Confocal Microscope. To be added without glucose sugar for comparison.
Fluid pool silica gel sealing is got up, moves to and observe under Laser Scanning Confocal Microscope, identical with the 2 of embodiment 1, it is not added with in the system of glucose, micro-pipe does not move;And add the system of glucose, As time goes on, micro-pipe MT can successfully slide on the kinesin surface modified, sliding speed average out to 50nm/s.
Embodiment 4, prepare the micro-pipe-kinesin transportation system of glucose responding
In step prepared by the micro-pipe-kinesin transportation system of the present embodiment glucose responding and parameter and embodiment 3,1 is essentially identical, and difference is in that step (1):
(1)(GA/GOD)nGA/CAT/GA/GOD microcapsule
In the step of the present embodiment and parameter and embodiment 3 essentially identical, be distinctive in that: when preparing GOD microcapsule, assemble one layer of catalase (CAT) more, obtain (GA/GOD)nGA/CAT/GA/GOD microcapsule, specific as follows:
(GA/GOD) that will obtain in (1) step of the 1 of embodiment 36Microsphere is distributed in glutaraldehyde (GA) aqueous solution containing 0.025wt% and adsorbs 1h, and precipitation is collected in centrifugation, washes 3 times; Being distributed to by the microsphere obtained in the 4mg/mLCAT aqueous solution containing 0.1M sodium chloride again and adsorb 3h, precipitation is collected in centrifugation, washes 3 times; Same operation, then adsorb GA, GOD successively at microsphere surface; Then in the microsphere obtained, add the disodium EDTA solution of 0.1M, concussion reaction 3h, molten except manganese carbonate particle, after centrifugal collecting precipitation, washing 3 times, obtain (GA/GOD)6GA/CAT/GA/GOD microcapsule (particle diameter is 3 ��m).
Described manganese carbonate particle, PAH, glutaraldehyde, GOD, CAT proportioning be mass ratio be 160mg:4mg:1mg:8mg:8mg.
(GA/GOD)nGA/CAT/GA/GOD microcapsule can effectively remove the hydrogen peroxide that catalytic oxidation of glucose produces, it is possible to the active oxygen of the concentration of hydrogen peroxide in reduction system and generation further as far as possible, reduces the active oxygen damage to micro-pipe or kinesin.
(2) preparation proteoliposome containing ATP synzyme
Identical with (2) of the 1 of embodiment 3;
(3) GOD microcapsule/ATP synzyme assembly system is prepared
Identical with (3) of the 1 of embodiment 3;
(4) micro-pipe-kinesin transportation system is prepared
Identical with (4) of the 1 of embodiment 3; Obtain micro-pipe-kinesin transportation system.
2, micro-pipe-kinesin transportation system is detected to glucose responding ability
Above-mentioned micro-pipe-kinesin transportation system will add the glucose of final concentration of 20wt%, mixing, fluid pool silica gel sealing is got up, moves to and observe under Laser Scanning Confocal Microscope. To be added without glucose sugar for comparison. It is shown that be not added with in the system of glucose, micro-pipe does not move; And add the system of glucose, As time goes on, micro-pipe MT can successfully slide on the kinesin surface modified, sliding speed average out to 26nm/s.
Claims (8)
1. a preparation method for the micro-pipe-kinesin transportation system of glucose responding, comprises the steps:
1) glucoseoxidase microsphere or microcapsule, proteolipid liquid solution containing ATP synzyme are prepared respectively;
The particle diameter of described glucoseoxidase microsphere or microcapsule is 1-4 ��m;
The final concentration of 100-500nM of ATP synzyme in the described proteolipid liquid solution containing ATP synzyme;
2) described glucoseoxidase microsphere or microcapsule are dispersed in the proteolipid liquid solution containing ATP synzyme, reaction, collect precipitation, obtain glucoseoxidase microsphere or microcapsule/ATP synzyme assembly system; Described glucoseoxidase microsphere is 10-16: 5 with the proteolipid weight proportioning containing ATP synzyme in the described proteolipid liquid solution containing ATP synzyme;
In described glucoseoxidase microcapsule, glucoseoxidase content is 1: 10-25 with the proteolipid weight proportioning containing ATP synzyme in the described proteolipid liquid solution containing ATP synzyme;
3) by micro-pipe, described glucoseoxidase microsphere or microcapsule/ATP synzyme assembly system, ADP, sodium dihydrogen phosphate, catalase and beta-mercaptoethanol mixing, dispersion liquid is obtained;
4) caseic aqueous solution, kinesin aqueous solution and described dispersion liquid are sequentially added in reaction vessel and mix, obtain micro-pipe-kinesin transportation system;
Described casein, described kinesin, described micro-pipe, described glucoseoxidase microsphere/ATP synzyme assembly system, described ADP, described sodium dihydrogen phosphate, described catalase, described beta-mercaptoethanol proportioning be g: 0.1-0.5 ��m ol: 0.1-0.5 ��m ol: 0.01-0.05 �� g: 1-5 �� g of 0.025mg: 1-1.5pmol: 1-5 �� g: 50-100 ��;
Described casein, described kinesin, described micro-pipe, described glucoseoxidase microcapsule/ATP synzyme assembly system, described ADP, described sodium dihydrogen phosphate, described catalase, described beta-mercaptoethanol proportioning be g: 0.1-0.5 ��m ol: 0.1-0.5 ��m ol: 0.01-0.05 �� g: 1-5 �� g of 0.03mg: 1-1.5pmol: 1-5 �� g: 1-5 ��.
