CN102174635B - Application of streptavidin combined functional fluorescent magnetic nano granules - Google Patents
Application of streptavidin combined functional fluorescent magnetic nano granules Download PDFInfo
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- CN102174635B CN102174635B CN 201010622840 CN201010622840A CN102174635B CN 102174635 B CN102174635 B CN 102174635B CN 201010622840 CN201010622840 CN 201010622840 CN 201010622840 A CN201010622840 A CN 201010622840A CN 102174635 B CN102174635 B CN 102174635B
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Abstract
The invention relates to the field of application of gene recombinant proteins and nano materials, in particular to application of streptavidin combined functional fluorescent magnetic nano granules. Apoptotic cells are detected by adopting the streptavidin combined functional fluorescent magnetic nano granules. Identification, separation and imaging of the apoptotic cells are realized by adopting the streptavidin combined functional fluorescent magnetic nano granules as tools and combing an enzyme linked immune technology; and visible enrichment, separation and imaging of the apoptotic cells are realized by fully using the specific combination effect and the signal amplification effect between streptavidin and biotin and the magnetic separation property and the fluorescent imaging effect of the streptavidin combined functional magnetic nano granules.
Description
Technical field
The present invention relates to the Application Areas of gene recombinant protein and nano material, is a kind of application of streptavidin combined function fluorescence magnetic nano particle specifically.
Background technology
Apoptosis is being played the part of pivotal player in the healthy survival processes that guarantees multicellular organism, normal development and the vital role of apoptosis disorder in pathological research to individuality, caused extensively and the in depth research of people to its mechanism and component, becoming present life science circle one of hot issue the most, also is the research field that life science circle and even various circles of society fall over each other to drop into.Persistently overheating along with this respect research emerged a large amount of new technology and method apoptosis detected, and the process of research has appearred also having accelerated greatly in commercial product.Wherein, the early detection of apoptotic cell, mainly the expression of passing through the cell surface phosphatidylserine can be passed through a series of Annexin V products as immune identifier, and combined with fluorescent labeling technique and flow cytometer detect.
Along with the fast development of nanotechnology, magnetic nano-particle has been subjected to paying close attention to widely at biomedical sector with its unique dimensional effect and magnetic field performance characteristic.By different functional modifications, the combined with fluorescent mark, technology such as enzyme linked immunological and signal amplification, increasing multifunction magnetic nanoparticle arises at the historic moment.Chinese patent " a kind of preparation method of streptavidin combined function fluorescence magnetic nano particle; 201010261766.7; 2010.08.20 ", a kind of streptavidin combined function fluorescence magnetic nano particle in conjunction with gene recombination technology and nanotechnology is provided, when providing new technology for biologically active substance, also visual enrichment, the separation for apoptotic cell provides new instrument.
Summary of the invention
The object of the invention is to provide a kind of application of streptavidin combined function fluorescence magnetic nano particle.
For achieving the above object, the technical solution used in the present invention is:
A kind of application of streptavidin combined function fluorescence magnetic nano particle adopts streptavidin combined function fluorescence magnetic nano particle to detect apoptotic cell.
The fluorescence magnetic nano particle that streptavidin is modified adds in the cell suspending liquid that contains the biotin labeling apoptotic cell, pass through enzyme linked immunoassay, the biotin labeled apoptotic cell of fluorescence magnetic nano particle specific combination that streptavidin is modified, and (being external magnet) isolated and is combined streptavidin combined function fluorescence magnetic nano particle with the apoptotic cell specificity under magneticaction.
The above-mentioned isolated streptavidin combined function fluorescence magnetic nano particle of being combined with the apoptotic cell specificity is carried out the free particle of centrifugal removal, namely obtain the apoptotic cell specificity in conjunction with streptavidin combined function fluorescence magnetic nano particle.
