CN102121030A - Fluorescent quantum dot marked chitosan DNA nano composite vector and preparation and application thereof - Google Patents
Fluorescent quantum dot marked chitosan DNA nano composite vector and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent quantum dot marked chitosan DNA nano composite vector and preparation and application thereof, and particularly aims at transduction of jatropha curcas genes. The method comprises the following steps of: preparing CdSe fluorescent quantum dots with good water solubility and good biological affinity, and preparing the chitosan DNA nano vector by using a complex coacervation method. The quantum dots are combined to the chitosan DNA nano vector under the action of static electricity to form a fluorescent nano complex using the vector as a center and encircled by the quantum dots. The preparation method is simple and feasible; the obtained complex is uniform and has enhanced fluorescent property and good stability; and when the complex is used for the transduction of jatropha curcas cells, fluorescent expression signals of intracellular report genes (GFP protein) can be successfully detected, and the complex can be widely used for the transduction of other plant genes.
Description
Technical field
The invention belongs to the applying nano biological technical field, relate to nano combined carrier of chitosan DNA of a kind of fluorescence quantum point mark and preparation method thereof, and the method that is applied to the Cortex jatrophae gene transfer.
Background technology
Nano-carrier has certain advantage at mediated gene transfer party mask: biological safety is good, and is biodegradable, and toxicity and immunogenicity are little; Protecting group is not because of being subjected to the destruction of various complements and enzyme; Have special construction and surface charge; At its surface energy coupling specificities targeted molecular.Many experts and scholar attempt utilizing nano material to carry out conversion of zooblast foreign gene and gene therapy as carrier both at home and abroad, have obtained gratifying progress.Also there is scientist to utilize nano-carrier to carry out the exploratory study that plant gene transforms recently.The hole nanoparticle was carrier during the botanist of U.S. Iowa State University and chemist successfully utilized, and with gene and stimulate the compound of this genetic expression, passed plant cell wall, and suitably the place discharges in cell.This breakthrough also transforms the more favourable instrument that provides to plant gene except nanosecond science and technology being brought into the field of plant biology.What the researchist was successful at present changes gene and compound in the plants such as Arabidopis thaliana, tobacco and corn over to.Domestic research to plant transgene is just at the early-stage, the Liu Jun of Hunan University etc. has prepared the poly-lysine starch nanometer granule, under ultrasonic-mediated, green fluorescence protein gene is imported in the Rhizome of Peltate Yam cell, effectively broken through the natural cover for defense of plant cell wall, realized that first this method is to genetically modified live body, detection in real time.
In the time of will foreign gene being imported the plant recipient cell with this nano particle gene carrier; there is the challenge of following aspect: when gene-nano-particle complex and cell carry out common cultivation; it is many that the barrier of cell walls makes exogenous origin gene integrator arrive the middle-chain of cellular genome; the nano particle that makes major part carry DNA is difficult to directly enter cell; be difficult to directly to take DNA and be diffused on the nuclear membrane, and pass Nuclear pore or in fission process, enter nucleus with expression alien gene.Utilizing fluorescent chemicals that plant gene carrier transduction process is carried out real-time tracing is the important directions that nano-carrier research strides forward.At present the report that the fluorescent chemicals labeled vector is carried out gene transfer research also seldom.And compare with traditional organic fluorescent dye, nano-quantum point has unique optical characteristics and application prospect: send different colours fluorescence, distinguishable or target compound that mark is different is realized unit molecule, multi-target detection; The photochemical stability height be difficult for photobleaching takes place, but long-time continuous is followed the tracks of imaging; Luminescent lifetime is long, is fit to the advantages such as biological detection of bands of a spectrum diversification and time seriesization.
Summary of the invention
One of purpose of the present invention is to utilize the quantum dot excellent fluorescence, and a kind of chitosan DNA fluorescence nano complex carrier and preparation method thereof is provided, and this method does not need harsh appointed condition, and easy and simple to handle, materials safety is easy to get, and is cheap.