2. method according to claim 1, it is characterised in that:
Step 1) in, the final concentration of 200nM of ATP synzyme in the described proteolipid liquid solution containing ATP synzyme;
Step 3) in, described micro-pipe is micro-pipe of the biotin modification of rhodamine labelling;
Described glucoseoxidase microsphere or microcapsule/ATP synzyme assembly system are glucoseoxidase microsphere or the microcapsule/ATP synzyme assembly system of Streptavidin modification.
3. method according to claim 1, it is characterised in that:
Step 2) in, described glucoseoxidase microsphere is 16: 5 with the proteolipid weight proportioning containing ATP synzyme in the described proteolipid liquid solution containing ATP synzyme;
In described glucoseoxidase microcapsule, glucoseoxidase content is 1: 25 with the proteolipid weight proportioning containing ATP synzyme in the described proteolipid liquid solution containing ATP synzyme;
Step 4) in,
Described casein, described kinesin, described micro-pipe, described glucoseoxidase microsphere/ATP synzyme assembly system, described ADP, described sodium dihydrogen phosphate, described catalase, described beta-mercaptoethanol proportioning be g: 0.5 ��m ol: 0.25 ��m ol: 0.04 �� g: 2.5 �� g of 0.025mg: 1.5pmol: 5 �� g: 80 ��;
Described casein, described kinesin, described micro-pipe, described glucoseoxidase microcapsule/ATP synzyme assembly system, described ADP, described sodium dihydrogen phosphate, described catalase, described beta-mercaptoethanol proportioning be g: 0.5 ��m ol: 0.25 ��m ol: 0.04 �� g: 2.5 �� g of 0.03mg: 1.35pmol: 5 �� g: 1 ��;
Described glucoseoxidase microcapsule/ATP synzyme assembly system is glucoseoxidase microcapsule/ATP synzyme assembly system that Streptavidin is modified.
4. according to described method arbitrary in claim 1-3, it is characterised in that:
Step 2) in, the time of described reaction is 30min;
Step 4) in, the joining day of described kinesin aqueous solution is 5min after caseic aqueous solution adds;
The joining day of described dispersion liquid is 5min after described kinesin aqueous solution adds.
5. method according to claim 1 and 2, it is characterised in that:
Step 1) in, described glucoseoxidase microsphere or microcapsule are the microsphere or the microcapsule that load polyelectrolyte, protein, polysaccharide or medicine.
6. the micro-pipe-kinesin transportation system of the glucose responding that prepared by method according to any one of claim 1-5.
7. micro-pipe of glucose responding described in claim 6-kinesin transportation system application in preparing nano-device.
8. the micro-pipe-kinesin transportation system of glucose responding described in claim 6 is as the application in transport agent.
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| CN1952126A (en) * | 2005-10-21 | 2007-04-25 | 株式会社Ntt都科摩 | Molecule transfer/delivery system and molecule transfer /delivery method |
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| JP4264745B2 (en) * | 2004-12-14 | 2009-05-20 | 独立行政法人産業技術総合研究所 | Enzyme and biotin modified anisotropic polymer microparticle, microtubule and microparticle complex using this, and ATP self-supply nanobiomachine |
| JP4258661B2 (en) * | 2005-04-14 | 2009-04-30 | 独立行政法人産業技術総合研究所 | Molecular motor system and use thereof |
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| CN1952126A (en) * | 2005-10-21 | 2007-04-25 | 株式会社Ntt都科摩 | Molecule transfer/delivery system and molecule transfer /delivery method |
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