Described with contain the streptavidin combined function fluorescence magnetic nano particle that biotin labeled apoptotic cell specificity is combined and be, 3.0-4.0 milligram streptavidin combined function fluorescence magnetic nano particle is dropped into the 1-3 milliliter to be contained in the PBS solution of streptavidin, the room temperature vibration is after 5-10 minute, after giving a baby a bath on the third day after its birth time with PBS, stand-by; The described PBS solution that contains streptavidin is that every milliliter of PBS solution includes the 0.01mg streptavidin.The described biotin labeling apoptotic cell that contains is that the Annexin V-biotin of 0.1mg/ml adds 1 milliliter and contains 1 * 10 approximately with 8-10 microlitre concentration
6In the suspension of individual cell, room temperature was cultivated after 10-15 minute, the centrifugal 5-6 of 280-300g minute, after then giving a baby a bath on the third day after its birth time with PBS, namely obtained containing the cell suspending liquid of biotin labeling apoptotic cell.Describedly separate to realize free streptavidin combined function fluorescence magnetic nano particle and after the separating of streptavidin combined function fluorescence magnetic nano particle that the apoptotic cell specificity is combined by magnetic, pass through 280-300g again, 5-6 minute centrifugal treating is removed free particle, obtains the streptavidin combined function fluorescence magnetic nano particle of being combined with the apoptotic cell specificity.
The advantage that the present invention has: the present invention adopts Streptavidin combined function fluorescence magnetic nano particle as instrument, identification separation and the imaging of apoptotic cell have been realized in conjunction with Enzyme-multiplied immune technique, specific combination effect and signal amplification between Streptavidin-vitamin H have been taken full advantage of, and the magnetic stalling characteristic of Streptavidin combined function magnetic nanoparticle and fluorescence imaging effect, realized visual enrichment, separation and the imaging of apoptotic cell.
Description of drawings
The cell suspending liquid smear image to be separated that Fig. 1 provides for the embodiment of the invention, a wherein, visual light imaging, b, fluorescence imaging.
The Streptavidin that Fig. 2 provides for the embodiment of the invention is modified the smear image of fluorescence magnetic nano particle, a wherein, visual light imaging, b, fluorescence imaging.
The particle that Fig. 3 provides for the embodiment of the invention and cell suspending liquid are cultivated the back after 10-15 minute altogether, suspension smear image, a wherein, visual light imaging, b, fluorescence imaging.
The magnetic separated free that Fig. 4 provides for the embodiment of the invention not in conjunction with the particle of cell and the mixture image of particle-apoptotic cell mixture, a wherein, visual light imaging, b, fluorescence imaging.
After the centrifugal removal free particles that Fig. 5 provides for the embodiment of the invention, particle-apoptotic cell mixture image, a wherein, visual light imaging, b, fluorescence imaging.
Embodiment
Embodiment 1
1, contain to 1 milliliter and to have an appointment 10
6In the PBS solution of individual cell, add 10 microlitre 0.1mg/ml AnnexinV-biotin (Medical and Biological Laboratories Co., LTD.Japan), room temperature was cultivated 10-15 minute altogether, centrifugal 5 minutes collecting cells of 300g, 0.5ml PBS/ time, it is inferior to give a baby a bath on the third day after its birth, and obtains containing the cell suspending liquid (referring to Fig. 1 a Fig. 1 b) of biotin labeling apoptotic cell;
2,4.0 milligrams of streptavidin combined function fluorescence magnetic nano particles are added in the PBS solution of 1 milliliter of 0.01mg/ml streptavidin, room temperature was cultivated 5 minutes altogether, magnetic separates, 0.5ml PBS/ time, it is inferior to give a baby a bath on the third day after its birth, and obtains the fluorescence magnetic nano particle that streptavidin is modified, and namely namely uses (referring to Fig. 2 a Fig. 2 b) fully;
The preparation process of streptavidin combined function fluorescence magnetic nano particle is 201010261766.7 referring to application number, a kind of preparation method's of streptavidin combined function fluorescence magnetic nano particle patent application case, be specially: 1) by the PCR reaction StrepII label gene is connected in the α of allophycocyanin APC and the N-terminal of β subunit apoprotein gene respectively, obtain StrepII-apcA and Strep II-apcB gene fragment respectively, stand-by;
2) Strep II-apcA gene fragment is inserted into the downstream of 6 * His gene in the carrier A that has 6 * His gene, simultaneously phycobilin biosynthetic enzyme genes hol and pcyA is inserted on the carrier, obtain pCDF-Strep II-apcA-hol-pcyA, stand-by;
Strep II-apcB gene fragment is inserted into the downstream of 6 * His gene in the carrier B that has 6 * His gene, simultaneously color base lyase genes cpcS and cpcU is inserted on the carrier, obtain pRSF-Strep II-apcB-cpcS-cpcU, stand-by;
3) with step 2) two expression vectors whiles of gained transformed into escherichia coli, the engineering bacteria that said gene is expressed in the screening acquisition simultaneously;
4) with separation and purification behind the above-mentioned engineering bacteria inducing culture, obtain two label reorganization APC fluorescence proteins;
5) the two label reorganization of gained APC fluorescence protein is mixed with the superparamagnetism silicon core-shell nanoparticles vibration that zine ion is modified, namely obtain streptavidin combined function fluorescence magnetic nano particle.
