CN102010872B - Method for preparing fluorescent magnetic nanoparticles with streptavidin combination function - Google Patents
Method for preparing fluorescent magnetic nanoparticles with streptavidin combination function Download PDFInfo
- Publication number
- CN102010872B CN102010872B CN2010102617667A CN201010261766A CN102010872B CN 102010872 B CN102010872 B CN 102010872B CN 2010102617667 A CN2010102617667 A CN 2010102617667A CN 201010261766 A CN201010261766 A CN 201010261766A CN 102010872 B CN102010872 B CN 102010872B
- Authority
- CN
- China
- Prior art keywords
- strep
- gene
- carrier
- apca
- apcb
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of nano materials and particularly relates to a method for preparing fluorescent magnetic nanoparticles with streptavidin combination function. The method comprises the following specific steps of: respectively connecting a Strep II label gene to N ends of alpha and beta sub-gene apoprotein genes to obtain Strep II-apcA and Strep II-apcB gene segments;embedding the Strep II-apcA gene segment to the downstream of a 6*His gene, and cloning to one expression vector together with a phycobilin biosynthetic enzyme gene; embedding the Strep II-apcB gene segment to the downstream of the 6*his gene, cloning to another vector together with a chromphore lyase gene, simultaneously converting the two expression vectors into escherichia coli, screening engineering bacteria, separating and purifying the engineering bacteria by protein, and oscillating and mixing the purified double-label recombinant APC fluorescent protein and zinc ion modified superparamagnetism silicon shell nanoparticles to obtain the fluorescent magnetic nanoparticles with the streptavidin combination function. The obtained double-label recombinant protein can biologically combine streptavidin without being modified chemically.
Description
Technical field
The invention belongs to technical field of nano material, relate to a kind of preparation method of streptavidin combined function fluorescence magnetic nano particle specifically.
Background technology
The eighties in 20th century, along with the birth and the development of scanning tunnel microscope (STM), AFM microcosmic such as (AFM) sign and manipulation technology, nanotechnology is arisen at the historic moment.Wherein, magnetic nano-particle has good superparamagnetism and the surface is prone to functionalization, has caused increasing concern.Superparamagnetic nanoparticle through finishing can be applied to biological fixnig, separation, modification and detection, thereby plays the effect of extracting or measure a series of bioactive compoundss, xenobiotic, the interior component of born of the same parents or cell itself.
In addition; The fluorescence labeling probe technology; As a kind of bioassay technique commonly used; Have highly sensitive, selectivity good, specificity is high, particularity is good, characteristics such as visual directly perceived, is widely used in Flow Cytometry, immunoassay, molecular biology, cell analysis and apoptosis, to the research in a plurality of fields such as identification of tumour cell.Fluorescent marker commonly used at present comprises organic fluorescent dye, fluorescence semiconductor quantum dots material, common rare-earth luminescent material and goes up conversion rare earth material etc.The small molecules organic dye has been widely used in the life sciences such as diagnostics and molecular imaging as biological fluorescent labeling.Yet most of organic fluorescence probes are to photo-labile, and the fluorescence spectrum broad, are subject to the interference of the fluorescent signal background of sample own.The nano-probe technology has overcome some shortcomings of organic dye like inorganic light-emitting quantum dot and fluorescence nano emulsion microballoon.But the inorganic light-emitting quantum dot is because its water-soluble relatively poor, viscosity is big, quantum yield is lower and contain deleterious cadmium metal compound, thereby receives certain restriction in fields such as biomedicines.Therefore, organic polymer nano particle and be that the inorganic nanoparticles etc. of representative has become one of research focus of namo fluorescence probe as the matrix of load fluorescence molecule with the silicon oxide.
The super-paramagnetism nano fluorescent grain is to have the multi-functional nanometer material that magnetic is handled separation function and fluorescent mark function simultaneously; Nano level particle diameter connects biomolecules such as its can serve as a mark thing and albumen, nucleic acid; And have magnetic resolution and characteristic such as carry, the fluorescence mass-energy that has of itself plays the effect of spike and detection simultaneously.