Two of purpose of the present invention provides the induction method of above-mentioned this chitosan DNA fluorescence nano complex carrier at plant transgene, especially for the method for Cortex jatrophae gene transfer.
For reaching above purpose, technical scheme of the present invention is:
A kind of nano combined carrier of chitosan DNA of fluorescence quantum point mark is that the binding substances with chitosan and dna vector is the center, coats the mixture of fluorescence quantum on every side; Described fluorescence quantum comprises CdSe, CdS or ZnSe quantum dot; And the compound of other II～VI family or III～V group element composition.
A kind of preparation method of the nano combined carrier of chitosan DNA of fluorescence quantum point mark is prepared into the chitosan dna vector with chitosan and dna vector earlier, adds CdSe, CdS or ZnSe quantum dot mixing again, filters to get final product.Detailed process is as follows:
With isopyknic chitosan be dissolved in dna solution in the 5mM sodium sulfate respectively 55 ℃ of preheatings 30 minutes, vortex mixing instrument mixed 30 seconds, formed the chitosan dna vector; Adding CdSe quantum dot (100uL, 0.5mM) in carrier soln, vortex mixing 2 minutes, ultra-filtration and separation is removed unreacted quantum dot, and filters with the filter-sterilizer of 0.22um millipore filtration, obtains complex carrier.
The molecular weight ranges of described chitosan is 10-30 ten thousand, is dissolving fully in 5.5 the 5mM sodium acetate soln at pH, and concentration is 200ug/mL; Dna solution concentration in the described 5mM of the being dissolved in sodium sulfate is 80ug/mL.
The preparation method of described CdSe quantum dot: be that to be stablizer with L-halfcystine (1.5mM) synthetic at aqueous phase, 30 ℃ of bath temperatures, water-bath time 90min, L-Cys: Cd: Se are (mol ratio 3: 2: 1), and the pH value is 8.
Described dna vector is pEGAD plasmid or pBI121 plasmid, and Jin Weike (China) biotechnology center is bought.
The nano combined carrier of chitosan DNA of the above-mentioned fluorescence quantum point mark for preparing can be applied to plant transgene; Particularly can be applied to the Cortex jatrophae transgenosis.Detailed process is as follows:
The Cortex jatrophae callus is cultivated a week in the 30mL fresh culture; Described then complex carrier joins the receptor solution that contains loose callus of 1mL, and ultrasonication 6 minutes (ultrasonic power 200W) was cultivated altogether through 2 days, finished transduction.
Advantage of the present invention:
1, the present invention combines fluorescence quantum first with chitosan DNA, prepares a kind of complex carrier, and this method does not need harsh appointed condition, and easy and simple to handle, materials safety is easy to get, and is cheap;
2, first the carrier of above-mentioned preparation is applied to the field of transgenic technology of plant, has particularly obtained success in the application aspect the Cortex jatrophae gene transfer; This carrier can not only effectively be broken through the natural cover for defense of plant cell wall, improves transformation efficiency, can also be to genetically modified live body, detection in real time; And this working method is also very simple.
3, compare with traditional organic fluorescent dye, the nano-quantum point of the present invention utilization has unique optical characteristics and application prospect: send different colours fluorescence, distinguishable or target compound that mark is different is realized unit molecule, multi-target detection; The photochemical stability height be difficult for photobleaching takes place, but long-time continuous is followed the tracks of imaging; Luminescent lifetime is long, is fit to the advantages such as biological detection of bands of a spectrum diversification and time seriesization.
Description of drawings
Fig. 1 is the fluorescence spectrum figure of chitosan-DNA nano particle, CdSe quantum dot and CdSe/ chitosan-DNA mixture;
Line a represents chitosan-DNA nano particle; Line b represents the CdSe quantum dot; Line c represents CdSe/ chitosan-DNA mixture.
Fig. 2 is the transmission electron microscope figure of chitosan-DNA nano particle, CdSe quantum dot and CdSe/ chitosan-DNA mixture;
A represents chitosan-DNA nano particle; B represents the CdSe quantum dot; C represents CdSe/ chitosan-DNA mixture.
