CN107759698A - A kind of application of EGFP CTA2 TAT fusion proteins in fluorescence probe is prepared - Google Patents
A kind of application of EGFP CTA2 TAT fusion proteins in fluorescence probe is prepared Download PDFInfo
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- CN107759698A CN107759698A CN201710842079.6A CN201710842079A CN107759698A CN 107759698 A CN107759698 A CN 107759698A CN 201710842079 A CN201710842079 A CN 201710842079A CN 107759698 A CN107759698 A CN 107759698A
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Abstract
The invention belongs to technical field of bioengineering, application of more particularly to a kind of EGFP CTA2 TAT fusion proteins in fluorescence probe is prepared.The TAT parts contained in the structure of described EGFP CTA2 TAT fusion proteins are that the fluorescence probe that cell-penetrating peptide can help to design enters cell interior through cell membrane；It can realize that the targeting to intracellular endoplasmic reticulum positions containing endoplasmic reticulum positioning sequence KDEL in CTA2 parts；The real-time microcosmic Tracing detection to endoplasmic reticulum in living cells and its corresponding physiological change can be achieved eventually through exciting EGFP to send green fluorescence in the fusion protein.With good biocompatibility, safe and non-toxic and the characteristics of with higher targeting and detection sensitivity compared with common endoplasmic reticulum coloring agent.
Description
Technical field
The invention belongs to technical field of bioengineering, more particularly to a kind of EGFP-CTA2-TAT fusion proteins prepare it is glimmering
Application in light probe.
Background technology
In recent decades, with the foundation of some new molecular recognition mechanisms, the exploitation of new material and application and section
The progress of skill, increasing biological fluorescent labeling are developed for the basic research in life science subject.Its pair studied
As the ion, small molecule and large biological molecule (enzyme, the core that are transitioned into from the various lewis' acids in homogeneous phase solution in organism
Acid, protein etc.).Researchers have that sensitivity is higher, the more preferable biological fluorescent labeling of selectivity except constantly pursuing
Construction method outside, more focus on by biological fluorescent labeling be applied to the in situ detections of important signal transducers in organism with
And their stress change in metabolic process of life are monitored in real time.This cause sensor based on biological fluorescent labeling into
For the important tool in research life research-on-research, they have also greatly promoted the development of life science, and help to study
Persons go to explore the tera incognita in life process.Develop and the biological fluorescent labeling for detecting intracellular object is main
Have：Fluorescence probe based on organic molecule fluorescence probe, nucleic acid or polypeptide, nano material probe and green fluorescent protein probe
Deng.
The technology of multi-functional fusion protein construction is quite ripe in laboratory research, EGFP have green fluorescence,
The identity for containing endoplasmic reticulum positioning sequence KDEL and TAT in CTA2 as cell-penetrating peptide is well-known.But this utilize egg
The large biological molecule albumen with good biocompatibility of white integration technology structure is current not yet as the method for fluorescence probe
Appear in the newspapers.
The content of the invention
The shortcomings that to overcome prior art and deficiency, it is an object of the invention to provide a kind of EGFP-CTA2-TAT fusions
Application of the albumen in fluorescence probe is prepared.