Summary of the invention
The object of the invention is to provide a kind of preparation method of streptavidin combined function fluorescence magnetic nano particle.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:
A kind of preparation method of streptavidin combined function fluorescence magnetic nano particle:
1) through 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 IIapcB gene fragment respectively, for use;
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, for use;
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, for use;
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), obtain two label reorganization APC fluorescence proteins with separation and purification behind the above-mentioned engineering bacteria inducing culture;
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, promptly obtain streptavidin combined function fluorescence magnetic nano particle.
When said step 1) made up Strep II-apcA gene fragment, adopting Synechocystis sp.PCC6803 genomic dna was template, is primer with A1 and A2, carries out pcr amplification; A1:5 ' AAGGATCCGAGTAACTGGTCACACCCACAATTCGAGAAAATGAGTATCGTCACGAA 3 '; A2:5 ' GCGAGCTCCTAGCTCATTTTTCCGAT3 ';
When said step 1) made up Strep II-apcB gene fragment, adopting Synechocystis sp.PCC6803 genomic dna was template, is primer with B1 and B2, carries out pcr amplification; B1:5 ' AAGGATCCGAGTAACTGGTCACACCCACAATTCGAGAAAATGCAAGACGCAATTAC 3 '; B2:5 ' CCGAGCTCCTAGCTCAAGCCAGAGC3 '.
Said step 2) carrier A that has 6 * His gene is the pCDFDuet carrier; The carrier B that has 6 * His gene is the pRSFDuet carrier.
Said step 2) Strep II-apcA gene fragment, Strep II-apcB gene fragment, pCDFDuet carrier and pRSFDuet carrier all carry out double digestion with restriction enzyme BamH I and Sac I in; Obtain pCDFDuet-Strep II-apcA on the pCDFDuet carrier after then Strep II-apcA gene endonuclease bamhi is connected to enzyme and cuts through the T4 ligase enzyme, obtain pRSFDuet-StrepII-apcB on the pRSFDuet carrier after Strep II-apcB gene enzyme segment sheet is connected to enzyme and cuts through the T4 ligase enzyme; Above-mentioned endonuclease bamhi is 3-7 with carrier according to the volume parts ratio: 1 ratio is connected;
Phycobilin biosynthetic enzyme genes hol and pcyA are connected with carrier pCDFDuet-Strep II-apcA after cutting through corresponding enzyme successively, obtain expression vector pCDF-Strep II-apcA-hol-pcyA; Color base lyase genes cpcS and cpcU are connected to pRSFDuet-StrepII-apcB after cutting through corresponding enzyme successively, obtain expression vector pRSF-Strep II-apcB-cpcS-cpcU; Above-mentioned endonuclease bamhi is 3-7 with carrier according to the volume parts ratio: 1 ratio is connected.
The superparamagnetism silicon core-shell nanoparticles that zine ion described in the said step 5) is modified is that shell is the superparamagnetism silicon core-shell nanoparticles of nuclear with the Z 250 with silicon-dioxide for what modify through zine ion.
The advantage that the present invention had:
1. the optical dye that adopts among the present invention is an allophycocyanin, and this GFP good water solubility, toxic side effect is little, fluorescence quantum yield is high.
2. the optical dye that adopts among the present invention has the spectral signature similar with natural allophycocyanin for the allophycocyanin of reorganization, has avoided natural allophycocyanin content limited simultaneously, extracts the shortcoming of difficulty.
3. the optical dye that adopts among the present invention at first, contains the His-tag label for the allophycocyanin of reorganization, is convenient to the selective separation purifying; Secondly, contain Strep II label, need not chemically modified and can biologically combine streptavidin.
4. the superparamagnetism silicon core-shell nanoparticles that adopts among the present invention is to be the nano particle of shell with silicon-dioxide, and this shell material itself has adsorptive power to zine ion, need not chemical reaction and just can combine zine ion.
5. the zine ion that adopts among the present invention is as particle and the proteic bridge that is connected, the harm of having avoided heavy metal ion such as traditional nickel ion, cupric ion, cobalt ion to bring.
6. utilize the sequestering action between zine ion and the Histidine, realized the selective binding to histidine-tagged protein, this combination can be competed wash-out with imidazoles, and promptly this GFP is that reversibility combines.