Fig. 3 is chitosan-DNA nano particle, the laser confocal microscope figure of CdSe quantum dot and CdSe/ chitosan-DNA nano-complex and with the Cortex jatrophae callus at ultrasonic 6 minutes, cultivate the laser confocal microscope figure after a week again;
A1, A represent chitosan-DNA nano particle,
B1, B represent the CdSe quantum dot;
C1, C represent CdSe/ chitosan-DNA nano-complex
A1, B1, C1 represent chitosan-DNA nano particle respectively, the laser confocal microscope figure of CdSe quantum dot and CdSe/ chitosan-DNA nano-complex;
A, B, C represent chitosan-DNA nano particle respectively, CdSe quantum dot and CdSe/ chitosan-DNA nano-complex with the Cortex jatrophae callus at ultrasonic 6 minutes, cultivate the laser confocal microscope figure after a week again.
Fig. 4 enters the Cortex jatrophae callus cell for the nano combined carrier of CdSe/ chitosan-DNA by ultrasonic transduction, the fluorescence pattern of the GFP protein gene expression of embedding and the micro-collection of illustrative plates of laser co-focusing are contrast with the fluorescence pattern and the micro-collection of illustrative plates of laser co-focusing of not ultrasonic transduction;
A represents the fluorescence pattern of GFP protein gene expression;
B represents the micro-collection of illustrative plates of laser co-focusing;
A1 representative is with the fluorescence pattern of ultrasonic transduction not;
The B1 representative is with the micro-collection of illustrative plates of the laser co-focusing of not ultrasonic transduction.
Specific embodiments
Following examples are intended to further specify the present invention, and are not used in restriction the scope of protection of present invention:
1. adopt water-soluble method to prepare the CdSe quantum dot, Fig. 1 (line b) shows that CdSe has strong absorption peak at the 525nm place.Adopt complex coacervation to prepare chitosan nano carrier, with 500uL200ug/mL chitosan and 500uL80ug/mLpDNA (pEGAD, this plasmid contains the GFP protein gene) solution water-bath 30 minutes under 55 ℃ of temperature respectively, equal-volume mixes the back and mixed 30 seconds on vortex mixing instrument, form the chitosan dna vector, with chitosan solution 0.22um millipore filtration membrane filtration.Fig. 1 (line a) show the chitosan dna vector at the 400nm place a faint emission peak.(100uL, 0.5mM) in carrier soln, vortex mixing two minutes, and filter with the millipore filtration of 0.22um obtains can be used in the nano-carrier mixture of transduction to add the CdSe quantum dot.With quantum dot combine of the faint emission peak disappearance of the nano combined carrier in back at 400nm place, increase by force at the absorption peak-to-peak of 525nm, shown in Fig. 1 (line c).Shown the gathering of carrier granule behind the mark and its fluorescence intensity strengthened to some extent.The variation of grain diameter is shown in Fig. 2 transmission electron microscope figure in addition, and composite particles is even behind the mark, and particle diameter is about 200nm, and forming nano-carrier is the fluorescence polymer of core.
With the Cortex jatrophae aseptic seedling stem as explant material, getting 0.5cm for every section is inoculated in the MS minimum medium, substratum adds 30g/L sucrose, 7.2g/L agar and hormones of different concentrations combination, the pH value transfers to 5.8, put into constant temperature culture chamber shaking culture, 25 ℃ of temperature, rotating speed 110rpm, loosen the inducing of callus, select after one month to increase fast and loosely organized yellow callus is organized as succeeding transfer culture, can carry out one time succeeding transfer culture every month, with the callus of Cortex jatrophae acceptor as the transduction of nano-carrier mixture.