The purpose of the present invention is achieved through the following technical solutions：
A kind of application of EGFP-CTA2-TAT fusion proteins in fluorescence probe is prepared, described EGFP-CTA2-TAT melt
The amino acid sequence of hop protein is as follows：
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGV
QCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY
NSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYKASEFELGGGGSMSNTCDEKTQSLGVKFLDEYQSKVKRQIFSGYQSDIDTHNRIKDELVDGR
KKRRQRRRPPQLEHHHHHH
The nucleotide sequence of the described EGFP-CTA2-TAT fusion proteins of coding is as follows：
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTA
AACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTG
CACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCT
ACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC
TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCT
GAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCT
ATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTG
CAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAG
CACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCG
GGATCACTCTCGGCATGGACGAGCTGTACAAGGCTAGCGAATTCGAGCTCGGAGGTGGTGGATCCATGAGCAATACG
TGCGATGAGAAGACACAAAGCCTGGGCGTGAAATTCTTAGACGAATATCAAAGCAAAGTGAAACGCCAAATCTTCAG
CGGATACCAAAGCGATATTGACACGCACAATCGCATCAAGGATGAGTTAGTCGACGGTCGTAAGAAACGTCGTCAGC
GTCGTCGTCCACCACAGCTCGAGCACCACCACCACCACCACTGA
The preparation method of described EGFP-CTA2-TAT fusion proteins, is comprised the following steps：
By Escherichia coli BL21 (DE3) bacterium containing purposeful recombinant plasmid pET22b-EGFP-CTA2-TAT
Kind is activated, expands culture；Then the induced expression of target protein is carried out, obtains wet thallus；Wet thallus, which suspends, to be cracked, and from
The heart, obtain supernatant protein solution；Supernatant protein solution carries out affinity chromatography, desalination of dialysing, freeze-drying, obtains EGFP-
CTA2-TAT fusion proteins；
The specific steps of described activation are preferably：
(1) by E.coli BL21 (DE3) the culture presevation liquid containing recombinant plasmid pET22b-EGFP-CTA2-TAT containing
There are in the LB fluid nutrient mediums of final concentration of 50 μ g/mL ammonia benzyl antibiotic 37 DEG C, 3~4h of 200r/min cultures；
(2) bacterium solution made from step (1) is drawn into flat board, 37 DEG C of 12~14h of solid culture；
(3) choose the single bacterium that step (2) culture obtains and fall within the LB liquid training containing final concentration of 50 μ g/mL ammonia benzyl antibiotic
37 DEG C, 12~14h of 200r/min cultures are supported in base；
(4) bacterium solution made from glycerine and step (3) is mixed, contains recombinant plasmid pET22b-EGFP- after being activated
CTA2-TAT E.coli BL21 (DE3) monoclonal strain bacterium solution；
The specific steps of described expansion culture are preferably：
(1) by E.coli BL21 (DE3) monoclonal containing recombinant plasmid pET22b-EGFP-CTA2-TAT after activation
Strain bacterium solution is with volume ratio 1:100 ratio is inoculated into the LB fluid nutrient mediums containing final concentration of 50 μ g/mL ammonia benzyl antibiotic,
37 DEG C, 12~14h of 200r/min cultures；
(2) by the bacterium solution of culture in step (1) according to volume ratio 1:50 are transferred to containing final concentration of 50 μ g/mL ammonia benzyl antibiosis
In the LB fluid nutrient mediums of element, 37 DEG C, 200r/min expansion cultures 4h；
The condition of described induced expression is preferably：
Using final concentration of 1mmoL/L IPTG, 37 DEG C, 200r/min inductions 5h；
The specific steps of described suspension cracking are preferably：
Wet thallus is suspended in lysis buffer, lysozyme is added and stirs；Above-mentioned mixed liquor is carried out repeatedly
Freeze thawing, until thalline fully cracks, it is no longer sticky；DNA enzymatic is added if bacterium solution is still sticky after the multigelation further to split
Solution, until thalline fully cracks, it is no longer sticky；
Described wet thallus and the mass volume ratio of lysis buffer are preferably 1g:10mL；
Described lysis buffer is preferably PBS；
The final concentration of described lysozyme is preferably 80 μ g/mL；
The concrete operations of described affinity chromatography are preferably：
Chromatographic column wet method filling material (Ni- Ago-Gels 6FF (His labels Purification Resin)), layer is carried out with PBS
Analyse column equilibration, supernatant protein solution loading；Loading is balanced with PBS to chromatographic column again after finishing；Balance knot