Description of drawings
Fig. 1 is an embodiment of the invention construction of recombinant plasmid synoptic diagram;
Fig. 2 be electrophoresis that the two label recombinant allophycocyanins of the embodiment of the invention are expressed characterize and detect with biological affinity and to scheme (wherein, swimming lane from left to right: 1, dye Marker in advance; 2, before the conversion bacterial strain inducing; 3, behind the conversion bacterial strain inducing; 4, sample behind the gel chromatography.Light blue dyeing in the coomassie (on), ZnSO
4Dyeing (in), Western dyeing (descending) imaging.Dye albumen Maker in advance, molecular weight from top to bottom is 95,72,55,43,26,17kDa);
Fig. 3 is absorption spectrum and fluorescence emission spectrogram (wherein, a, the extinction spectrum of the two label recombinant allophycocyanins of the embodiment of the invention; B, fluorescence spectrum (λ
Ex=600nm));
Fig. 4 is the transmission electron microscope photo of embodiment of the invention nano particle;
Fig. 5 is the fluorescence imaging figure of embodiment of the invention multifunctional nanoparticles;
Fig. 6 combines the fluorescence imaging figure of streptavidin (FITC-Streptavdin) for embodiment of the invention multifunctional nanoparticles.
Embodiment
Embodiment
A. the clone of gene
(the National Center for Biotechnology Information from the U.S. state-run bioinformation center; NCBI) DB (http://www.ncbi.nlm.nih.gov/) obtains apcA, apcB, cpcS, cpcU, the ho1 of Synechocystis sp.PCC6803, the sequence of pcyA gene; And Strep II label gene order in the reference, design primer thus.Used primer and reaction conditions such as table 1.
With Synechocystis sp.PCC6803 genomic dna is template, carries out the PCR clone with each primer in the below table 1 respectively and obtains Strep II-apcA gene fragment, phycobilin biosynthetic enzyme hol and pcyA gene fragment, StrepII-apcB gene fragment, color base lyase cpcS and cpcU gene fragment respectively.
The PCR reaction system: distilled water 18.7 μ l contain Mg
2+Damping fluid 2.5 μ l, dNTP 0.5 μ l, upstream primer 1 μ l, downstream primer 1 μ l, Taq enzyme 0.3 μ l, Synechocystis sp.PCC 6803 genomes 1 μ l.
Because in design of primers; Strep II label gene being connected to 5 ' end of the upstream primer of apcA and apcB gene, is exactly the apcA and apcB gene fragment (abbreviation Strep II-apcA and Strep II-apcB) that N end connects Strep II gene so pcr amplification obtains.
Table 1
B. construction of recombinant plasmid
The StrepII-apcA gene fragment is inserted in the pCDFDuet carrier of Novagen company.The step of said gene insertion vector is: go on foot the PCR product fragment that the Strep II-apcA gene fragment that obtains through pcr amplification is carried out electrophoresis and reclaimed Strep II-apcA to last one; Gained fragment restriction enzyme BamH I and Sac I carry out double digestion; Simultaneously carrying out enzyme to carrier pCDFDuet with same restriction enzyme cuts; Reclaim the endonuclease bamhi of Strep II-apcA and carrier pCDFDuet respectively with TIANgel Midi Purification Kit (plain agar sugar gel DNA reclaims test kit, day root); Two bar segment that reclaim were mixed in 3: 1 by volume, under the effect of T4 ligase enzyme 16 ℃, connected 16h; Change intestinal bacteria over to connecting product; Utilization contains antibiotic flat screen selects circular single bacterium colony, and the picking mono-clonal is cultivated, and pcr amplification also detects and sequence verification with agarose gel electrophoresis; It is consistent with aim sequence to draw the bacterium colony that picking goes out, and this bacterial strain is protected kind for use.