3, the fluorescence magnetic nano particle input that Streptavidin is modified contains in the cell suspending liquid of biomarker apoptotic cell, room temperature is cultivated 10-15 minute (referring to Fig. 3 a Fig. 3 b) altogether, separates the mixture (referring to Fig. 4 a Fig. 4 b) of the mixture of fluorescence magnetic nano particle that the Streptavidin of not being combined with cell obtain dissociating modifies and particle-biotin labeling apoptotic cell by magnetic under the external magneticaction;
4, magnetic is separated the fluorescence magnetic nano particle of the free Streptavidin of not being combined with the cell modification that obtains and the mixture of particle-apoptotic cell mixture, be suspended in 0.5 milliliter of PBS solution, centrifugal 5 minutes of 300g, supernatant reclaims the fluorescence magnetic nano particle that the free Streptavidin of not being combined with cell is modified, be precipitated as particle-apoptotic cell mixture, with particle-apoptotic cell mixture smear, fluorescence imaging under fluorescent microscope (referring to Fig. 5 a Fig. 5 b).
Embodiment 2
Difference from Example 1 is:
Described with contain the streptavidin combined function fluorescence magnetic nano particle that biotin labeled apoptotic cell specificity is combined and be, 3 milliliters of 3.0 milligrams of streptavidin combined function fluorescence magnetic nano particles inputs are contained in the PBS solution of streptavidin, the room temperature vibration is after 10 minutes, after giving a baby a bath on the third day after its birth time with PBS, stand-by; The described PBS solution that contains streptavidin is that every milliliter of PBS solution includes the 0.01mg streptavidin.
The described biotin labeling apoptotic cell that contains is that the AnnexinV-biotin of 0.1mg/ml adds 1 milliliter and contains 1 * 10 approximately with 8 microlitre concentration
6In the suspension of individual cell, room temperature was cultivated after 10-15 minute, and centrifugal 6 minutes of 280g after then giving a baby a bath on the third day after its birth time with PBS, namely obtains containing the cell suspending liquid of biotin labeling apoptotic cell.
Describedly separate to realize free streptavidin combined function fluorescence magnetic nano particle and after the separating of streptavidin combined function fluorescence magnetic nano particle that the apoptotic cell specificity is combined by magnetic, pass through 280g again, 6 minutes centrifugal treating is removed free particle, obtains the streptavidin combined function fluorescence magnetic nano particle of being combined with the apoptotic cell specificity.
Claims (6)
1. the application of the fluorescence magnetic nano particle modified of a streptavidin, it is characterized in that: the fluorescence magnetic nano particle that adopts streptavidin to modify detects apoptotic cell, and wherein this is applied as non-diagnosis and therapeutic purpose;
Wherein the preparation process of fluorescence magnetic nano particle is:
1) by the PCR reaction Strep II label gene is connected in the α of allophycocyanin APC and the N-terminal of β subunit apoprotein gene respectively, obtains Strep II-apcA and Strep II-apcB gene fragment respectively, stand-by;
2) Strep II-apcA gene fragment is inserted into the downstream of 6 * His gene in the carrier A that has 6 * His gene, simultaneously phycobilin biosynthetic enzyme genes ho1 and pcyA is inserted on the carrier, obtain pCDF-Strep II-apcA-ho1-pcyA, stand-by;
Strep II-apcB gene fragment is inserted into the downstream of 6 * His gene in the carrier B that has 6 * His gene, simultaneously color base lyase genes cpcS and cpcU is inserted on the carrier, obtain pRSF-Strep II-apcB-cpcS-cpcU, stand-by;
3) with step 2) two expression vectors whiles of gained transformed into escherichia coli, the engineering bacteria that said gene is expressed in the screening acquisition simultaneously;
4) with separation and purification behind the above-mentioned engineering bacteria inducing culture, obtain two label reorganization APC fluorescence proteins;
5) the two label reorganization of gained APC fluorescence protein is mixed with the superparamagnetism silicon core-shell nanoparticles vibration that zine ion is modified, namely obtain the fluorescence magnetic nano particle.