Protein ladder elution is carried out after beam, the eluent of gradient elution is respectively：PBS containing final concentration of 20mM imidazoles,
PBS containing final concentration of 60mM imidazoles, the PBS containing final concentration of 100mM imidazoles, containing final concentration of 200mM
The PBS of imidazoles, the PBS containing final concentration of 300mM imidazoles, the PBS bufferings containing final concentration of 500mM imidazoles
Liquid；Collect the collection liquid that the PBS containing final concentration of 100mM imidazoles affords；
The concrete operations of described dialysis desalination are preferably：
The collection liquid that PBS containing final concentration of 100mM imidazoles affords is utilized respectively PBS, body
PBS that fraction is 50%, water carry out dialysis desalination, per changing a dialyzate in 8h；Wherein, water can dialyse twice
Ensure that desalination is more thorough；
The present invention is had the following advantages relative to prior art and effect：
EGFP-CTA2-TAT fusion proteins as fluorescence probe biocompatibility is good, in acellular poison effect and targeting
Matter net, wherein, EGFP-CTA2-TAT fusion proteins not only have the dyeing function of common endoplasmic reticulum coloring agent, and EGFP is excited
Light is green fluorescence, and green belongs to the most sensitive light color of human eye, substantially increases the sensitivity of detection；EGFP-CTA2-TAT
TAT in fusion protein structure partly belongs to cell-penetrating peptide, and film activity is worn so as to maintain cytoactive, Neng Goubang with good
The fluorescence probe of design is helped to enter cell interior through cell membrane；CTA2 parts in EGFP-CTA2-TAT fusion protein structures
In containing endoplasmic reticulum positioning sequence KDEL can realize targeting positioning to intracellular endoplasmic reticulum, reduce to other intracellular membrane structures
The probability of dyeing.Eventually through exciting EGFP to send green fluorescence, can be achieved to intracellular endoplasmic reticulum and its corresponding physiological change
Real-time microcosmic Tracing detection.Therefore, the living cells that EGFP-CTA2-TAT fusion proteins can be good as a kind of biocompatibility
The green fluorescence probe of interior targeting positioning endoplasmic reticulum.
Brief description of the drawings
Fig. 1 is pET22b-EGFP-CTA2-TAT plasmid figures.
The SDS-PAGE detection figures of collection liquid when Fig. 2 is different gradient elutions；Wherein, 1：PBS delays after protein sample loading
Fliud flushing balances period collection liquid；2：PBS elution period collection liquid containing final concentration of 20mM imidazoles；3：Containing final concentration of
The PBS elution period collection liquid of 100mM imidazoles；4：The PBS elution period containing final concentration of 500mM imidazoles receives
Liquid collecting.
Fig. 3 is cell-penetrating activity the result analysis chart.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
In embodiment, E.coli BL21 (DE3) strain containing recombinant plasmid pET22b-EGFP-CTA2-TAT is in Shen
Please number be " 201410578270.0 ", application entitled " a kind of oligomeric protein and its preparation side with Brain targeting effect
Disclosed in method, purposes ", recombinant plasmid contained by the strain is that EGFP-CTA2-TAT genetic fragments insertion pET22b plasmids are polyclonal
Obtained by Nde I and the Xho I of site (Fig. 1)；
The formula of used medium is as follows in embodiment：
LB fluid nutrient mediums (1L formulas)：Tryptone 10g, yeast extract 5g, sodium chloride 10g；
LB solid mediums (1L formulas)：Tryptone 10g, yeast extract 5g, sodium chloride 10g, agar powder 15g.
The actication of culture of embodiment 1 and glycerol tube preservation
(1) prepared by flat board：LB solid medium dispensings, sterilize 121 DEG C, 30min；Ultraviolet 30min ultra-clean work is opened in advance
Alcolhol burner is lighted as platform, flat board semi-loop is placed around alcolhol burner；Sterilized LB solid mediums are cooled in superclean bench
About 60 DEG C, ammonification benzyl antibiotic (the μ g/mL of final concentration 50) is blown and beaten rapidly, shaken up；Complete to be down flat plate before culture medium solidifying, be finished down
Flat board is half-open, and workbench opens wind, and culture medium solidifying just covers preservative film and wraps preservation.