Clone's phycobilin biosynthetic enzyme genes hol and pCDFDuet-Strep II-apcA are carried out double digestion with restriction enzyme Nde I and EcoR V; (plain agar sugar gel DNA reclaims test kit with TIANgel Midi Purification Kit; It root) reclaims hol endonuclease bamhi and carrier segments respectively; With endonuclease bamhi and carrier segments 3-7 by volume: 1 mixes, and 16 ℃ connect 16h under the effect of T4 ligase enzyme, changes the connection product over to intestinal bacteria; Utilization contains antibiotic flat screen selects circular single bacterium colony; The picking mono-clonal is cultivated, and pcr amplification also detects and sequence verification with agarose gel electrophoresis, obtains pCDFDuet-Strep II-apcA-hol carrier.Then phycobilin biosynthetic enzyme genes pcyA and pCDFDuet-Strep II-apcA-hol carrier are carried out double digestion with restriction enzyme EcoR V and Xho I; (plain agar sugar gel DNA reclaims test kit with TIANgel Midi Purification Kit; It root) reclaims pcyA endonuclease bamhi and carrier segments respectively; With endonuclease bamhi and carrier segments 3-7 by volume: 1 mixes, and 16 ℃ connect 16h under the effect of T4 ligase enzyme, changes the connection product over to intestinal bacteria; Utilization contains antibiotic flat screen selects circular single bacterium colony; The picking mono-clonal is cultivated, and pcr amplification also detects and sequence verification with agarose gel electrophoresis, obtains the pCDF-StrepII-apcA-hol-pcyA carrier.
As stated above Strep II-apcB gene fragment is inserted in first expression cassette of pRSFDuet carrier of Novagen company and obtained pRSFDuet-Strep II-apcB carrier; In second expression cassette that simultaneously clone's color base lyase genes cpcS and cpcU is connected in pRSFDuet-Strep II-apcB carrier successively, the gained plasmid is called pRSF-Strep II-apcB-cpcS-cpcU, and is as shown in Figure 1.
C. the screening of the conversion of recombinant plasmid and reorganization bacterium
With plasmid pCDF-Strep II-apcA-hol-pcyA and pRSF-Strep II-apcB-cpcS-cpcU while transformed into escherichia coli BL21; It is following that plasmid is transformed into colibacillary mode: single bacterium colony of picking coli strain BL21 from the flat board of fresh coating; Be inoculated in 5ml LB liquid nutrient medium, 37 ℃ of concussion overnight cultures; Get 100 μ l bacterium liquid, be forwarded to 5ml LB liquid nutrient medium under the aseptic condition, 37 ℃ of concussions are cultured to OD
600Be about 0.6, place 10min on ice; Centrifugal 5min (5000g, 4 ℃) supernatant discarded, the collecting precipitation thalline; With 1ml 0.1mol/L CaCl
2The resuspended thalline of sterile solution, centrifugal 30s (10000g, 4 ℃) supernatant discarded, bacterial sediment is with the 0.1mol/L CaCl of 100 μ l precoolings
2Resuspended; Add each 1 μ l of above-mentioned gained plasmid pCDF-Strep II-apcA-hol-pcyA and pRSF-StrepII-apcB-cpcS-cpcU; Ice bath 30min behind the mixing, 42 ℃ of heat shock 90s add 400 μ l LB liquid nutrient mediums behind the ice bath 1-2min; 15min is cultivated in the 60rpm concussion under 37 ℃ of conditions; Rotating speed increases to the 100-140rpm concussion and cultivates 30min, and coating bacterium liquid is containing on two kinds of antibiotic LB solid plates of kantlex and spectinomycin, is inverted in 37 ℃ of incubators to forming visible mono-clonal bacterial plaque.