2. press the application of the fluorescence magnetic nano particle of the described streptavidin modification of claim 1, it is characterized in that: the fluorescence magnetic nano particle that streptavidin is modified adds in the cell suspending liquid that contains the biotin labeling apoptotic cell, pass through enzyme linked immunoassay, the biotin labeled apoptotic cell of fluorescence magnetic nano particle specific combination that streptavidin is modified is isolated under magneticaction and is combined the fluorescence magnetic nano particle that streptavidin modifies with the apoptotic cell specificity.
3. press the application of the fluorescence magnetic nano particle of the described streptavidin modification of claim 2, it is characterized in that: above-mentioned isolated fluorescence magnetic nano particle of being combined the streptavidin modification with the apoptotic cell specificity is carried out the free particle of centrifugal removal, namely obtain the fluorescence magnetic nano particle that the apoptotic cell specificity is modified in conjunction with streptavidin.
4. press the application of the fluorescence magnetic nano particle of the described streptavidin modification of claim 2, it is characterized in that: described is 3.0-4.0 milligram fluorescence magnetic nano particle to be dropped into the 1-3 milliliter contain in the PBS solution of streptavidin with containing the fluorescence magnetic nano particle that streptavidin that biotin labeled apoptotic cell specificity is combined modifies, the room temperature vibration is after 5-10 minute, after giving a baby a bath on the third day after its birth time with PBS, stand-by; The described PBS solution that contains streptavidin is that every milliliter of PBS solution includes the 0.01mg streptavidin.
5. the application of the fluorescence magnetic nano particle of modifying by the described streptavidin of claim 2 is characterized in that: the described biotin labeling apoptotic cell that contains is to be that the Annexin V-biotin of 0.1mg/ml adds 1 milliliter and contains 1 * 10 with 8-10 microlitre concentration
6In the suspension of individual cell, room temperature was cultivated after 10-15 minute, the centrifugal 5-6 of 280-300g minute, after then giving a baby a bath on the third day after its birth time with PBS, namely obtained containing the cell suspending liquid of biotin labeling apoptotic cell.
6. press the application of the fluorescence magnetic nano particle of the described streptavidin modification of claim 2, it is characterized in that: describedly separate to realize fluorescence magnetic nano particle that free streptavidin is modified and after the separating of fluorescence magnetic nano particle that the streptavidin that the apoptotic cell specificity is combined is modified by magnetic, pass through 280-300g again, 5-6 minute centrifugal treating is removed free particle, the fluorescence magnetic nano particle that the streptavidin that obtains being combined with the apoptotic cell specificity is modified.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1431070A (en) * | 2003-01-21 | 2003-07-23 | 武汉大学 | Method for preparing water-soluble nano particles |
| CN101178362A (en) * | 2006-11-08 | 2008-05-14 | 北京金迪克生物技术研究所 | Establishment and application of novel E-LAMP |
| CN101445834A (en) * | 2008-12-30 | 2009-06-03 | 东南大学 | Magnetic particle and single base extension based SNP automatic detection method |
| CN101864291A (en) * | 2010-05-26 | 2010-10-20 | 上海大学 | Fluorescent nanoparticles Ru(bpy)3/SiO2, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1431070A (en) * | 2003-01-21 | 2003-07-23 | 武汉大学 | Method for preparing water-soluble nano particles |
| CN101178362A (en) * | 2006-11-08 | 2008-05-14 | 北京金迪克生物技术研究所 | Establishment and application of novel E-LAMP |
| CN101445834A (en) * | 2008-12-30 | 2009-06-03 | 东南大学 | Magnetic particle and single base extension based SNP automatic detection method |
| CN101864291A (en) * | 2010-05-26 | 2010-10-20 | 上海大学 | Fluorescent nanoparticles Ru(bpy)3/SiO2, preparation method and application thereof |
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