3. callus is cultivated a week in 30 milliliters of fresh cultures.Then, the chitosan DNA nano-carrier that CdSe is quantum dot-labeled joins the receptor solution that contains callus of 1mL, and the nano-carrier solution behind the mark vibrated 30 minutes on 37 ℃ shaker, changed aseptic bottle over to, with Cortex jatrophae callus altogether after ultrasonic 6 minutes, carry out sterile culture.Cultivated altogether through 2 days, finish transduction.The Cortex jatrophae callus does not produce fluorescence phenomenon in the chitosan nano carrier water culture, and the mixture fluorescence intensity strengthens behind the mark, transforms the back and produces certain fluorescent signal at the histocyte different positions, sees Fig. 3 laser confocal microscope figure.The proteic nano-carrier of embedding GFP enters vegetable cell, and discharges GFP albumen, makes it obtain expressing, and sees Fig. 4.
Claims (10)
1. the nano combined carrier of chitosan DNA of a fluorescence quantum point mark is characterized in that, described complex carrier is that the binding substances with chitosan and dna vector is the center, coats the mixture of fluorescence quantum on every side.
2. complex carrier according to claim 1 is characterized in that, described fluorescence quantum comprises CdSe, CdS or ZnSe quantum dot.
3. the preparation method of the nano combined carrier of chitosan DNA of a fluorescence quantum point mark is characterized in that, earlier chitosan and dna vector is prepared into the chitosan dna vector, adds CdSe, CdS or ZnSe quantum dot mixing again, filters to get final product.
4. preparation method according to claim 3 is characterized in that, with isopyknic chitosan be dissolved in dna solution in the 5mM sodium sulfate respectively 55 ℃ of preheatings 30 minutes, vortex mixing instrument mixed 30 seconds, formed the chitosan dna vector; Add 100uL, the CdSe quantum dot of 0.5mM in carrier soln, vortex mixing 2 minutes, ultra-filtration and separation is removed unreacted quantum dot, and filters with the filter-sterilizer of 0.22um millipore filtration, obtains complex carrier.
5. preparation method according to claim 3 is characterized in that, the molecular weight ranges of described chitosan is 10-30 ten thousand, is dissolving fully in 5.5 the 5mM sodium acetate soln at pH, and concentration is 200ug/mL; Dna solution concentration in the described 5mM of the being dissolved in sodium sulfate is 80ug/mL.
6. preparation method according to claim 3 is characterized in that, the preparation method of described CdSe quantum dot: be that L-halfcystine with 1.5mM is that stablizer is synthetic at aqueous phase, 30 ℃ of bath temperatures, water-bath time 90min, L-Cys: Cd: Se are mol ratio 3: 2: 1, and the pH value is 8.
7. preparation method according to claim 3 is characterized in that, described dna vector is pEGAD plasmid or pBI121 plasmid.
8. the nano combined carrier of chitosan DNA of claim 3 or 4 or 5 or 6 or 7 fluorescence quantum point marks that prepare is applied to plant transgene.
9. the nano combined carrier of chitosan DNA of claim 3 or 4 or 5 or 6 or 7 fluorescence quantum point marks that prepare is applied to the Cortex jatrophae transgenosis.
10. application method according to claim 9 is characterized in that, the Cortex jatrophae callus is cultivated a week in the 30mL fresh culture; Described then complex carrier joins the receptor solution that contains loose callus of 1mL, ultrasonication 6 minutes, and ultrasonic power 200W cultivated altogether through 2 days, finished transduction.
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| CN104316460A (en) * | 2014-09-16 | 2015-01-28 | 济南大学 | Making method and application of TiO2-CdSe nanocomposite photoelectric biosensor |
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| CN103316692A (en) * | 2013-06-24 | 2013-09-25 | 江苏大学 | Preparation method and application of CdS/CNTs composite photocatalyst |
| CN104316460A (en) * | 2014-09-16 | 2015-01-28 | 济南大学 | Making method and application of TiO2-CdSe nanocomposite photoelectric biosensor |
| CN104316460B (en) * | 2014-09-16 | 2016-03-02 | 济南大学 | A kind of TiO 2the preparation method of-CdSe nano composite material photoelectricity biology sensor and application thereof |
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