20 single bacterium colonies of picking are inoculated in 5ml respectively and contain in 0.05mg/ml kantlex and the antibiotic LB liquid nutrient medium of 0.05mg/ml spectinomycin, the concussion overnight cultures; Therefrom taking out 100 μ l respectively is forwarded to 5ml and contains in 0.05mg/ml kantlex and the antibiotic LB liquid nutrient medium of 0.05mg/ml spectinomycin; 37 ℃ of concussions are cultured to OD
600Be 0.8-1.0, add IPTG (final concentration is 1mM) abduction delivering, lucifuge, 28 ℃, 150rpm expressed 8-10 hour.Getting 1ml abduction delivering liquid, centrifugal (5000g 10min), collects thalline; Thalline is uniformly dispersed with 50 μ l zero(ppm) water, adds 50 μ l2 * SDS-PAGE sample-loading buffer, in boiling water, boils 10min; It is centrifugal that (10000g 10min) gets supernatant 10 μ l and carries out the SDS-PAGE electrophoresis.Get separation gel behind the electrophoresis and be soaked in 20mM ZnSO
4Behind the 10min, Fig. 2 middle part is seen in the UV-irradiation imaging in the solution.Get separation gel subsequently and carry out the coomassie brilliant blue staining imaging, see Fig. 2 top.Visible by figure, protein expression contrast in the bacterial strain before and after inducing find between 17-26KDa, to have target protein to express obviously, and this target protein has color base.Bacterial strain that can expression target protein as shown in the figure, volume(tric)fraction be in the glycerine solution of 15-20%-20 ℃ protect kind.
Wherein, comprised the various albumen of expressing in the thalline in the supernatant after centrifugal, but can be by ZnSO
4Be coloured to picture, just have only the albumen of band color base.Do not contrast with inducing thalline, induce to start T7 promoter expression target protein.Be coloured to picture contrast through two kinds, can infer that the target protein that size is about 21KDa and 20KDa has color base.
D. the recombinate 5L scale fermentation of bacterium
The program that the 5L fermentor tank express to merge Phycocyanins, C-is following: the bacterial classification 10 μ l aseptic inoculations deposited of going bail for are in 5ml liquid LB substratum, and 12h are cultivated in 37 ℃ of concussions, transfer then in the triangular flask that contains 300ml liquid LB substratum, and 6h are cultivated in 37 ℃ of concussions.The fermentation of engineering bacteria is carried out in the automatic fermentor tank of 5L, the inoculum size of volume ratio 5%, and culture temperature is made as 37 ℃ before inducing, and rotating speed is increased progressively by 300-500rpm with fermentation time, and pH is controlled to be 7.0 automatically with ammoniacal liquor; Behind the fermentation beginning 4h, carry out feed supplement with the speed of 0.08g/ (Lmin) glucose.When fermentation begins about 7h, be 28 ℃ with the tank body temperature regulation, add inductor IPTG to final concentration 1mM, the reduction rotating speed is to 150rpm, inducing culture 10-12h.
E. the separation and purification of two label recombinant allophycocyanins
Above-mentioned fermented liquid is centrifugal, and (5000g 10min) has been expressed the thalline of target protein, and wet thallus is resuspended in the PBS of the pH that contains the 20mM imidazoles 7.4 damping fluid, resuspended 50g wet thallus in every liter of damping fluid, ultrasonication cell 30min.12000g, centrifugal 30min under 4 ℃ of conditions collects supernatant, is splined on Ni
2+Affinity chromatographic column.After the PBS damping fluid flushing chromatographic column with the pH that contains the 30mM imidazoles 7.4 of 5 times of column volumes, with the PBS elution buffer wash-out of the pH 7.4 that contains the 300mM imidazoles, elutriant is removed imidazoles through the G-25 post with 50mM potassium phosphate salt damping fluid (pH 7.0).Cross the Superdex200 gel chromatographic columns then, with 50mM potassium phosphate salt damping fluid (pH 7.0) wash-out, elution speed is 10ml/h, collects A
650nm/ A
620nm＞1.3 elutriant promptly gets target protein solution.
Get target protein 50 μ l and 50 μ l, 2 * SDS-PAGE sample-loading buffer, in boiling water, boil 10min, centrifugal (10000g 10min) gets supernatant 10 μ l and carries out the SDS-PAGE electrophoresis.Gel behind the electrophoresis is soaked in 20mM ZnSO
4Solution 10min, Fig. 2 middle part is seen in the uv irradiating imaging, then gel is carried out coomassie brilliant blue R250 dyeing, scanning imagery is seen Fig. 2 top.Purifying gained albumen can be dyed the uv irradiating imaging by zinc, explains that this protein band has color base.With albumen Marker contrast, the molecular weight of target protein is about 21 and 20kDa respectively.The natural allophycocyanin α of existing bibliographical information and β molecular weight subunit are about 19 and 18KDa respectively.Two labels that reorganization is introduced make target protein distinguish about 2KDa than the molecular weight of native protein subunit.