(2) bacterium is connect：One pipe 5mL LB fluid nutrient mediums, first add 2.5 μ L ammonia benzyl antibiotic mother liquors (mother liquid concentration 100mg/
ML E.coli BL21 (DE3) the culture presevation liquid for), adding 50 μ L to contain recombinant plasmid pET22b-EGFP-CTA2-TAT again；37
DEG C, 200r/min culture 4h；
(3) flat board is drawn：After the abundant calcination of oese alcolhol burner, bacterium solution made from step (2) is stained with, " Z " word method draws flat board；
37 DEG C of 12~14h of incubator culture of preservative film package；
(4) monoclonal is chosen：Single bacterium made from choosing step (3) is fallen within containing 2.5 μ L ammonia benzyl antibiotic mother liquor (mother liquid concentrations
37 DEG C of 5mL LB fluid nutrient mediums, 200r/min cultures 12h 100mg/mL)；
(5) culture presevation (volume fraction is 60% glycerol tube)：, bacterium solution made from 50 μ L glycerols and 50 μ L steps (4)
Mixing, E.coli BL21 (DE3) monoclonal strain containing recombinant plasmid pET22b-EGFP-CTA2-TAT after being activated
Bacterium solution；Sealing compound seals, and makes marks and is frozen in -20 DEG C.
The expansion culture of the strain of the mesh of embodiment 2 and the induced expression of target protein
(1) recombinant plasmid pET22b-EGFP-CTA2- will be contained after the obtained activation of embodiment 1 in superclean bench
TAT E.coli BL21 (DE3) monoclonal strain bacterium solution is with volume ratio 1:100 ratio is inoculated into containing ammonia benzyl antibiotic (eventually
The μ g/mL of concentration 50) LB fluid nutrient mediums in；37 DEG C are subsequently placed in, 200r/min incubator overnight culture 12h；
(2) on superclean bench by the bacterium solution being incubated overnight in step (1) according to volume ratio 1:50 are transferred to containing ammonia benzyl
In antibiotic (the μ g/mL of final concentration 50) and the conical flask of LB fluid nutrient mediums；It is subsequently placed in 200r/min, 37 DEG C of shaking tables expand training
Support 4h；
(3) IPTG of the final concentration of 1mmoL/L of bacterium solution addition after cultivating will be expanded in step (2)；37 DEG C of shaking table,
200r/min induces 5h；
(4) 4 DEG C of bacterium solution after step (3) induced expression, 10000r/min are centrifuged into 10min, collects thalline and abandon supernatant training
Base is supported, wet thallus is obtained and weighs its quality.
The extraction and purifying of embodiment 3EGFP-CTA2-TAT fusion proteins
(1) wet thallus made from embodiment 2 is suspended in (wet thallus in lysis buffer (PBS)：Cracking buffering
Liquid=1g：10mL)；Addition lysozyme (the μ g/mL of final concentration 80) is simultaneously uniform with magnetic stirrer；Above-mentioned mixed liquor is put into
Refrigerator carries out multigelation, if still sticky addition DNA enzymatic further cracks bacterium solution after multigelation；
(2) when the thalline in step (1) fully cracks, guarantee is no longer sticky, uses 4 DEG C of refrigerated centrifuge, 10000r/
Min centrifuges 10min, discards precipitation and collects supernatant, obtains supernatant protein solution；
(3) chromatographic column wet method filling material (Ni- Ago-Gels 6FF (His labels Purification Resin)), chromatograph device is connected
(nucleic acid-protein detector, peristaltic pump, chromatographic column) each adapter, chromatography column equilibration is carried out with PBS；Utilize peristaltic pump
Supernatant protein solution loading made from step (2) is made the abundant hanging column of destination protein by (0.8rpm), and pillar reach saturation it
Stopping loading afterwards prevents protein solution from wasting；
(4) loading is balanced with PBS to chromatographic column again after finishing；
(5) protein ladder elution is carried out after balance terminates, the eluent of gradient elution is respectively：Containing final concentration of 20mM
The PBS of imidazoles, the PBS containing final concentration of 60mM imidazoles, the PBS containing final concentration of 100mM imidazoles,
PBS containing final concentration of 200mM imidazoles, the PBS containing final concentration of 300mM imidazoles, containing final concentration of 500mM
The PBS of imidazoles；Collect each efflux, and separately sampled progress SDS-PAGE detections.As a result as shown in Fig. 2 containing dense eventually
Spend collection liquid (protein solution) the destination protein content highest afforded for the PBS of 100mM imidazoles and purity also most
It is high.