F. the biological affinity Western of two label recombinant allophycocyanins detects
After c and the described SDS-PAGE electrophoresis end of e; To dye Marker in advance is reference (17-26kDa), adhesive tape is cut to suitable size (between the 17-26KDa), under constant voltage 12V condition; Change NC film (Nitrocellulose Transfer Membrane, Whatman Protran BA 85) 1.5h.NC film behind the commentaries on classics film is used confining liquid, and confining liquid is w/v 5% skim-milk, w/v 0.1%tween-20, and the PBS of pH 7.4, room temperature is shaken 2h.After abandoning confining liquid, wash film with PBST, 5min/ time, it is inferior to give a baby a bath on the third day after its birth, and PBST is w/v 0.1%tween-20, the PBS of pH 7.4.The streptavidin of horseradish peroxidase is spent the night for 4 ℃ than 1: 1000 usefulness confining liquid dilution back immersion NC film with reference to proportional volume.Wash film with PBST, 15min/ time, wash four times.Get the NC film then, wrap, be fixed in the metal splint with preservative film.In the darkroom, cut an X-ray sheet bigger than film, overlay on the film, behind the exposure 15min, be placed in the developing solution and develop, after waiting image to occur, take out mating plate at Shui Zhongchong once, be placed in the stationary liquid fixedly 5min, after the washing, drying forms images sees Fig. 2 bottom again.The target protein of expressing in the thalline reaches the streptavidin that all can combine horseradish peroxidase through the target protein behind the gel chromatography, has proved the biological affinity of two label proteins.
The spectroscopic properties of g. two label recombinant allophycocyanins
Shown in Fig. 3 (a), the gained target protein mainly contains two absorption peaks in the visible spectrum district of 400-700nm in the embodiment of the invention, i.e. the acromion at the main peak at 650nm place and 620nm place.Shown in Fig. 3 (b), the gained target protein is under the optical excitation of 600nm in the embodiment of the invention, and emission peak is at the 660nm place.Absorption spectrum is consistent with the natural allophycocyanin tripolymer spectral signature of bibliographical information with fluorescence spectrum, explains that the two label recombinant allophycocyanins of this purpose are suitable as the fluorescent probe dyestuff.
H. the preparation of zine ion modifying magnetic nano particle: adopt microemulsion method to prepare superparamagnetism silicon core-shell nanoparticles (can prepare nano particle Selective separation of proteinswith pH-dependent magnetic nanoadsorbents.Nanotechnology 18 (2007) 365604.) according to record in the document; Then add 10mg superparamagnetism silicon core-shell nanoparticles in every milliliter of 0.5mM zinc sulfate solution; Behind the ultra-sonic dispersion 5 minutes; Shaking table vibration 30 minutes; Form zine ion and modify superparamagnetism silicon core-shell nanoparticles, wash four times after particle subsequent use, and utilize transmission electron microscope to carry out diameter characterization (referring to Fig. 4).
I. the preparation of multifunctional nanoparticles: with the two label recombinant allophycocyanins of gained among the e in zine ion modifying magnetic nano particle mixing; Vibration 30min; The magnetic branch supernatant of leaving away; The superparamagnetic nano particle cleaned standby seam with streptavidin combined function that obtains utilizes microscope that particle is carried out fluorescence imaging (referring to Fig. 5).
J. multifunctional nanoparticles combines the streptavidin ability to detect: gained multifunctional nanoparticles among the i and the resonance of FITC-streptavidin are swung 30min; The magnetic branch supernatant of leaving away; The biology that obtains has combined the superparamagnetic nano particle cleaned standby seam of streptavidin, utilizes microscope that particle is carried out fluorescence imaging (referring to Fig. 6).
Claims (5)
1. the preparation method of a streptavidin combined function fluorescence magnetic nano particle is characterized in that:
1) through 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, for use;
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, for use;
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, for use;
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), obtain two label reorganization APC fluorescence proteins with separation and purification behind the above-mentioned engineering bacteria inducing culture;
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, promptly obtain streptavidin combined function fluorescence magnetic nano particle.
2. press the preparation method of the described streptavidin combined function of claim 1 fluorescence magnetic nano particle; It is characterized in that: when said step 1) makes up the StrepII-apcA gene fragment; Adopting Synechocystis sp.PCC6803 genomic dna is template; With A1 and A2 is primer, carries out pcr amplification; A1:5 ' AAGGATCCGAGTAACTGGTCACACCCACAATTCGAGAAAATGAGTATCGTCACGAA 3 '; A2:5 ' GCGAGCTCCTAGCTCATTTTTC CGAT3 ';
When said step 1) made up Strep II-apcB gene fragment, adopting Synechocystis sp.PCC6803 genomic dna was template, is primer with B1 and B2, carries out pcr amplification; B1:5 ' AAGGATCCGAGTAACTGGTCACACCCACAATTCGAGAAAATGCAAGACGCAATTAC 3 '; B2:5 ' CCGAGCTCCTAGCTCAAGCCAGAGC3 '.
3. by the preparation method of the described streptavidin combined function of claim 1 fluorescence magnetic nano particle, it is characterized in that: the carrier A that said step 2) has 6 * His gene is the pCDFDuet carrier; The carrier B that has 6 * His gene is the pRSFDuet carrier.
4. press the preparation method of claim 1 or 2 described streptavidin combined function fluorescence magnetic nano particles; It is characterized in that: Strep II-apcA gene fragment, Strep II-apcB gene fragment, pCDFDuet carrier and pRSFDuet carrier all carry out double digestion with restriction enzyme BamH I and Sac I said step 2); Obtain pCDFDuet-Strep II-apcA on the pCDFDuet carrier after then Strep II-apcA gene endonuclease bamhi is connected to enzyme and cuts through the T4 ligase enzyme, obtain pRSFDuet-Strep II-apcB on the pRSFDuet carrier after Strep II-apcB gene endonuclease bamhi is connected to enzyme and cuts through the T4 ligase enzyme;
Phycobilin biosynthetic enzyme genes ho1 and pcyA are connected with carrier pCDFDuet-Strep II-apcA after cutting through corresponding enzyme successively, obtain expression vector pCDF-Strep II-apcA-ho1-pcyA; Color base lyase genes cpcS and cpcU are connected to pRSFDuet-Strep II-apcB after cutting through corresponding enzyme successively, obtain expression vector pRSF-Strep II-apcB-cpcS-cpcU;
5. according to the preparation method of the streptavidin combined function fluorescence magnetic nano particle described in the claim 1, it is characterized in that: the superparamagnetism silicon core-shell nanoparticles that the zine ion described in the said step 5) is modified is that shell is the superparamagnetism silicon core-shell nanoparticles of nuclear with the Z 250 with silicon-dioxide for what modify through zine ion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102617667A CN102010872B (en) | 2010-08-20 | 2010-08-20 | Method for preparing fluorescent magnetic nanoparticles with streptavidin combination function |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102617667A CN102010872B (en) | 2010-08-20 | 2010-08-20 | Method for preparing fluorescent magnetic nanoparticles with streptavidin combination function |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102010872A CN102010872A (en) | 2011-04-13 |
| CN102010872B true CN102010872B (en) | 2012-07-11 |
Family
ID=43841202
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102617667A Expired - Fee Related CN102010872B (en) | 2010-08-20 | 2010-08-20 | Method for preparing fluorescent magnetic nanoparticles with streptavidin combination function |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102010872B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103267841B (en) * | 2013-05-15 | 2014-03-12 | 湖南农业大学 | Method for preparing ELISA (enzyme-linked immuno sorbent assay) enzyme-labelled antigen through utilizing avidin binding peptide and avidin combining principle |
| CN109115741A (en) * | 2018-08-23 | 2019-01-01 | 海南大学 | A kind of multicolour fluorescent magnetic chemistry nose sensor quickly detects pathogenic microorganism |
| CN114516911B (en) * | 2022-03-15 | 2024-06-18 | 东莞市东南部中心医院 | Magnetic nano chemotactic factor SDF-1 and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101440370A (en) * | 2008-10-17 | 2009-05-27 | 中国科学院海洋研究所 | Preparation of allophycocyanin fluorescent protein |