(6) collection liquid for affording the PBS containing final concentration of 100mM imidazoles in step (4) is put into advance
In the bag filter activated；It is utilized respectively pure PBS, the PBS that volume fraction is 50%, clear water is to protein solution
Dialysis desalination is carried out, interior per 8h to change a dialyzate, clear water dialysis ensures that desalination is more thorough twice；
(7) after the destination protein solution dialysed threading plate is sealed on request, putting refrigerator into makes it fully freeze；It is accurate
The sample got ready is put into the good freeze dryer of advance precooling and freezed under -80 DEG C of vacuumized conditions, obtains EGFP-CTA2-TAT
Fusion protein, EGFP-CTA2-TAT fusion proteins (the cotton-shaped albumen of silk) on plate are scraped after the completion of lyophilized and sterilized in cleaning
In EP pipes, and make marks and frozen in -20 DEG C.
Embodiment 4
Solution (10mg/mL) is made in EGFP-CTA2-TAT fusion proteins made from embodiment 3 and adds prostate gland cancer cell
In PC-3 (being purchased from Shanghai cell institute of the Chinese Academy of Sciences, conventionally cultivate), in 37 DEG C of CO2Culture is incubated in incubator altogether
1h；It is incubated culture and removes culture medium later, and cell is carefully washed three times with PBS；The cell washed is transferred to fluorescence and shown
The intracellular fluorescence distribution situation of micro- Microscopic observation.
As a result as shown in figure 3, EGFP-CTA2-TAT fusion proteins have wear film activity well and endoplasmic reticulum can be navigated to
The characteristics of upper progress fluorescence developing.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Guangdong University of Technology
<120>A kind of application of EGFP-CTA2-TAT fusion proteins in fluorescence probe is prepared
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 319
<212> PRT
<213> Artificial Sequence
<220>
<223>The amino acid sequence of EGFP-CTA2-TAT fusion proteins
<400> 1
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ala
225 230 235 240
Ser Glu Phe Glu Leu Gly Gly Gly Gly Ser Met Ser Asn Thr Cys Asp
245 250 255
Glu Lys Thr Gln Ser Leu Gly Val Lys Phe Leu Asp Glu Tyr Gln Ser
260 265 270
Lys Val Lys Arg Gln Ile Phe Ser Gly Tyr Gln Ser Asp Ile Asp Thr
275 280 285
His Asn Arg Ile Lys Asp Glu Leu Val Asp Gly Arg Lys Lys Arg Arg
290 295 300
Gln Arg Arg Arg Pro Pro Gln Leu Glu His His His His His His
305 310 315
<210> 2
<211> 960
<212> DNA
<213> Artificial Sequence
<220>
<223>Encode the nucleotide sequence of EGFP-CTA2-TAT fusion proteins
<400> 2
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggct 720
agcgaattcg agctcggagg tggtggatcc atgagcaata cgtgcgatga gaagacacaa 780
agcctgggcg tgaaattctt agacgaatat caaagcaaag tgaaacgcca aatcttcagc 840
ggataccaaa gcgatattga cacgcacaat cgcatcaagg atgagttagt cgacggtcgt 900
aagaaacgtc gtcagcgtcg tcgtccacca cagctcgagc accaccacca ccaccactga 960
Claims (9)
- A kind of 1. application of EGFP-CTA2-TAT fusion proteins in fluorescence probe is prepared, it is characterised in that：Described EGFP- The amino acid sequence of CTA2-TAT fusion proteins is as shown in SEQ ID NO.1.
- 2. application of the EGFP-CTA2-TAT fusion proteins according to claim 1 in fluorescence probe is prepared, its feature exist In：The nucleotide sequence of the EGFP-CTA2-TAT fusion proteins described in claim 1 is encoded as shown in SEQ ID NO.2.