| US20100008862A1 (en) * | 2008-07-10 | 2010-01-14 | Aihua Fu | Fluorescent magnetic nanoprobes, methods of making, and methods of use |
-
2010
- 2010-08-20 CN CN2010102617667A patent/CN102010872B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100008862A1 (en) * | 2008-07-10 | 2010-01-14 | Aihua Fu | Fluorescent magnetic nanoprobes, methods of making, and methods of use |
| CN101440370A (en) * | 2008-10-17 | 2009-05-27 | 中国科学院海洋研究所 | Preparation of allophycocyanin fluorescent protein |
Non-Patent Citations (3)
| Title |
|---|
| E. K. M. Uedaa et al..Current and prospective applications of metal ion–protein binding.《Journal of Chromatography A》.2003,第988卷(第1期),第3页第2栏. * |
| V T Oi et al..Fluorescent phycobiliprotein conjugates for analyses of cells and molecules.《JCB》.1982,第93卷(第3期),第981-986页. * |
| 杨雨.光学活性重组别藻蓝蛋白的初步研究.《中国优秀硕士学位论文全文数据库 基础科学辑》.2007,(第4期),第54-63页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102010872A (en) | 2011-04-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jacob et al. | Magnetotactic bacteria and magnetosomes–Scope and challenges | |
| Arakaki et al. | Formation of magnetite by bacteria and its application | |
| Fliermans et al. | Isolation of Legionella pneumophila from nonepidemic-related aquatic habitats | |
| Sekar et al. | Phycobiliproteins as a commodity: trends in applied research, patents and commercialization | |
| Zhang et al. | Semicontinuous culture of Magnetospirillum gryphiswaldense MSR-1 cells in an autofermentor by nutrient-balanced and isosmotic feeding strategies | |
| Šafaříková et al. | Biosorption of water-soluble dyes on magnetically modified Saccharomyces cerevisiae subsp. uvarum cells | |
| CN102010872B (en) | Method for preparing fluorescent magnetic nanoparticles with streptavidin combination function | |
| Xu et al. | Surface expression of protein A on magnetosomes and capture of pathogenic bacteria by magnetosome/antibody complexes | |
| Chang et al. | Coagulation–membrane filtration of Chlorella vulgaris at different growth phases | |
| CN103777016B (en) | A kind of by fluorescent target to cell detection virus and the method for bacterium | |
| Brussaard et al. | Virioplankton dynamics and virally induced phytoplankton lysis versus microzooplankton grazing southeast of the Kerguelen (Southern Ocean) | |
| Berland et al. | The antibacterial substances of the marine alga Stichochrysis immobilis (chrysophyta) 1 2 | |
| CN108753670A (en) | Multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis and preparation method | |
| CN109593523A (en) | A kind of preparation method and application of hypha,hyphae nitrogen sulphur auto-dope carbon dots | |
| Burkholder et al. | Studies on B vitamins in relation to productivity of the Bahia Fosforescente, Puerto Rico | |
| Shukia et al. | Antarctic cyanobacteria as a source of phycocyanin: an assessment | |
| hong Gao et al. | Imaging of jasmonic acid binding sites in tissue | |
| CN104877011B (en) | The protein transduction domain in fish nervous necrosis virus source as well as preparation method and application thereof | |
| CN101509007B (en) | Synthesis of LfcinB15-Mag12 encoding gene and expression method in colon bacillus | |
| CN102094029A (en) | Method for preparing allophycocyanin tripolymer fluorescent protein | |
| CN102703509A (en) | Method for increasing genetic transformation of improved Shewanella oneidensis MR-1 | |
| CN101838661A (en) | Preparation method of high stability phycocyanin fuse fluorescent protein | |
| CN109187970B (en) | It is a kind of quickly to detect aquatic products disease gold mark nucleic acid test strip and preparation method thereof | |
| Mathuriya et al. | Magnetotactic bacteria: performances and challenges | |
| Froneman | The importance of phytoplankton size in mediating trophic interactions within the plankton of a southern African estuary |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120711 Termination date: 20170820 |