- 3. application of the EGFP-CTA2-TAT fusion proteins according to claim 1 in fluorescence probe is prepared, its feature exist In：The preparation method of described EGFP-CTA2-TAT fusion proteins, is comprised the following steps：Escherichia coli BL21 (DE3) strain containing purposeful recombinant plasmid pET22b-EGFP-CTA2-TAT is entered Row activation, expand culture；Then the induced expression of target protein is carried out, obtains wet thallus；Wet thallus, which suspends, to be cracked, and is centrifuged, Obtain supernatant protein solution；Supernatant protein solution carries out affinity chromatography, desalination of dialysing, freeze-drying, obtains EGFP- CTA2-TAT fusion proteins.
- 4. application of the EGFP-CTA2-TAT fusion proteins according to claim 3 in fluorescence probe is prepared, its feature exist In：Described activation concretely comprises the following steps：(1) E.coli BL21 (DE3) the culture presevation liquid containing recombinant plasmid pET22b-EGFP-CTA2-TAT is being contained into end Concentration for 50 μ g/mL ammonia benzyl antibiotic LB fluid nutrient mediums in 37 DEG C, 200r/min cultivate 3~4h；(2) bacterium solution made from step (1) is drawn into flat board, 37 DEG C of 12~14h of solid culture；(3) choose the single bacterium that step (2) culture obtains and fall within the LB fluid nutrient mediums containing final concentration of 50 μ g/mL ammonia benzyl antibiotic In 37 DEG C, 200r/min cultivate 12~14h；(4) bacterium solution made from glycerine and step (3) is mixed, contains recombinant plasmid pET22b-EGFP- after being activated CTA2-TAT E.coli BL21 (DE3) monoclonal strain bacterium solution.
- 5. application of the EGFP-CTA2-TAT fusion proteins according to claim 3 in fluorescence probe is prepared, its feature exist In：Described expansion culture concretely comprises the following steps：(1) by E.coli BL21 (DE3) monoclonal strain containing recombinant plasmid pET22b-EGFP-CTA2-TAT after activation Bacterium solution is with volume ratio 1:100 ratio is inoculated into the LB fluid nutrient mediums containing final concentration of 50 μ g/mL ammonia benzyl antibiotic, and 37 DEG C, 200r/min cultivate 12~14h；(2) by the bacterium solution of culture in step (1) according to volume ratio 1:50 are transferred to containing final concentration of 50 μ g/mL ammonia benzyl antibiotic In LB fluid nutrient mediums, 37 DEG C, 200r/min expansion cultures 4h.
- 6. application of the EGFP-CTA2-TAT fusion proteins according to claim 3 in fluorescence probe is prepared, its feature exist In：The condition of described induced expression is：Using final concentration of 1mmoL/L IPTG, 37 DEG C, 200r/min inductions 5h.
- 7. application of the EGFP-CTA2-TAT fusion proteins according to claim 3 in fluorescence probe is prepared, its feature exist In：Described suspension cracking concretely comprises the following steps：Wet thallus is suspended in lysis buffer, lysozyme is added and stirs；Above-mentioned mixed liquor is subjected to multigelation, Until thalline fully cracks, it is no longer sticky.
- 8. application of the EGFP-CTA2-TAT fusion proteins according to claim 3 in fluorescence probe is prepared, its feature exist In：The concrete operations of described affinity chromatography are：Chromatographic column wet method filling material, chromatography column equilibration is carried out with PBS；Supernatant protein solution loading；After loading finishes Chromatographic column is balanced with PBS again；Balance carries out protein ladder elution, the eluent of gradient elution after terminating Respectively：PBS containing final concentration of 20mM imidazoles, the PBS containing final concentration of 60mM imidazoles, containing final concentration of The PBS of 100mM imidazoles, the PBS containing final concentration of 200mM imidazoles, the PBS containing final concentration of 300mM imidazoles Buffer solution, the PBS containing final concentration of 500mM imidazoles；Collect the PBS elution containing final concentration of 100mM imidazoles Obtained collection liquid.
- 9. application of the EGFP-CTA2-TAT fusion proteins according to claim 3 in fluorescence probe is prepared, its feature exist In：The concrete operations of described dialysis desalination are：The collection liquid that PBS containing final concentration of 100mM imidazoles affords is utilized respectively PBS, volume integral Count the PBS for 50%, water carries out dialysis desalination, dialyzate of interior replacing per 8h.
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